Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

An extended one-generation study with sodium trifluoroacetate is currently ongoing and is anticipated to be finalized by the end of October 2021.

Link to relevant study records

Referenceopen allclose all

Endpoint:
reproductive toxicity, other
Remarks:
Dose Range finder for the extended one-generation reproductive performance study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From 2020-06-17 to 2020-08-29 (day of necropsy)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
no guideline required
Principles of method if other than guideline:
Due to the preliminary nature of this study no specific regulations or guidelines are applicable.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Trifluoroacetic acid (TFA) is a corrosive liquid. To identify the systemic hazards of the substance, testing with the neutral salt sodium trifluoroacetate is considered appropriate to avoid local corrosive effects at the site of administration.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited, UK
- Strain: Wistar rat, RccHan™;WIST
- Sex: females; stock males used for mating
- Age of F0 animnals at the start of the study (GD0): 77 to 83 days old
- Weight range of F0 animals at the start of the study (GD0): 174 to 204 g
- Housing:
♦ During acclimatisation, up to 4 animals per sex per cage in solid bottomed polycarbonate cages, with bedding (softwood based bark-free fiber).
♦ During mating, 1 male/1 female in polycarbonate cages with a stainless steel mes lid and floor.
♦ After mating, during gestation, birth and lactation, the females were individually housed in solid bottomed polycarbonate cages, with bedding (softwood based bark-free fiber).
♦ The selected F1 animals were pair housed as siblings in solid bottomed polycarbonate cages, with bedding (softwood based bark-free fiber).
- Diet: SDS VRF1 Certified, powdered diet, ad libitum.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals, ad libitum.
- Acclimation period: 5 days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC.
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes: not reported, Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 June 2020 To: 29 August 2020
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and stability and homogeneity at a concentration of 50 to 10000 ppm was determined as part of another study (Covance Study no. LK56VW).

DIET PREPARATION
- Rate of preparation of diet (frequency): Once weekly
- Mixing appropriate amounts with (Type of food): The test substance was mixed with SDS VRF1 Certified, powdered diet. The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. The amount of test substance required for the premix was added to an equal amount of plain diet and stirred. An amount of plain diet equal to the weight of the mixture was added and the mixture was stirred again until visibly homogenous. The doubling up process was repeated until approximately half the premix diet was added. At this stage the mixture was ground with a mechanical grinder. The mixture was made up to the weight of the premix with plain diet. The premix was then mixed using a turbula mixer for 200 cycles. This premix was diluted with further quantities of plain diet using the doubling up process to prepare the test mixes. Each formulation was mixed using a Turbula mixer for 200 cycles.
- Storage temperature of food: Ambient (15 to 25°C). A stability of 22 days under these conditions was confirmed.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Duration of pairing: Nominally four days
- Proof of pregnancy: vaginal plug/sperm in vaginal smear referred to as Day 0 of pregnancy (GD 0)
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No formulation analysis was performed in this study.
Duration of treatment / exposure:
- F0 females: From Day 6 after mating up to weaning of the F1 offspring.
- F1 animals: From mid lactation (when offspring would be expected to start to consume diet) until Day 35 of age.
During the lactation phase, from Day 1 to Day 21, females received reduced dietary concentrations of 700, 1700 or 4200 ppm. Due to the preliminary nature of this study no specific regulations or guidelines are applicable.
Frequency of treatment:
Continuously via the diet
Dose / conc.:
1 400 ppm (nominal)
Remarks:
Main phase
Dose / conc.:
3 400 ppm (nominal)
Remarks:
Main phase
Dose / conc.:
8 400 ppm (nominal)
Remarks:
Main phase
Dose / conc.:
700 ppm (nominal)
Remarks:
Lactation phase
Dose / conc.:
1 700 ppm (nominal)
Remarks:
Lactation phase
Dose / conc.:
4 200 ppm (nominal)
Remarks:
Lactation phase
No. of animals per sex per dose:
F0 generation: 6 females per group
F1 generation: 6 males and 6 females per group. The offspring with the lowest within-litter identification per sex from each selected litter was selected to form the F1 generation, after exclusion of grossly atypical animals (where possible, one male and one female from each litter). Selected animals were microchipped on Day 18-20 of age and separated from littermates on Day 21 of age.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels of 1400, 3400, and 8400 ppm, corresponding to approximately 100, 250 and 625 mg/kg/day, were selected based on the results of a combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening (OECD TG422, CitoxLab 2012) supported by a 90-day repeat dose study in rats (Bayer 2007, Report no: SA 06080).
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal to monitor general health at the following time points:
♦ F0 females: Gestation days 0, 5, 12, 18 and 20. Lactation days 1, 7, 14 and 21.
♦ Selected F1 generation: once each week from from PND 21 to PND 35.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 Females: Gestation days 0, 3, 6, 10, 14, 17 and 20 and Lactation days 1, 4, 7, 11, 14, 18 and 21. F1 selected animals: Twice weekly from PND 21 to PND 35.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured as follows:
♦ F0 females: Gestation days 0-2, 3-5, 6-9, 10-13, 14-16 and 17-19. Lactation days 1-3, 4-6, 7-10, 11-13, 14-17 and 18-20.
♦ Selected F1 generation: Twice weekly from PND 21 to PND 35.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

OTHER:
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Oestrous cyclicity (parental animals):
Not examined.
Sperm parameters (parental animals):
Not examined.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring during littering phase:
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatmentd
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1 to 21 of age.
- Sex ratio of each litter: Recorded on Days 1, 4 (before and after culling) and 11, 14, 17 and 21 of lactation
- Individual offspring body body weights: Days 1, 4, 7, 11, 14, 17 and 21 of age.

GROSS EXAMINATION OF DEAD PUPS:
yes, where possible, a fresh macroscopic examination with an assessment of stomach for milk content was performed. Abnormal tissues were retained in an appropriate fixative.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: Surviving F0-females were sacrificed on LD21. F0-females that failed to produce viable litters were sacrificed on day 25 after mating. Mothers of litters that dyed before weaning were killed on day on which the last offspring died.

GROSS NECROPSY
- After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The liver was weighed and samples fixed for all F0 females. These samples were not processed histologically, but retained against any future requirement for microscopic examination. For F0-females the number of implantation sites was recorded. In addition, for females whose litters died before LD 21 the appearance of mammary tissue was checked.
Postmortem examinations (offspring):
SACRIFICE
- Unselected F1 offspring were sacrificed at culling on PND 4, at scheduled kill on LD 21, or selected spares after establishment of selected F1 generation.
- Selected F1 animals were sacrificed on Day 35 of age

GROSS NECROPSY
- Culled offspring with no clinical signs on Day 4 of age were killed and discarded without necropsy examination. Unselected offspring at scheduled kill on Day 21 of age and selected offspring on Day 35 were subject to complete macroscopic examination. Abnormal tissues retained in an appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGTHS
The liver was weighed and samples fixed for all F0 females. These samples were not processed histologically, but retained against any future requirement for microscopic examination. The number of implantation sites were recorded for each females.
Statistics:
The following sequence of statistical tests was used for body weight, food consumption, implantations, litter size, sex ratio, post implantation survival index and organ weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett, 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied.
If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pre-treatment data, Kruskal-Wallis’ test (Kruskal and Wallis, 1952; 1953) was used to test for group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests were made. For all other analyses the H1 approximate test, the non parametric equivalent of the F1 test, was applied.
- For live birth and viability indices dichotomized to 1 when 100% and 0 otherwise, if the Cochran Armitage test was significant at the 5% level, then the direction of the trend was established and one-tailed step-down testing in this direction was performed. If the Cochran-Armitage test was not significant at the 5% level, then a Chi-square test was applied. If the Chi-square test was significant at the 5% level, the treatment groups were compared using pairwise comparisons of each dose group against the Control using Fisher’s exact tests; otherwise, no further comparisons were made.
Reproductive indices:
♦ Gestation index (%) = (Number of live litters born) / (Number of pregnant) x 100
Offspring viability indices:
♦ Post-implantation survival index (%) = (Total number of offspring born) / (Total number of uterine implantation sites) x 100
♦ Live birth index (%) = (Number of live offspring on Day 1 after littering) / (Total number of offspring born) x 100
♦ Viability index (%) = (Number of live offspring on Day 4 before culling) / (Number live offspring on Day 1 after littering) x 100
♦ Lactation index (%) = (Number of live offspring on Day 21 after littering) / (Number of live offspring on Day 4 (after culling) x 100
♦ Percentage males = (Number of males in litter) / (Total number of offspring in litter) x 100. The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after culling) 11, 14, 17 and 21 of age.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs at routine physical examination that could be attributed to administration of Sodium Trifluoroacetate.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following the start of treatment on GD6 animals receiving Sodium Trifluoroacetate showed low body weight gain up to GD10 (p<0.01); a dose response was apparent. At 8400 ppm the overall body weight gain for females from GD6 to GD20 was marginally low at approximately 92% of Controls although this difference did not attain statistical significance; body weight gain at 1400 or 3400 ppm over the same period was similar to Controls.
Absolute body weights for all treated females were low when compared with Controls during lactation with the difference at 4200 (8400) ppm attaining statistical significance (p<0.05) on Lactation Day 1 (LD1); a dose response was not apparent. However, overall body weight gains for treated females showed no adverse effect of treatment. Detailed results are provided in Table 1 (see attached document).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 8400 ppm food consumption was low from GD6 to GD19 at approximately 88% of Controls; with the mean value for GD6-9 attaining statistical significance (p<0.05). Food consumption at 1400 or 3400 ppm over the same period was similar to Controls. During lactation food consumption for females receiving 4200 (8400) ppm was low at approximately 87% Controls, with the difference attaining statistical significance (p<0.05) on LD4 to LD6; food consumption at 700 (1400) or 1700 (3400) ppm was similar to Controls.
Achieved dose levels in F0 females are provided in Table 2 (see attached document).
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
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
- Gestation index: The duration of gestation and the gestation index were unaffected by administration of Sodium Trifluoroacetate at dose levels up to and including 8400 ppm
- Litter size/Survival indices: Live birth index was slightly low at 4200 (8400) ppm, with viability index on Day 4 unaffected, resulting in a marginally low mean litter size prior to litter standardisation on Day 4 of age. Offspring survival from Day 4 of age was unaffected by administration of the test item. Litter size and survival at 700 (1400) or 1700 (3400) ppm showed no differences that could be related to treatment.
- Sex ratio: There was no effect of maternal treatment on sex ratio at dose levels up to and including 8400/4200ppm.
Clinical signs:
no effects observed
Description (incidence and severity):
The clinical condition of offspring was unaffected by administration of Sodium Trifluoroacetate to maternal animals at dose levels up to and including 4200 (8400) ppm.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Unselected offspring (LD1-21): On Day 1 of age the mean absolute body weight for both male and female offspring were low when compared with Controls (p<0.05) at 4200 (8400) ppm and marginally low at 1700 (3400) ppm. Body weight gain up to Day 4 of age was essentially similar across the groups, however during Days 4-7 of age weight gain for offspring at all dose levels was significantly low when compared with Controls (p<0.05); a dose response was apparent for males but not females. Thereafter, body weight gains remained marginally low at 4200 (8400) ppm. Overall body weight gain (LD1-21) for offspring at 4200 (8400) ppm was low when compared with Controls (p<0.05; approximately 87% of Controls); at 700 (1400) or 1700 (3400) ppm overall body weight gain was similar to Controls.
Selected offspring (Day 21-35): At weaning on Day 21 of age mean bodyweight for males and females at 8400 ppm was low at approximately 89% of Controls (p<0.05); mean bodyweights at 1400 or 3400 ppm were similar to Controls. Subsequent body weight gain from Day 21 to Day 24 of age was similar to Controls for both male and females at all dietary concentrations. From Day 24 of age selected male animals showed low body weight gain when compared with Controls and the overall weight gain at 8400 ppm from Day 21 to Day 35 of age was approximately 79% of Controls; males receiving 1400 or 3400 ppm showed weight gain that was similar to Controls. From Day 24 to Day 28 female animals at 3400 or 8400 ppm showed slightly but significantly low body weight gain when compared with Controls (p<0.05). The overall body weight gain for selected females from Day 21 to Day 35 of age was low at approximately 85% of Controls for females at 3400 ppm however this difference was not apparent at 1400 or 8400 ppm. Detailed results are provided in Table 4 (see attached document).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall food consumption from Day 21 to Day 34 of age was marginally low for animals at 3400 or 8400 ppm at approximately 89/90% of Controls; food consumption at 1400 ppm was similar to Controls. Achieved dose levels in F1 animals are provided in Table 5 (see attached document).
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
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight relative liver weight for both male and female animals were high when compared with Controls (p<0.01) at all dietary concentrations; a dose response was apparent. Detailed results are provided in Table 6 (see attached document).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring that died prematurely and those at scheduled termination on Day 21 of age did not reveal any findings that could be attributed to Sodium Trifluoroacetate. Macroscopic examination of selected F1 animals on Day 35 of age did not reveal any findings that could be attributed to administration of Sodium Trifluoroacetate.
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
no

