Trifluoroacetic acid

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-10-09