Proof of absorption

Blood samples were collected 0.5-1hr, 4.5-5hr and 8.5-9hr after light was switched on. Blood was collected during the gestation period (GD 17) and the lactation period (LD11) from n=6/females per dose group. The first data point at 0.5-1hr was also used as a 24-h value in the TK calculations[1].


TFA groups:

F0: At GD 17 mean maximum concentrations of 214, 307 and 485 mg/L were measured at the 0.5-1 hour sampling time point on GD 17. AUC242values were 4819, 6892 and 10285 µg*h/mL for the low, mid-and high-dose group. Compared to the external applied / achieved dose level the internal dose (described as AUC24) showed a sub-proportional increase, indicating that the absorption is affected.

Comparable results were found for the measurements on LD 11. Mean maximum concentrations were 171 mg/L at 4.5-5 hours at the low dose and 279 and 405 mg/L at 0.5-1 h in the mid- and high-dose group. AUC24 values were 4032, 6334 and 9304 µg*h/mL.

 

F1: For F1 animals on PND 4 and 21 only one sample was taken, therefore it is not possible to calculate an AUC24.

In pooled samples from PND 4 males and females TFA plasma concentrations were much lower without showing a dose-correlation. Mean cMaxvalues on PND 4 (measured for pooled samples from males and females) were comparable in all dose groups, with values of 36.4, 33.7 and 34.2 mg/L at the low, mid- and high dose. At PND 21 (unclear when plasma samples were taken) TFA plasma concentrations in F1 weanlings were similar to the values of the first sampling timepoint at 0.5-1 hr in dams. Mean plasma concentrations were 140, 289 and 342 mg/L in males, and 142, 276 and 394 mg/L in females at the low- mid- and high-dose, respectively.

 

In dams, relatively high initial plasma concentrations were noted at the 0.5-1 hr sampling time point across all dose groups which did not decline relevantly within the sampling interval of ≈ 9 hours, assuming either a strong oral absorption and/or a slow clearance from blood (probably saturation of excretion/metabolism). Increasing the external dose from 97.5 to 230 mg/kg bw was associated with a slightly less than dose-proportional increase in the cMaxand the AUC24. Internal exposure still increased from ≈ 230 to 547 mg/kg bw, but in a clearly less-than dose proportional manner.

[1] The AUC24 is calculated, as described by Jochemsen et. al. 1993, by taking the concentrations measured during the daylight hours and using the concentration measured at lights on again at 24 hours post the original time point to cover night-time exposure.

Jochemsen R, Bazot D, Brillanceau MH and Lupart M (1993). Assessment of drug exposure in rat dietary studies. Xenobiotica, 23(10), 1145-1154

Conclusions:
On the basis of this preliminary reproductive study in rats, dose levels of 10, 50 and 250 mg/kg bw/day were selected for the subsequent Extended One-generation Reproductive Study.
Executive summary:

This study was performed to assess the influence of sodium trifluoroacetate on reproductive performance when administered continuously via the diet to female Han Wistar rats, and to establish suitable treatment levels for the Extendend one-generation reproductive performance study. In the F0 generation, three groups of six female rats received Sodium Trifluoracetate at dietary concentrations of 1400, 3400 or 8400 ppm orally, during gestation from Day 6 after mating. During the lactation phase, from Day 1 to Day 21, females received reduced dietary concentrations of 700, 1700 or 4200 ppm. A similarly constituted Control group received untreated basal diet. In the F1 generation, six males and six females were treated from weaning to their scheduled termination on Day 35 of age at the same dietary concentrations as the F0 generation main phase. A similarly constituted Control group received untreated basal diet.

During the study, for the F0 generation, clinical condition, body weight, food consumption, gestation length and parturition observations, organ weight and macroscopic pathology investigations were undertaken. For the F1 generation, clinical condition, body weight, food consumption, organ weight and macroscopic pathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio and body weight for all offspring were also assessed. In addition, to determine systemic exposure, plasma concentrations of TFA were determined at each dosage level.

Results

F0 Maternal responses

During gestation females receiving 1400, 3400 or 8400 ppm the mean achieved dose levels were 97.5, 230 or 547 mg/kg/day and during the first two weeks of lactation the achieved dose levels for females receiving 700, 1700 or 4200 ppm were 110.2, 262 or 578 mg/kg/day. Clinical condition, gestation length, gestation index and macropathology of F0 females was unaffected by administration of Sodium Trifluoroacetate. At 8400 ppm the overall body weight gain and food consumption for females from GD6 to GD20 was low at approximately 92% and 88% of Controls respectively; body weight gain and food consumption at 1400 or 3400 ppm over the same period was similar to Controls. During lactation the absolute body weights for all treated females were low when compared with Controls; however, overall body weight gains for treated females showed no adverse effect of treatment.  During lactation food consumption for females receiving 4200(8400) ppm was low at approximately 87% Controls; food consumption at 700(1400) or 1700(3400) ppm was similar to Controls. On Day 21 of lactation the mean body weight relative liver weights for females that received 1700(3400) or 4200(8400) ppm were slightly high at approximately 111% of Controls; however, these differences did not attain statistical significance. Liver weight at 700(1400) ppm was similar to Controls.

F1 Litter Responses

General condition and macropathology for offspring were unaffected by administration of Sodium Trifluoroacetate to maternal animals at dose levels up to and including 4200 (8400) ppm. Live birth index was slightly low at 4200 (8400) ppm, with viability index on Day 4 unaffected, resulting in a marginally low mean litter size prior to litter standardisation on Day 4 of age. Offspring survival from Day 4 of age was unaffected by administration of the test item.

Litter size and survival at 700(1400) or 1700(3400) ppm showed no differences that could be related to treatment and there was no effect of maternal treatment on sex ratio at dose levels up to and including 8400/4200ppm.

On Day 1 of age the mean absolute body weight for both male and female offspring were low when compared with Controls (p<0.05) at 4200(8400) ppm and marginally low at 1700(3400) ppm. Overall body weight gain (LD1-21) for offspring at 4200(8400) ppm was low when compared with Controls (p<0.05; approximately 87% of Controls); at 700(1400) or 1700(3400) ppm overall body weight gain was similar to Controls.

F1 Generation Responses

The mean achieved dose levels for selected animals from weaning up to Day 34 of age was 202, 476 and 1290 mg/kg/day for males and 228, 557 and 1369 mg/kg/day for females at 1400, 3400 and 8400 ppm, respectively. Clinical condition and macropathology of F1 females were unaffected by administration of Sodium Trifluoroacetate. At weaning on Day 21 of lactation mean bodyweight for males and females at 8400 ppm was low at approximately 89% of Controls (p<0.05); mean bodyweights at 1400 or 3400 ppm were similar to Controls. At 8400 ppm the overall weight gain from Day 21 to Day 35 of age was approximately 79% of Controls for males and 85% of Controls for females; overall weight gain for animals receiving 1400 or 3400 ppm was similar to Controls.

Overall food consumption from Day 21 to Day 34 of age was marginally low for animals at 3400 or 8400 ppm at approximately 89/90% of Controls; food consumption at 1400 ppm was similar to Controls.

The mean body weight relative liver weight for both male and female animals were high when compared with Controls (p<0.01) at all dietary concentrations; a dose response was apparent.

In dams, relatively high initial plasma concentrations were noted at the 0.5-1 hr sampling time point across all dose groups which did not decline relevantly within the sampling interval of ≈ 9 hours, assuming either a strong oral absorption and/or a slow clearance from blood (probably saturation of excretion/metabolism). Increasing the external dose from 97.5 to 230 mg/kg bw was associated with a slightly less than dose-proportional increase in the cMaxand the AUC24. Internal exposure still increased from ≈ 230 to 547 mg/kg bw, but in a clearly less-than dose proportional manner.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
other justification
Justification for data waiving:
other:
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes
Limit test:
no
Justification for study design:
The study is being performed in rats according to OECD guideline 443 in compliance with GLP including a premating exposure period of ten weeks for the parental (P0) generation; The test substance is administered by the oral, dietary route. The basic configuration of EOGRTS is performed as based on the toxicological profile of the substance there are no concern-driven scientific triggers for the performance of the F2 generation (extension of Cohort 1B), developmental neurotoxicity (DNT; cohorts 2A and 2B) and/or developmental immunotoxicity (DIT; cohort 3) cohorts.

- Premating exposure duration for parental (P0) animals: A premating exposure period of 10 weeks is used to cover the full spermatogenesis and folliculogenesis before the mating.

- Basis for dose level selection: The highest dose level was selected with the aim to induce some systemic toxicity, but not death or severe suffering of the animals. The dose levels were selected based on the dose-range-finding study (Covance 2020, Study no. 8437241), plasma TK-data, as well as further repeat dose toxicity studies conducted with the test compound. In the dose-ranger finder study, body weight gains were dose-related statistically significantly reduced during the first days of gestation at all dose levels. At 3400 ppm (229 mg/kg/day) body weights were reduced by 38% although there was no effect on food consumption at this dose. Liver weights were dose-related increased in F0 and liver weights were statistically significantly high in F1 animals. These findings correlate with observation in the 90-day rat study (Bayer 2007, Report no: SA 06080) where histopathological liver findings were observed (hypertrophy and necrotic foci). In addition, glucose and bilirubin vales were statistically significantly decreased, and urinary volume was increased at 100 mg/kg/day and above. Plasma TK-data of the dose range finding study (Covance 2020, Study no. 8437241) demonstrate very high plasma concentrations as well as a sub-proportional increase, indicating that the absorption is affected. The dose levels for the final study were set at 120, 600 and 3000 ppm in diet, corresponding to approx. 10, 50 and 250 mg/kg/day.

- Inclusion/exclusion of extension of Cohort 1B: According to column 2 (specific rules for adaptation from column 1) point 8.7.3 of the amended REACH Annex X, extension of cohort 1B to include the F2 generation shall be proposed by the registrant based on the following conditions being met (a and any of b(i), b(ii) or b(iii)). See also: Chapter R.7a: Endpoint specific guidance Version 5.0 - December 2016:
A. The substance has uses leading to significant exposure of consumers or professionals, taking into account, inter alia, consumer exposure from articles
No – The substance has no uses leading to significant exposure of consumers or professionals. The substance has only industrial uses.
B (i). The substance displays genotoxic effects in somatic cell mutagenicity tests in vivo which could lead to classifying it as Mutagen Category 2, or
No – The substance is not classified as Mutagen Category 1A or 1B or 2.
B (ii). There are indications that the internal dose for the substance and/or any of its metabolites will reach a steady state in the test animals only after an extended exposure, or
No – TFA is rapidly absorped by oral route. TFA is fully miscible in water, has a log Pow of 0.79 and does not trigger any bioaccumulation potential. Therefore there are no indications that the internal dose for the substance will reach a steady state in the test animals only after an extended exposure.
B (iii) There are indications of one or more relevant modes of action related to endocrine disruption from available in vivo studies or non-animal approaches
No - There are no indications based on the available study results that endocrine disruption is a relevant mode of action for the substance. In particular, no effects on reproductive organs or tissues or effects on the thyroid were evidenced in the available repeated dose toxicity studies. The substance also does not have a structural similarity to steroid hormones.

Therefore, based on the above considerations, the registrant does not believe that there is a basis for extending cohort 1B to include the F2 generation.

- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: The registrant does not believe there is a need to include cohorts 2A and 2B in the test design. This is based on:
• Neurobehavioural observations (arena and Functional Observational Battery testing) and motor activity assessment performed as part of the subchronic toxicity study, did not indicate any neurotoxic potential of the test material.

- Inclusion/exclusion of developmental immunotoxicity Cohort 3: The registrant does not believe there is a need to include cohort 3 in the test design. This is based on:
• the substance has not caused biologically significant changes in haematology/clinical chemistry and/or organ weight associated with immunotoxicity such as reduced leucocyte count in combination with reduced spleen weight in repeated dose studies
• the substance has not caused significant effects to immunology organs such as thymus atrophy in repeated dose studies

- Route of administration: The oral, dietary route has been selected as it is the most appropriate route of administration for substances to focus on the detection of hazardous properties on reproduction.
Specific details on test material used for the study:
Trifluoroacetic acid (TFA) is a corrosive liquid. To identify the systemic hazards of the substance, testing with the neutral salt sodium trifluoroacetate is considered appropriate to avoid local corrosive effects at the site of administration.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo (UK) Limited
- Strain: RccHan; WIST
- Age at study initiation: Females and males, approx. 22-28 Days
- Weight range: 20 g per sex
- Housing:
♦ From arrival to pairing and after mating (males), 4 animals per sex per cage in solid bottomed polycarbonate cages, with bedding (Softwood based bark-free fiber).
♦ During mating, 1 male/1 female in polycarbonate cages with a stainless steel mes lid and floor.
♦ After mating, during gestation, birth and lactation, the females will be individually housed in solid bottomed polycarbonate cages, with bedding (Softwood based bark-free fiber).
♦ Offspring (from weaning to selection): 1 litter / cage
♦ Selected F1-animals: up to 4 animals /sex/cage in solid bottom polycarbonate cages with softwood-based bark-free fiber bedding.
- Diet: SDS VRF1 Certified, powdered diet, ad libitum (except when diet removed overnight for blood sampling for hematology or blood chemistry or during urine collection)
- Water: Potable water from the public supply, ad libitum via polycarbonate bottles with sipper tubes (except when removed overnight during urine collection).
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Maintained within the range of 20-24ºC.
- Humidity (%): Maintained within the range of 40-70%.
- Air supply: not reported¸ Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 October 2020 To: July 2021 (expected end date of the in-life phase)
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and stability and homogeneity at a concentration of 50 to 10000 ppm was determined as part of another study (Covance Study no. LK56VW).

DIET PREPARATION
- Rate of preparation of diet (frequency): Once weekly
- Mixing appropriate amounts with (Type of food): The test substance will be mixed with SDS VRF1 Certified, powdered diet.
- Storage temperature of food: Ambient (15 to 25°C) - 22 days stability
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug/sperm in vaginal smear referred to as Day 0 of pregnancy (GD 0)
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At specified intervals during treatment, the test formulations will be analyzed for achieved concentration of the test item on 3 suitable occasions. The analytical method was validated in the previous Covance Study No. LK56VW and involves extraction from the diet by mechanical shaking with water, dilution of the extracts initially with solvent and finally with control diet extract, followed by quantification with LC-MS/MS chromatographic assay.
Duration of treatment / exposure:
In the F0 generation, three groups of 25 rats/sex received Sodium Trifluoracetate at dietary concentrations of 120, 600 or 3000 ppm, corresponding to nominal dose levels of 10, 50 and 250 mg/kg bw/day, during 10 weeks premating, gestation and lactation. To compensate for the higher food intake during lactation and dietary concentrations were reduced to 60, 300 and 1500 ppm during lactation. A similarly constituted Control group received untreated basal diet.
In the F1 generation, 20 rats/sex/group were treated from weaning at PND 21 until termination at an approximate age of 13 weeks (cohort 1A) or 14 weeks (cohort 1B). From PND 21 to 35 dietary concentration were the same as during the lactation phase, i.e. 60, 300, and 1500 ppm, afterwards until termination dietary levels were 120, 600 and 3000 ppm. A similarly constituted Control group received untreated basal diet.
Frequency of treatment:
Continuously via the diet
Dose / conc.:
120 ppm (nominal)
Remarks:
Main phase
Dose / conc.:
600 ppm (nominal)
Remarks:
Main phase
Dose / conc.:
3 000 ppm (nominal)
Remarks:
Main phase
No. of animals per sex per dose:
Parent generation: 25/sex/group
Cohort 1A: One male and/or one female per litter will be selected (20/sex/group)
Cohort 1B: One male and/or one female pup per litter will be selected (20/sex/group)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dose levels 120, 600 and 3000 ppm, corresponding to 10, 50 and 250 mg/kg/day were selected based on the dose-range-finding study (Covance study no. 8437241), plasma TK-data, as well as further repeat dose toxicity studies conducted with the test compound. In the dose-range finding study body weight gains were dose-related statistically significantly reduced during the first days of gestation at all dose levels. At 3400 ppm (229 mg/kg/day) body weights were reduced by 38% although there was no effect on food consumption at this dose. Liver weights were dose-related increased in F0 and liver weight were statistically significantly high in F1 animals. These findings correlate with observation in the 90-day rat study (Report no: SA 06080) where histopathological liver findings were observed (hypertrophy and necrotic foci). In addition, glucose and bilirubin vales were statistically significantly decreased, and urinary volume was increased at 100 mg/kg/day and above. Preliminary plasma TK-data of the dose range finding study (Study No. 8437241) demonstrating very high plasma concentrations, as well as indications that elimination and metabolism is already affected. To account / compensate for the higher food intake during lactation and in the early post-weaning phase, dietary levels were reduced to 60, 300 and 1500 ppm during lactation and for F1 offspring from PND 21 to PND 35.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- F0 and selected F1 generation: Twice daily observations were conducted for moribundity, mortality, and clinical signs by observing the animals in their cages. From GD 20 females will be inspected three times daily for evidence of parturition. The progress and completion of parturition wwill be monitored, numbers of live and dead offspring will be recorded and any difficulties observed were recorded. The gestation index will be calculated.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations will be done once per week for all F0 and selected F1 generation animals. Observations were made for F0 animals on Gestation days 0, 5, 12, 18 and 20 and on Lactation days 1,7, 14 and 21.

BODY WEIGHT: Yes
- F0 males: Day on which treatment commences, weekly thereafter and before necropsy.
- F0 females: Day on which treatment commences, weekly thereafter until mating is detected, on gestation days 0, 7, 14, and 20; lactation days 1, 4, 7, 14, 18 and 21, and before necropsy.
- F1 selected animals: Day 25 of age and then weekly from nominal 4 weeks of age (at the formal start of the F1 generation).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was measured as follows:
F0-Males: Each week until paired for mating
F0-Females: Each week until paired for mating; Gestation days 0-6, 7-13, and 14-19; Lactation days 1-3, 4-6, 7-13, 14-17 and 18-20
Selected F1: Twice weekly from nominal 4 weeks of age.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

CLINICAL PATHOLOGY
- Blood samples from F0 adults will be taken at termination after overnight fasting (2 x 0.5 mL for hematology; 0.7 mL for clinical chemistry) and the following parameters assessed:
- Haematology: Haematocrit, hemoglobin, erythrocyte count, total leucocyte count, differential leucocyte count, platelet count, MCH, MCV, MCHC, prothrombin time and activated partial thromboplastin time.
- Clinical chemistry: ALP, ASAT, ALAT, -GT, gluciose, bilirubin, cholesterol, triglycerides, non-esterified fatty acids, creatinine, urea, protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, phosphorous

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

OTHER: T4 AND TSH HORMONE DETERMINATIONS: See Table 3 (Any other information on materials and methods)

Oestrous cyclicity (parental animals):
Estrous cycle assessment is done by dry vaginal smears for 15 days before pairing and by wet smears after mating until evidence of mating.
Sperm parameters (parental animals):
Sperm analysis will be done for F0 animals at scheduled termination.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On Day 4 of age, litters containing more than eight offspring will be reduced to eight by random culling, leaving, whenever possible, five male and five female offspring in each litter.

PARAMETERS EXAMINED
- Litter evaluation: Litter size will be recorded daily from postnatal day (PND) 1 to PND 21. The sex ratio of each litter will be recorded on PND 1, 4 (before and after culling) and PND 21.
- Mortality and clinical signs: Twice daily observations will be conducted for moribundity, mortality, and clinical signs by observing the animals in their cages. Detailed physical examinations will be done once per week
- Body weight: Individual offspring body weights will be recorded on PND 1, 4, 7, 14, and 21 for all offsprings. In addition, on PND 22 for unselected F1-offspring, and on PND 25 for selected F1 offspring and then weekly until termination.
- Food consumption: For selected F1 animals, twice weekly from nominal 4 weeks of age.
- Nipple retention in male pups: Nipple count will be done on male offspring on PND 13.
- T4 and TSH hormone determinations: See Table 3 (Any other information on materials and methods). Samples from F1-adults and F1 offspring at Day 22 will be analysed for T4 by LC-MS/MS. Samples from F1-offspring at Day 4 will be stored frozen pending analysis. Serum samples for TSH analysis will be stored frozen until analysis. TSH analysis is done using Millipore Luminex multiplex kits.
- Sexual maturation: Males will be examined daily from Day 38 of age for the completion of balano preputial separation. Body weight recorded on day of completion of separation. Females will be examined daily from Day 25 of age until vaginal opening occurs. Body weight recorded on day of vaginal opening. The sex ratio of each litter will be recorded on PND 1, 4 (before and after culling) and PND 21.
- Estrous cycle: For Cohort 1A females estrous cycle monitoring is done by wet smears following onset of vaginal patency until the first cornified (estrus) smear is recorded. In addition, for at least 3 days prior to the start of the necropsy phase and on the day of termination. Dry smears were taken for 2 weeks from nominal Day 75 of age [relative to the formal commencement of the F1 generation at nominal Day 28 of age (28±2 days of age)]. For Cohort 1B females estrous cycle monitoring is done by wet smears for at least 3 days prior to the start of the necropsy phase and on the day of termination.
- Clinical pathology: Blood samples from F1-cohort 1A adults will be taken at termination after overnight fasting (2 x 0.5 mL for hematology; 0.7 mL for clinical chemistry) and the following parameters assessed:
• Haematology: Haematocrit, hemoglobin, erythrocyte count, total leucocyte count, differential leucocyte count, platelet count, MCH, MCV, MCHC, prothrombin time and activated partial thromboplastin time.
• Clinical chemistry: ALP, ASAT, ALAT, -GT, gluciose, bilirubin, cholesterol, triglycerides, non-esterified fatty acids, creatinine, urea, protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, phosphorous
- Urinalysis: Urine will be collected at termination from 10 F1 cohort 1A rats/sex/dose. The following parameters will be assessed: appearance, volume, pH, specific gravity, glucose, ketone, bilirubin (bile pigments) blood pigments, protein, sodium, potassium, chloride. In addition, microscopic examination of sediment for epithelial cells, leucocytes, erythrocytes, crystals, casts, spermatozoa and other abnormal components.
- Sperm Analysis: Sperm analysis will be done for selected F1 animals at scheduled termination.
- Immunox analysis: Ten males and ten females per group from Cohort 1A, from as many litters as possible, will be selected for immunophenotyping (See Table 2 for the cell markers that will be assessed).
Postmortem examinations (parental animals):
SACRIFICE
Surviving F0-females will be sacrificed on PND 28. F0-females that failed to produce viable litters and F0-females with total litter loss will be sacrificed with the first cohort of females with live litters. F0-females that are failing to mate will be terminated as soon as possible if an estrous smear is seen after completion of the mating period, or in case no estrous smear is seen will be terminated on day 25 after the last day of pairing.

GROSS NECROPSY
All F0 females will be subject to a detailed necropsy. For F0-females the number of implantation sites will be recorded.

HISTOPATHOLOGY/ORGAN WEIGHTS
Selected tissues and any abnormalities of F0 are fixed and examined. Details will be provided in the final report. The tissues indicated in Table 1 will be weighed.
Postmortem examinations (offspring):
SACRIFICE
Unselected F1 offspring will be sacrificed at culling on PND 4, and on LD 22. Selected F1 animals of Cohort 1A will be sacrificed at approximately 13 weeks of age and F1-cohort 1B adults are sacrificed at approximately 14 weeks of age.

GROSS NECROPSY
All F1 animals of the selected F1 generation will be subject to a detailed necropsy.

HISTOPATHOLOGY/ORGAN WEIGHTS
Selected tissues and any abnormalities of unselected F1 and selected F1 of cohort 1A and 1B are fixed and examined. Details will be provided in the final report.
The tissues indicated in Table 1 will be weighed.
Statistics:
The following data types will be analyzed at each timepoint separately, where required, in support of interpretation:
• Body weight, using absolute weights and gains over appropriate study periods.
• Food consumption, over appropriate study periods.
• Estrous cycles, vaginal opening to first estrus and pre-coital interval.
• Mating performance and fertility.
• Gestation length
• Litter size and survival indices.
• Sexual maturation, age and body weight at completion.
• Clinical pathology
• Thyroid hormones (TSH and T4)
• Organ weights, absolute and body weight relative
• Sperm analysis, motility, morphology and count.

Methods
For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group. For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary. Under the advice of the Associate Director, Global Statistics, or other qualified Statistician, alternative or additional methods may be carried out if deemed appropriate following data review. Details will be included in the study report.
Reproductive indices:
• Gestation Index
Offspring viability indices:
The following indices will be calculated:
• Post-implantation survival index
• Live birth index
• Viability index (PND 4)
• Lactation index (LD 21)
Conclusions:
Since the in-life phase of this study is ongoing, no results can be provided at this point of time. Results will be provided after the report is finalized.
Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

An extended one-generation study with sodium trifluoroacetate is currently ongoing and is anticipated to be finalized by the end of October 2021.

In a preliminary EOGRTS (Covance, 2020), sodium trifluoroacetate was administered via the diet to four groups of 6 mated F0 female Wistar rats at levels of 0, 1400, 3400 and 8400 ppm from GD 6 throughout gestation and 0, 700, 1700 and 4200 ppm during lactation until PND 21. The females were allowed to litter and the F1 offspring were treated via the diet at the same dose levels as the pregnant females until PND 35, at which time the study was terminated. To determine systemic exposure, plasma concentrations of sodium trifluoroacetate were determined at each dose level in F0 femals at Gestation Day 17 and Lactation Day 11 and in F1 animals on PND4 and 21.

The calculated achieved maternal dose levels during the gestation phase were 97.5, 230 and 547 mg/kg bw/day. At 97.5 and 230 mg/kg/day litter size at birth, viability of offspring and offspring morphology were similar to those of the controls. At 547 mg/kg/day, litter size at birth was similar, but the number of pups surviving to the designated PND 1 was slightly reduced, mainly due to one litter in which 6/9 pups died within 24 hours of birth. Four of the pups that died were available for examination and, other than a lack of milk in the stomach, no abnormal findings were recorded. Birth weight and body weight gain until PND 35 were reduced but subsequent survival and terminal necropsy of offspring did not show any adverse effects. Mean maximum plasma concentrationsof 214, 307 and 485 mg/L were measured at the 0.5-1 hour sampling time point on GD 17. AUC values were 4819, 6892 and 10285 µg*h/mL for the low, mid-and high-dose group. Compared to the external applied / achieved dose level the internal dose (described as AUC) showed a sub-proportional increase, indicating that the absorption is affected.

In addition, a GLP compliant combined oral repeated dose toxicity study with reproduction/developmental screening study according to GLP and OECD 422 with potassium trifluoroacetate (TFAK) is available which was tested as a reaction mass with potassium trifluoromethanesulpinate (TFSK). The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day active ingredient, The NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day active ingredient, The NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day active ingredient in the absence of any treatment-related effect on pups at this dose-level. In this study, about 50% (49.9%) of the active ingredients is TFAK, the remaining being TFSK. Therefore, the actual TFAK parental toxicity NOAEL, reproductive performance NOEL and toxic effect on progeny are > 500 mg/kg bw/day (CiTox Lab, 2012).

In the available subchronic toxicity study (Bayer, 2007), in which sodium trifluoroacetate was administered to male and female rats at 160, 1600 and 16000 ppm via dietary administration, no histopathological effects were seen on any reproductive organ.

Effects on developmental toxicity

Description of key information

The experimental phase of the developmental toxicity study in rabbits is finalized and the final report of the study is anticipated to be finalized by the end of March 2021. Increased incidence of major and associated minor abnormalities were observed in fetuses/litters across all treatment groups predominantly affecting the eyes and skeletal system. These results are preliminary (pending the finalization of the study report) and evaluation of the relationship of these effects to treatment with sodium trifluoroacetate as the test substance is ongoing. Since similar findings were not observed in a developmental toxicity study in rats at comparable dose levels (Baxter, 2010; Chunsheng, 2020), the findings seem to be rabbit specific. Therefore we consider the necessity for further investigations to elucidate the reason for the species differences and potential mechanism essential to fully understanding the relevance of the findings. A currently ongoing Extended One-Generation study with sodium trifluoroacetate in rats will also provide further information.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Crl:CD (SD) IGS BR)
- Age at study initiation: approximately 10 to 12 weeks of age at receipt on GD 1 to 3
- Weight at study initiation: The weight range was 245 to 299 grams at start of dosing
- Fasting period before study: No
- Diet: Certified Global 16% Protein Rodent Diet No. 2016C (pellet, Harlan), ad libitum
- Water: Water (New Jersey-American Water Company), ad libitum
- Acclimation period: The study animals arrived at the test facility on GD1-3 and were acclimated until GD6.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Daily Average Range, 20 to 23°C
- Humidity (%): Daily Average Range, 47 to 53%
- Air changes (per hr): no information
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: A dosing volume of 5 mL/kg was applied for all animals, which was adjusted based on the latest body weight. No neutralization of the dose formalations was performed. Fresh dose formulations were prepared once daily. The pure test article has a pH of 1.1 and pKa of 0.3, and reacts strongly with water. Therefore, a PAPR with an OV/SD/HV/CL/HE organic vapour filter was used during preparation and dose administration. Due to evaporation properties of the formulations, the containers were covered with parafilm or similar covering during preparation, as well as, during dosing to ensure accurate and consistent concentration results.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine homogeneity, stability, and concentration of the test and/or control materials were carried out at the testing facility.
Details on mating procedure:
The female rats were inseminated at the vendor facility. Gestation Day 0 (GD0) was the day of detection of a copulatory plug in situ and/or sperm in a vaginal smear. The weight range was 245 to 299 grams at start of dosing
Duration of treatment / exposure:
Gestation day 6 up to and including gestation day 19
Frequency of treatment:
Daily
Duration of test:
14 treatment days.
Dose / conc.:
37.5 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 mated female rats
Control animals:
yes, concurrent vehicle
Maternal examinations:
VIABILITY:
Animals were observed in their cages twice daily (once in the morning and once in the afternoon) for mortality, morbidity and signs of severe toxic or pharmacologic effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals on study were examined daily from GD 4 through terminal euthanasia (GD 20). Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluations of respiration. During the stabilization period, and during the post-dosing phase, these evaluations were performed once daily. During the treatment period, these examinations were performed prior to dosing (within approximately 30 minutes) and at approximately 2 hours after dosing. Observations after dosing were limited to recording transient signs directly associated with dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded for all animals on GD 4 and daily from GD 6 through 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Feed was available without restriction 7 days/week. A weighed quantity of feed was presented to each animal daily from GD 4 to GD 18. After 2 or 3 days, any feed remaining was weighed.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: A macroscopic post-mortem evaluation was performed on GD 20, during which corpora lutea, implantation data and uterine weights were recorded. In addition, liver and kidney weights were determined.
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
- Number of corpora lutea;
- Weight of intact gravid uterus;
- Location and number of implantation sites, resorptions, fetal death, dead fetuses;
- Gross evaluation and weight of placentas.
Fetal examinations:
The fetuses were removed, weighed (live and recently dead fetuses), sexed and examined externally for defects. Approximately half of the fetuses were examined for soft tissue abnormalities. The other half was processed for staining with Alizarin Red S and examined for skeletal abnormalities and ossification state.
Statistics:
Statistical analyses were performed using the individual animal (or litter) as the basic experimental unit. For litter/fetal findings, the litter was the treated experimental unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.The following statistical methods have been used for the analysis of the results: Williams' test, Dunnett's test, Shirley's test, Steel's test, Kruskal-Wallis test, Wilcoxon rank sum tests
Indices:
Sex ratio, Pre implantation loss, Post implantation loss, Gravid uterine adjusted body weights, Mean litter placental and fetal weights
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test article-related clinical observations
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females survived to termination of the study
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Description (incidence and severity):
Test article-related organ weight increases were observed in liver and kidney (see Table below in 'any other information on results')
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female each in the control and high-dose groups was not pregnant, although this was not attributed to the administration of the test article.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment related effects on maternal toxicity at the highest dose level
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effect on embryo-fetal development in rats.
Abnormalities:
no effects observed
Developmental effects observed:
no

Analysis of initial mixes confirmed that the preparation procedure used for this study produced homogeneous mixtures and that the test material was stable in the vehicle, under refrigerated conditions, for at least seven days. The formulations were prepared daily for dosing, and no storage was required. Analyses conducted during the treatment period confirmed that dose solutions of appropriate concentration were administered.

Body weight in pregnant rats (body weight on GD20 was adjusted by subtracting weight of uterus with fetuses):

 

Body weight (g)

Kidney (g)

Liver (g)

Dose (mg/kg)

GD4

GD6

GD12

GD16

GD20

GD20-adjusted

0

264

284

310

336

383

309

 1.97

15.6 

37.5

265

284

310

339

376

306

2.05

15.7

75

261

278

303

333

384

299

2.05

15.7

150

266

282

307

337

386

308

2.08*

17.1*

Number of live fetuses, mean number of live fetuses per litter and mean body weight of fetuse:

Dose (mg/kg)

Fetuses/Litters

Fetuses per litter

Body weight (g)

0

244/21

12

4.0

37.5

279/22

13

4.1

75

294/22

13

4.0

150

251/21

12

4.0

Conclusions:
The No-Observed-Adverse-Effect-Level (NOAEL) of Trifluoroacetic acid for maternal and embryo-fetal development toxicity in rats was considered to be 150 mg/kg/day.
Executive summary:

A GLP compliant prenatal developmental toxicity study with Trifluoroacetic acid was conducted in rats according to OECD Guideline 414. This study was designed to assess any maternal and/or embryo-fetal toxicity of the test article, Trifluoroacetic Acid (TFA), in the rat, following daily oral (gavage) administration during the period of major organogenesis, from implantation to the day of closure of the hard palate, Gestation Days (GD) 6 to 19, with Cesarean section on GD 20. Eighty-eight time-mated female Sprague-Dawley rats were distributed into 4 groups, each containing 22 rats. The animals were administered TFA at 0 (Control Article – distilled water), 37.5, 75 and 150 mg/kg bw/day TFA at a dose volume of 5 mL/kg by oral gavage once daily from GD 6 to 19. The following parameters were evaluated for all animals: viability, detailed physical examinations (immediately prior to dosing [within approximately 30 minutes] and approximately 2 hours after dosing), body weights, including gravid uterine adjusted body weights, food consumption and organ weights. A macroscopic postmortem evaluation was performed on GD 20, during which corpora lutea, implantation data and uterine weights were recorded. The fetuses were removed, weighed (live and recently dead fetuses), sexed and examined externally for defects. Approximately half of the fetuses were examined for soft tissue abnormalities. The other half were examined for skeletal abnormalities and ossification state. Placentas were examinedand weighed.

All females survived until termination. One female each in the control and high-dose groups was not pregnant, although this was not attributed to the administration of the test article. Dosing was well-tolerated by all females, and doses up to 150 mg/kg bw/day had no adverse effect on body weight, body weight gain, food consumption, pregnancy, c-section parameters, fetal, placental, and uterine weights, organ weights, or fetal abnormalities, variations or ossification parameters. In conclusion, under the conditions of this study, the maternal and the embryo-fetal no-observed-adverse-effect-level (NOAEL) were established at 150 mg/kg bw/day TFA. Due to the non-adverse, test article-related organ weight increases, the maternal and embryo-fetal no-observed-effect-levels (NOEL) were established at 75 mg/kg bw/day (maternal) and 150 mg/kg bw/day (embryo-fetal).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 Sep 2019 to 24 Aug 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP Guideline study
Justification for type of information:
For the Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate a new chemical substance notification in China has been prepared under the Regulation ‘Measures on the Environmental Management of New Chemical Substances' (Decree No. 7 of the Ministry of Environmental Protection of the P.R. China, also known as ‘China REACH’). Under this regulation an prenatal developmental toxicity study according to OECD 414 is part of the data requirements for substances that are produced or imported in volumes > 100 t/y. For this reason an OECD 414 study was performed in 2020 at Shenyang Research Institute of Chemical Industry. The results of the study are included in the dossier and serve as a key study related to the endpoint developmental toxicity. More information about the read-across justification is included in the Reporting format as attached to the respective IUCLID entry (section 13).
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
2019-02-11
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF (Beijing) Biotechnology Co., Ltd
- Age at study initiation: 11 weeks of age at arrival at the test facility
- Weight at study initiation: 267.07 - 404.02 g
- Fasting period before study: No
- Housing: animals were housed in plastic cages (L46.0xW31.5xH20.0 cm). During the acclimatization period, the animals were housed in groups of max. 2. Mated females were housed individually in cages.
- Diet (e.g. ad libitum): SPF rodent growth and breeding feed supplied by Shenyang Mao Hua biological science and Technology Co., Ltd, ad libitum
- Water (e.g. ad libitum): Purified drinking water, ad libitum
- Acclimation period: 33 days. Clinical observations were performed daily during acclimatization period, and were continued until the start of the test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled at 20 - 25°C (actual range: 22.6 - 25.6°C)
- Humidity (%): controlled 40 - 70% (actual range 34-82%)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 Nov 2019 To: 5 Dec 2019
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item in the vehicle (drinking water) were prepared daily before the administration. The test item concentrations were based on the active ingredient of the reaction mass (Potassium triflinate, TFSK: 14.2%, Potassium trifluoroacetate, TFAK: 14.8%). A dosing volume of 10 mL/kg was applied for all animals, which was adjusted based on the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATION VERIFICATION: The concentration of the test item was analyzed in the first and last week of the test item preparation with a validation analytical method (G1999B0040).

- Separation method: Ionic Chromatography (IC) (ICS-5000+ EG)
- Detection method: Conductivity detector.
- Conditions:
Analytical column: Dionex IonPac AS11-HC RFIC, 4 mm x 250 mm, 13 µm
Pre-column: Dionex IonPac AS11-HC RFIC, 4 mm x 50 mm, 13 µm
Column temperature: 30°C
    Flow rate: 1.0 mL/min
    Injection volume: 50 μL
    Mobile phase: 10mM potassium hydroxide aqueous solution
Retention time: About 11.2 and 13.7 min.
Run time: 17.0 minutes
- Data processing: the analytical data obtained were processed using the software of Chromeleion (c) Dionex. Since the test item is a reaction mass of two materials, the two main chromatography peaks in the chromatogram were considered as the target peak during sample analysis. The quantitative analysis was based on the sum of the two main peaks. The RSD of retention time was obtained from the first chromatography peak and the RSD of peak area was obtained from the sum of the two mean peaks in the system suitability test.
- External calibration and linearity range: A stock solution of 1.0 g active ingredient/L was diluted with ultra-pure water to obtain a series of standard solutions of 70.00, 60.00, 50.00, 40.00 and 30.00 mg/L. The accuracy of calibration of 5 concentrations should be within 90-110% by regression analysis and the coefficient of correlation (R) should be more than 0.99.
- System suitability/Method validation: To demonstrate the validity of the method, each analytical sequence consisted of at least:
+ A blank control (drinking water only), two repeats at a time.
+ Low dose (10 mg/ml), five repeats at a time.
+ Middle dose (30 mg/ml), five repeats at a time.
+ High dose (100 mg/ml), five repeats at a time.
Acceptance criteria: At the retention time of the target peak, the peak area of the blank matrix should be less than 10% of that of the lowest concentration standard solution. The determined concentrations of the low-, mid- and high-dose formulations should be 85-115% of the nominal concentration, and the RSD should not exceed 10%.
- Sample analysis: The dose formulations (nominal concentrations: 10, 30 and 100 mg/mL) were sampled and diluted within the linear range (30-70 mg/L), and determined by Ion Chromatography. The results of the determind concentration were in accordance with the target concentrations (accuracy 94.8 - 101.2%).
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 2 (or one) females : one male
- Length of cohabitation: until a sperm positive smear was detected.
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy
Duration of treatment / exposure:
Females were dosed daily in the morning from GD5 to GD19 by oral gavage.
Frequency of treatment:
Once daily
Duration of test:
15 treatment days
Dose / conc.:
100 mg/kg bw/day
Remarks:
Active ingredient
Dose / conc.:
300 mg/kg bw/day
Remarks:
Active ingredient
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Active ingredient
No. of animals per sex per dose:
27 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route in rats is available for the Reaction mass of TFSK/TFAK. No effects were observed on reproductive organs, reproductive performances and progeny at the maximum dose level tested (1000 mg active ingredient/kg bw/day). The No Observed Adverse Effect Level (NOAEL) for parental toxicity, reproductive performance (mating and fertility) and effects on progeny was therefore considered to be 1000 mg/kg/day. For this reason the maximum dose level of 1000 mg a.i./kg bw/day was selected as the highest dose level in the OECD 414 study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed daily by cage-side observations until the termination. After dosage, dams were observed for morbidity and mortality twice daily (once daily on non-working days)

BODY WEIGHT: Yes
- Time schedule for examinations: All pregnant rats were weighed on GD0, then they were weighed once per 3 days during the dosing period (GD5-19), and on the day of scheduled necropsy (GD20).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: During the period of administration, all pregnant rats were provided with a known quantity of feed on the day before body-weight determination, and the remaining feed were weighed on the next day (24h ± 1.5h). The daily food consumption of each animal was calculated.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All rats were killed on GD20, and the uterus was removed for examination immediately.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included: the whole gravid uteri and total placentas were weighed. The number of corpora lutea, absorbed fetuses (early and late separately), dead fetuses (early and late separately) and viable fetuses were counted.
Fetal examinations:
- External examinations: Yes, the sex and body weight of each viable fetus were determined. Each fetus was examined for external alterations, including head, trunk, limbs and tail.
- Soft tissue examinations: Yes, one-half live fetuses of each litter were immersed in modified Davidson's fixative (the recipe for 100 ml of fixative: 14 ml ethanol, 6.25 ml glacial acetic acid, 37.5 ml saturated 37% formaldehyde and 37 ml distilled water) for one week, rinsed twice with tap water and stored in 70% isopropyl alcohol for soft tissue examination. Limbs and tail of the fetus were cut down first, then four chips were cut to examine the structural alterations of the head. Finally, thorax and abdomen of the fetus were opened to examine the size, shape and position of the organs.
- Skeletal examinations: Yes, the other half of each litter was prepared and examined for skeletal alterations. After removal of the skin and soft tissue, the remainder of the fetus was stained by using Alizarin red staining method, and stored in 70% glycerol for examination. This procedure include the skull, vertebra, sternum, ribs, limb bones and pelvis. At the same time the number of sternal ossification points, namely the number of ossified sternal segments, was recorded.
Statistics:
Average and standard deviation of the data for each group were calculated including body weight, food consumption, body weight change corrected for gravid uterine weight, number of corpora lutea and implantations, number of viable fetuses and body weight of fetuses, number of absorptive and dead fetuses.
Data statistics: If variance homogeneity test was P >0.05, Anova was used. If variance homogeneity tests P ≤0.05, Kruskal Wallis test was used. The data about ratio of viable fetuses, dead fetus and absorptive fetus, and incidence of anomalies were evaluated by the chi-square test. Anova and non-parametric analysis was done with Provantis 9.4.3.0 software, the chi-square test was done with SPSS 17.0 software.
Indices:
Pre-implantation loss (%): (No. of corpora lutea - No. of implantations) / No. of corpora lutea ×100 %
Post-implantation loss (%): (No. of implantations - No. of live fetuses) / No. of implantations ×100 %
Sex ratio distribution (%): Number of male fetuses / Number of fetuses x 100%
External abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%
Visceral abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%
Skeletal abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed in the course of the study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No deaths were observed in the course of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the administration period, the body weight and body weight change of pregnant rats in dose groups had no significant difference compared with the control group (P>0.05). The mean corrected body weight gain on GD20 (body weight minus gravid uterine weight) and net body weight change (body weight on GD20 minus body weight on GD5 minus gravid uterine weight) during the gestation period in the pregnant rats in all dose groups had no significant difference compared with the control group (P>0.05). Detailed results are provided in Table 1 see 'Any other information on results'.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
During the administration period, the food consumption of the pregnant rats in all dose groups had no statistically significant difference compared with the control group (P>0.05). Detailed results are provided in Table 2 see 'Any other information on results'.
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:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The percentage of absorbed fetuses and the pre-implantation loss of high-dose group had a significant decrease compared with the control group (P≤0.05 or P≤0.01), but without toxicological significance and that had no statistically difference in the low- and mid- dose group (P>0.05). Detailed results are provided in Table 3 see 'Any other information on results'.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not specified
Details on maternal toxic effects:
In all dose groups, no statistically significant difference was observed in the mean numbers of corpora lutea, implantation sites and total placental weights, and the gravid-uterine weights compared with the control group (P>0.05). Detailed results are provided in Table 3 see 'Any other information on results'.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
other: No effects observed at the highest dose level tested.
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No statistically significant difference in the body weight of live fetuses was observed in all dose groups compared with the control group (P>0.05).
Detailed results are provided in Table 4 see 'Any other information on results'.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
In the high-dose group there was a statistically significant increase in the percent of live fetuses (P≤ 0.05).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No statistically significant difference in the sex distribution of live fetuses was observed in all dose groups compared with the control group (P>0.05).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
One fetus in the mid-dose group showed sign of narrow tail and 1 fetus in the high-dose group showed sign of tail absent. Two fetuses in the control group showed sign of small brain, and 1, 0, 1 and 3 fetuses in the control group, low-, mid- and high-dose groups showed signs of uronephrosis or small kidney in unilateral or bilateral kidneys. Five, 0, 5 and 4 fetuses in the control group, low-, mid- and high-dose groups showed signs of parietal imcomplete ossification, and one fetus showed signs of wavy rib, but the frequency of these abnormalities in the dose groups had no significant difference compared with the control group (P>0.05), which were considered as incidental findings. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations; some examined fetuses had less than six sternal ossification points,but the mean number of sternal ossification points in all dose groups had no statistically difference compared with the control group (P>0.05).
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No effects observed at the highest dose tested.
Abnormalities:
no effects observed
Developmental effects observed:
no

Deviations from protocol: During the test the relative humidity and the realitve temperature in the animal room were beyond the controlled range of respectively 40 -70% (actual 34 -82%) and 20 -25°C (actual 22.6 -25.6°C). As the deviated relative humidity and temperature lasted for a short term, and no related adverse effects were observed in the animals, the deviaions are not considered to affect the quality or integrety of the study.

Table 1: Body weight and body weight gain of pregnant rats (g)

Day(s) relative to mating

0 mg/kg d

100 mg/kg d

300 mg/kg d

1000 mg/kg d

Body weight gain (g)

5-20 [a]

Mean

SD

N

121.5

20.1

27

130.1

24.4

27

124.0

23.5

27

122.6

22.7

27

Corrected body weight on GD20 (g)

[a]

Mean

SD

N

378.25

27.98

27

381.26

33.47

27

375.95

25.77

27

375.19

32.46

27

Corrected body weight gain (g)

[a]

Mean

SD

N

54.327

17.186

27

56.334

21.815

27

52.323

16.921

27

49.599

17.193

27

Net body weight change (g)

[a]

Mean

SD

N

25.02

12.77

27

28.44

13.52

27

24.71

20.83

27

22.37

12.81

27

[a] – Anova&Dunnett

Table 2: Food consumption of animals (g/day)

 

Day(s) Relative to mating

7 → 8

10 → 11

13 → 14

16→ 17

0 mg/kg d

Mean

SD

N

28.15

4.60

27

27.77

4.76

27

29.77

4.98

27

32.31

4.67

27

100 mg/kg d

Mean

SD

N

26.71

3.96

27

28.65

4.82

27

28.70

4.12

27

32.38

5.32

27

300 mg/kg d

Mean

SD

N

25.99

4.22

27

27.77

4.26

27

29.61

4.75

27

32.12

5.38

27

1000 mg/kg d

Mean

SD

N

25.39

4.44

27

27.92

7.17

27

27.27

4.29

27

31.71

4.76

27

Table 3: Developmental endpoint results

 

0 mg/kg d

100 mg/kg d

300 mg/kg d

1000 mg/kg d

Mated female

(n)

28

28

28

28

Pregnant female

(n)

27

27

27

27

Corpora Lutea [a]

Mean

SD

Sum

16.9

3.7

456

17.2

2.8

465

16.7

2.9

451

16.6

3.2

449

Implantation[a]

Mean

SD

Sum

16.1

3.5

436

16.4

2.8

442

16.1

2.7

434

16.1

3.1

436

Gravid Uterus [a] (g)

Mean

SD

96.4771

22.7950

101.6460

19.9209

99.3060

16.2687

100.2396

17.2214

Total placental weight[a] (g)

Mean

SD

14.1308

3.2056

14.7799

2.9829

14.6335

2.5385

14.7466

2.7125

Live fetus[a]

Mean

SD

Sum

15.1

3.7

409

15.5

3.0

419

15.4

2.7

415

15.7

3.0

424

Percent[c] (%)

93.8

94.8

95.6

97.2*

Absorbed fetus[a]

Mean

SD

Sum

0.9

1.2

25

0.8

1.1

23

0.7

1.0

19

0.4

0.5

10

Percent[c] (%)

5.7

5.2

4.4

2.3**

Dead fetus[k]

Mean

SD

Sum

0.1

0.3

2

0.0

0.0

0

0.0

0.0

0

0.1

0.4

2

Percent[c] (%)

0.5

0.0

0.0

0.5

Pre-implantation loss[c] (%)

4.4

4.9

3.8

2.9

Post-implantation loss[c] (%)

6.2

5.2

4.4

2.8*

[a] – Anova&Dunnett

[k] – Kruskal-Wallis&Wilcoxon

[c] – Chi-square test: *p ≤ 0.05, **p ≤ 0.01

Table 4: Fetal examination results

 

0 mg/kg d

100 mg/kg d

300 mg/kg d

1000 mg/kg d

Body weight of fetus[a](g)

Mean

SD

3.974

0.308

4.068

0.273

4.019

0.264

3.965

0.322

Number of male fetuses

203

210

231

222

Number of female fetuses

206

209

184

202

Sex distribution (males/total) [c] (%)

49.63

50.12

55.66

52.35

[c] – Chi-square test

 

Conclusions:
The No-Observed-Adverse-Effect-Level (NOAEL) of the Reaction mass of TFSK/TFAK for maternal and embryo-fetal development toxicity in rats was considered to be 1000 mg/kg/day (corresponding to 510 mg TFAK/kg/day).
Executive summary:

A GLP compliant prenatal developmental toxicity study with the Reaction mass of TFSK/TFAK was conducted in Sprague Dawley rats according to OECD Guideline 414. Mated females (27/group) were treated once daily with the Reaction mass of TFSK/TFAK by oral gavage at dose levels of 100, 300, and 1000 mg active ingredient/kg bw/day during gestation (from Gestation Day 5 to 19). A control group of 27 females receiving the vehicle (water) was included in the study.

The study animals were observed daily for mortality and clinical signs. Body weight and food consumption of the dams were also recorded. On GD 20, the study animals were necropsied and the uterine was removed for determination of gravid uteri weight and placental weight. The number of corpora lutea, absorbed fetuses, dead fetuses and viable fetuses was determined. The fetuses were removed, weighed, sexed and examined externally for defects. Approximately half of the fetuses were examined for soft tissue abnormalities. The other half was examined for skeletal abnormalities and ossification state.

No deaths or treatment-related clinical signs were observed during the study. No effects on mean body weights, body weight change and food consumptions were observed in the treated groups when compared to the control group. No adverse effect was observed in the prenatal reproductive parameters. The fetal examination showed no adverse effect in body weight and sex distribution of live fetuses in all dose groups. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations.

Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) of the Reaction mass of TFSK/TFAK for maternal and embryo-fetal development toxicity in rats was considered to be 1000 mg/kg/day (corresponding to 510 mg TFAK/kg/day)

.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 Jun 2020 to 05 Nov 2020 (Experimental completion date)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK)
- Age at study initiation: 18 to 22 weeks old
- Weight at study initiation: 2.47 to 4.52 kg
- Housing: the animals were single housed in stainless steel cages elevated off the floor. From Day 20 after mating, cage paper was placed into each cage to allow expression of nesting behavior.
- Diet: Teklad 2930, pelleted diet, 200 g/animal/day
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation: Five days before commencement of treatment (Days 1 to 5 of gestation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 15-21ºC.
- Humidity (%): Monitored and maintained within the range of 45-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 14 hours light: 10 hours dark

IN-LIFE DATES: From: 23 June 2020 To: 24 July 2020
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Formulations (w/w) were prepared at least once weekly. The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer. No correction was made for the purity of the test substance. The formulation solutions were stored at 2-8°C. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATION VERIFICATION
The formulations for first and last preparation were sampled, 1 x 10 mL (accurately weighed), from the middle of the formulation. Two aliquots from the sample were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M021/20). The remainder of the samples were retained for contingency.

STABILITY AND HOMOGENEITY
The suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix in Covance Study Number LC59WX. The stability at ambient temperature (15 to 25°C) and refrigerated (2 to 8°C) is 24 hours and 15 days, respectively.
Details on mating procedure:
Natural mating with New Zealand White bucks of established fertility at the supplier’s facility. Males and females were not closely related. After mating each female was injected intravenously with 25 i.u. luteinising hormone. The day of mating was considered Gestation Day 0 (GD0). The study animals arrived at the test facility on GD1 and were acclimated until GD5.
Duration of treatment / exposure:
Females were exposed from GD6 to GD28 by oral gavage.
Frequency of treatment:
Once daily.
Duration of test:
23 treatment days.
Dose / conc.:
180 mg/kg bw/day
Dose / conc.:
375 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
24 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for this study were selected based on the results of a preliminary embryo-fetal study in the New Zealand White rabbit with Sodium Trifluoroacetate in water at dose levels of 250, 500 or 750 (1000) mg/kg/day (Covance Study Number YQ44HR). In that study an initial dose at 1000 mg/kg/day resulted in some unanticipated adverse signs (unsteady, underactive behaviour). Consequently, the decision was made to reduce this dose level to 750 mg/kg/day for the remainder of the treatment period; at 750 mg/kg/day these signs improved, until no longer present by Day 12. For females treated at 250 or 500 mg/kg/day, isolated incidences of unsteady gait and decreased activity were similarly observed however to a much lesser magnitude and as such were not considered to be adverse. Therefore a high dose level of 750 mg/kg/day was considered to be a suitable high dose for this main embryo-fetal development study. The low and intermediate dose levels of 180 and 375 mg/kg/day (respectively) were selected to allow evaluation of any dose related trends.
Maternal examinations:
VIABILITY:
A viability check was performed near the start and end of each working day. Animals were killed if they exhibited evidence for pregnancy loss.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 1, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 1, 3 and daily from Day 6 to 29 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 to Day 28 after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: All surviving study animals were euthanized and subjected to necropsy and caesarean section on GD 29. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The liver was weighed for animals killed at scheduled intervals and preserved in 10% Neutral Buffered Formalin.
Animals that experienced pregnancy lost were sacrificed on the day of detection and also subjected to necropsy.
Ovaries and uterine content:
For females surviving to term, the ovaries and uterine content was examined after termination. Examinations included:
• Gravid uterine weight (including cervix and ovaries)
• Number of corpora lutea
• Number of implantation sites
• Number of resorption sites (classified as early or late)
• Number of fetuses (live and dead).
Blood sampling:
Not included.
Fetal examinations:
All live fetuses were individually weighed and examined for external abnormalities. Fetus with external malformation was sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded. Approximately 50% of eviscerated fetuses were decapitated; heads were fixed in Bouin’s fluid and subject to free-hand serial sectioning for subsequent soft tissue abnormalities. Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit and stained with Alizarin Red for subsequent skeletal examination.
Statistics:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett, 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon, 1945) were made. For all other analyses the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant,
suggesting that the dose-response was not monotone, Steel's test (Steel, 1959) was performed instead.
For liver weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the liver weight.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Indices:
- Pre-implantation loss (%): (No. of corpora lutea - No. of implantations) / No. of corpora lutea ×100%
- Post-implantation loss (%): (No. of implantations - No. of live fetuses) / No. of implantations ×100%
- Sex ratios of the live fetuses were calculated as the percentage of males per litter.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Signs observed in association with dose administration were limited to one female at 375 mg/kg/day that showed underactive behavior and a dilated pupil on Days 27 and 28 and one female at 750 mg/kg/day that showed irregular breathing and an unsteady gait on GD7. Although these incidences were isolated and inconsistent, these observations are similar to those seen on the preliminary study and as such, a relationship to treatment cannot be discounted.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females died prior to scheduled termination. On GD27, a control female was observed to have a convulsion shortly after dosing and subsequently died. Macroscopic examination revealed a perforated trachea and consequently this death was attributed to dosing trauma. On GD27, a female receivinmg 750 mg/kg/day was killed for reasons of animal welfare following evidence for pregnancy loss. Macroscopic examination of this female revealed an enlarged spleen and uterine examination revealed eight corpora lutea and two resorbing implantation sites. Enlarged spleen was not apparent for any other females on study and as this pregnancy loss was an isolated incidence it was not attributed administration of Sodium Trifluoroacetate.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Group mean body weight gain during the treatment period (GD6-28) was unaffected by treatment at dose levels up to and including 750 mg/kg/day. Adjusted body weights on Day 29 after mating were calculated from the body weight at termination minus the gravid uterine weight. Body weight change values for the period Day 6-29 were also presented, after being adjusted for the contribution of the gravid uterus. On Day 29 of gestation, the gravid uterine weight for females that received 375 or 750 mg/kg/day was low when compared with Controls (p<0.05 and p<0.01, respectively). A decreased maternal adjusted body weight gain of 20, 48 and 56% of control was observed in the 180, 375 and 750 mg/kg bw/day groups, respectively. Detailed results are provided in Table 1 (see attached document).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
From Day 6 of gestation (Day 1 of treatment) food consumption was low in all treated groups when compared with Controls. Food intake remained low at 180 mg/kg/day until GD14, at 375 mg/kg/day until GD15 and at 750 mg/kg/day until GD16. At the end of the treatment period females receiving TFA showed high food consumption when compared with Controls (GD26-28). Detailed results are provided in Table 2 (see attached document).
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:
effects observed, treatment-related
Description (incidence and severity):
Body weight adjusted liver weights for females at scheduled termination on GD29 were high when compared with Controls (p<0.05 at 180 mg/kg/day; p<0.01 at 375 and 750 mg/kg/day); a dose response was apparent.
Detailed results are provided in Table 3 (see attached document).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination on Day 29 after mating revealed an irregular surface of the liver in one female, that received 375 mg/kg/day; there were no other findings that could be attributed to treatment with Sodium Trifluoroacetate.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the liver, minimal to moderate bile duct hyperplasia/fibrosis and minimal generalized hepatocellular hypertrophy was seen in females of all treated groups and exhibited a dose relationship. Bile duct hyperplasia can be a response to insult to the bile duct epithelium (Sahota et al., 2019), and is indicative of test article excretion in the bile. The bile duct finding appeared to have no adverse effect on maternal clinical condition, but in the absence of blood chemistry it is unknown whether there were any liver changes possibly attributable to the bile duct change. Therefore the hyperplasia/fibrosis, at the moderate severity which was observed at the mid and high dose is considered as adverse. Detailed results are provided in Table 4 (see attached document).
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female, administered 750 mg/kg/day, was killed on Gestation Day 27 due to suspected abortion. At necropsy, an enlarged spleen was noted. At microscopic examination of the liver, slight multifocal bile duct hyperplasia/fibrosis and minimally decreased generalized hepatocyte cytoplasmic rarefaction in the liver were seen. Bile duct hyperplasia/fibrosis was seen as a test article related finding in other females of this dose group. These findings were not considered to have contributed to the death of this animal, with the main contributory factor being due to suspected abortion.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Mean corpora lutea, the subsequent number of implantations and litter size were low for females that received 750 mg/kg/day; however, as treatment commenced at or around the time of implantation, this difference is considered unrelated to treatment. Post-implantation loss (%) was slightly high at 750 mg/kg/day but this difference did not achieve statistical significance. Detailed results are provided in Table 5 (see attached document).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Mean number of resorptions (early or late) showed no effect of treatment. Detailed results are provided in Table 5 (see attached document).
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male fetal weights were significantly reduced at 375 and 750 mg/kg/day (p<0.01) and female fetal weights were significantly reduced at 750 mg/kg/day (p<0.05); a dose response was apparent.
Detailed results are provided in Table 5 (see attached document).
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Mean live litter size at 750 mg/kg bwt/day was slightly but significantly lower than that of the controls (6.1** cf. 8.3, p<0.01). This was due to a combination of the lower mean number of ova shed, as indicated by the numbers of corpora lutea, which occurred before the start of treatment (8.3** cf. 10.5, p<0.01) and was therefore not relevant for the interpretation of the study, and a slight non-significant increase in the extent of post-implantation loss (14.9% cf. 9.7%). Detailed results are provided in Table 5 (see attached document).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 5 (see attached document).
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Mean live litter size at 750 mg/kg bwt/day was slightly but significantly lower than that of the controls (6.1** cf. 8.3, p<0.01). This was due to a combination of the lower mean number of ova shed, as indicated by the numbers of corpora lutea, which occurred before the start of treatment (8.3** cf. 10.5, p<0.01) and was therefore not relevant for the interpretation of the study, and a slight non-significant increase in the extent of post-implantation loss (14.9% cf. 9.7%). Overall mean fetal weights and consequently mean total litter weights were reduced at 375 and 750 mg/kg/day (p<0.01); a dose response was apparent. Detailed results are provided in Table 5 (see attached document).
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two of 173 fetuses with external malformation of omphalocele was observed in two litters of the mid-dose. Gastroschisis was observed in one mid-dose fetus and one high-dose fetus. These findings were considered incidental due to low incidence (within the range of historical control data) and and since there was no correlation to the dose. Detailed results are provided in Table 6 (see attached document). It has to be noted that the report is still in a draft status, since evaluation of the fetal observations is not finalized. In addition, the presented Historical control data (HCD) is only from a limited time period and need to be extended.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Multiple cervical/thoracic/lumbar/caudal vertebral and rib abnormalities; with associated minor abnormalities affecting the vertebrae, ribs, sternum and costal cartilages) were increased at 375 and 750 mg/kg/day. However, most of the abnormalities showed no dose-correlation and/or were within the range of historical control data. Therefore, the relation to treatment is uncertain. Fused/partially fused ribs were observed in one, four and eight fetuses at 180, 375 and 750 mg/kg/day, respectively; these were considered related to the treatment with sodium trifluoroacetate. Detailed results are provided in Table 6 (see attached document). It has to be noted that the report is still in a draft status, since evaluation of the fetal observations is not finalized. In addition, the presented Historical control data (HCD) is only from a limited time period and need to be extended.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
A number of visceral foetal abnormalities were recorded in all groups:
• An increased incidence of major eye abnormalities was observed at all dose levels. Multiple folded retina were observed in one, five and nine fetuses of one, four and eight litters at 180, 375 and 750 mg/kg/day, respectively; Absent aqueous/vitreous humour was observed in one, six and eight fetuses in one, four and six litters at 180, 375 and 750 mg/kg/day, respectively. Microphthalmia was observed in one foetus in each treated group. Absent lens was observed in one fetus of the highest dose group.
• Cardiovascular abnormalities, comprising of transposition of ascending aorta/pulmonary trunk; narrow/dilated ascending aorta and pulmonary trunk; double outlet ventricle(s) and ventricular septal defect were recorded in one or maximum two fetuses across all dose levels. Because these abnormalities occurred at low incidence, in concurrent controls (Transposition of ascending aorta/pulmonary trunk and Muscular ventricular septal defect) and/or were noted previously in historical controls and showed no clear dose response relationship, the relationship with treatment is uncertain.
• Kidney abnormalities in fetuses of treated dams consisted of fused/displaced kidney (one fetus at 180 and 750 mg/kg/day), absent kidney and absent ureter (one fetus at 375 mg/kg/day). Because these abnormalities occured singly and/or are within the range of historical control data, a relationship with the treatment is uncertain.
• Cleft plate was observed in one high-dose fetus. This finding was considered incidental due to low incidence and with no dose-relationship. This is also covered by historical control data.
Detailed results are provided in Table 6 (see attached document). It has to be noted that the report is still in a draft status, since evaluation of the fetal observations is not finalized. In addition, the presented Historical control data (HCD) is only from a limited time period and need to be extended.
Key result
Dose descriptor:
NOAEL
Effect level:
< 180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations
Developmental effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified

Results of Dose Formulations Analysis

The mean achieved concentrations of formulation samples taken from the first and last dose preparations were within 2% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis.

Conclusions:
The No-Observed-Adverse-Effect-Level (NOAEL) of sodium trifluoroacetate for maternal toxicity in rabbits was considered to be 180 mg/kg/day, and the NOAEL of sodium trifluoroacetate for embryo-fetal developmental toxicity in rabbits was considered to be <180 mg/kg/day.
Executive summary:

A GLP compliant prenatal developmental toxicity study with sodium trifluoroacetate was conducted in New Zealand White rabbits according to OECD Guideline 414. Groups of 24 time-mated female New Zealand White rabbits were treated daily with Sodium trifluoroacetate by oral gavage from gestation day (GD) 6 to GD28 at dose levels of 0, 180, 375 and 750 mg/kg bodyweight/day. Clinical condition, food consumption and bodyweights of the dams were recorded during the study and the females were killed on GD 29. At termination, the ovaries were examined for the numbers of corpora lutea, the gravid uteri were weighed and the contents were examined for numbers of implantations, early and late resorptions, and live and dead foetuses. Foetal and placental weights were recorded. At necropsy, all foetuses were examined externally and internally for thoracic and abdominal visceral changes. The heads were removed from one half of the foetuses and fixed to permit examination of internal head structures. The torsos and the remaining intact foetuses were processed, stained with alizarin red S and examined for skeletal changes.

In this study, 19/24, 21/24, 24/24, and 24/24 mated females were pregnant at 0, 180, 375 or 750 mg/kg/day, respectively. There were no treatment-related adverse effects upon group mean bodyweights or body weight gain; food consumption showed a slight dose-related reduction during the first part of the treatment period but was similar or slightly greater than that of the controls during the latter part of treatment. Adjusted body weights on Day 29 after mating were calculated from the body weight at termination minus the gravid uterine weight. On Day 29 of gestation, the gravid uterine weight for females that received 375 or 750 mg/kg/day was low when compared with Controls (p<0.05 and p<0.01, respectively). The maternal adjusted body weight gain was decreased by 20, 48 and 56% compared to control at 180, 375 and 750 mg/kg bw/day, respectively. Body weight adjusted liver weights for females at scheduled termination on GD29 were high when compared with Controls; a dose response was apparent. Histopathological examination of the liver revealed minimal to moderate bile duct hyperplasia/fibrosis and minimal generalized hepatocellular hypertrophy in all treated group; a dose response was apparent.

Litter data as assessed by the number of implantations, resorptions, live young and sex ratio, and the extent of pre-/post-implantation loss did not show any differences that could be attributed to maternal treatment. Mean placental weights were unaffected by maternal treatment. Mean live litter size at 750 mg/kg bwt/day was slightly but significantly lower than that of the controls (6.1** cf. 8.3, p<0.01). This was due to a combination of the lower mean number of ova shed, as indicated by the numbers of corpora lutea, which occurred before the start of treatment (8.3** cf. 10.5, p<0.01) and was therefore not relevant for the interpretation of the study, and a slight non-significant increase in the extent of post-implantation loss (14.9% cf. 9.7%). Overall mean fetal weights and consequently mean total litter weights were reduced at 375 and 750 mg/kg/day (p<0.01); a dose response was apparent.

Preliminary evaluation of fetal findings revealed an increased incidence of major and associated minor abnormalities, predominantly affecting the eyes and multiple cervical/thoracic/lumbar/caudal vertebral and rib abnormalities at 375 and 750 mg/kg/. At 180 mg/kg/day, identical abnormalities involving the eye were observed but at lower incidence.

Based on the maternal liver pathology that showed moderate bile duct hyperplasia/fibrosis at 375 and 750 mg/kg/day, 180 mg/kg/day was considered to be the maternal no observed adverse effect level (NOAEL). Due to the fetal abnormalities that were observed at all dose levels, a NOAEL for embryo-fetal developmental toxicity could not be established in this study.

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

A GLP compliant prenatal developmental toxicity study with Trifluoroacetic acid was conducted in Sprague Dawley rats according to OECD Guideline 414. Mated females were treated with Trifluoroacetic acid by oral gavage at dose levels of 37.5, 75, and 150 mg/kg/day during gestation (from Gestation Day 6 to 19). A control group of 22 females was included and the animals were given the vehicle (distilled water). The study animals were observed daily for mortality and clinical signs. On GD 20, all the surviving study animals were necropsied and examined macroscopically. The ovaries and uteri were examined for determination of litter data. The fetuses were removed, weighed (live and recently dead fetuses), sexed and examined externally for defects. All the live fetuses were weighed and examined for external abnormalities; their crown-rump length was measured and their sex was determined. Approximately half of the fetuses were examined for soft tissue abnormalities. The other half was examined for skeletal abnormalities and ossification state. All females survived until termination. Dosing was well-tolerated by all females, and doses up to 150 mg/kg/day had no adverse effect on body weight, body weight gain, food consumption, pregnancy, c-section parameters, fetal, placental, and uterine weights, organ weights, or fetal abnormalities, variations or ossification parameters. Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) of Trifluoroacetic acid for maternal and embryo-fetal development toxicity in rats was considered to be 150 mg/kg/day.

In a GLP compliant prenatal developmental toxicity study in Sprague Dawley rats, four groups of 27 mated animals were treated by oral gavage with the reaction mass of potassium triflinate (TFSK) and potassium trifluoroacetate (TFAK) at dose levels of 100, 300 and 1000 mg/kg bw/day from GD 5 to GD 19. The study was terminated on GD 20. The calculated dose levels for TFAK were 51, 153 and 510 mg/kg bw/day. No deaths or treatment-related clinical signs were observed during the study. No effects on mean body weights, body weight change and food consumptions were observed in the treated groups when compared to the control group. No adverse effect was observed in the prenatal reproductive parameters. The fetal examination showed no adverse effect in body weight and sex distribution of live fetuses in all dose groups. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations. Based on these results, the NOAEL of the Reaction mass of TFSK/TFAK for maternal and embryo-fetal development toxicity in rats was considered to be 1000 mg/kg/day (corresponding to 510 mg TFAK/kg/day).

A developmental toxicity study in pregnant New Zealand white rabbits was conducted with sodium trifluoroacetate, based on OECD Test Guideline 414. Twenty-four pregnant rabbits per dose level received daily treatment of 99.9% sodium trifluoroacetate by oral gavage from gestational days 6 to 28 at dose levels of 0, 180, 375 and 750 mg/kg bw/day. Data collected from the pregnant rabbits included postdose observations, clinical signs, mortality, detailed physical examinations, body weight and food consumption throughout gestation. At termination, maternal gross pathology and reproductive parameters (gravid uterine weight, number of corpora lutea, implantations, and early and late resorptions) were assessed and livers collected and processed for histopathology. Evaluations of fetuses included live and dead fetuses, fetal weights, sex and external, visceral and skeletal examinations. The most notable maternal finding was a decreased maternal adjusted body weight gain of 20, 48 and 56% of control in the 180, 375 and 750 mg/kg bw/day groups, respectively. The mean number of live fetuses was reduced by 73% of control in the high-dose and by 87% in the mid-dose. Total litter weight and male fetal weights were reduced in the mid- and high-dose and the female fetal weights were reduced in the high-dose. Increased incidences of major and associated minor abnormalities were observed in fetuses/litters across all treatment groups predominantly affecting the eyes and skeletal system. An evaluation of the relationship of these effects to treatment with sodium trifluoroacetate is ongoing. Other findings were observed, yet within the historical control data and/or not showing a dose response-relationship. As such appreciation of the relevance of these findings is unclear and requires further confirmations. The No-Observed-Adverse-Effect-Level (NOAEL) of sodium trifluoroacetate for maternal toxicity in rabbits was considered to be 180 mg/kg/day, and the NOAEL of sodium trifluoroacetate for embryo-fetal developmental toxicity in rabbits was considered to be <180 mg/kg/day.

Justification for classification or non-classification

A conclusion on the classification and labelling of TFA will be postponed until the final results of the studies investigating the effects on fertility and development are available.

Additional information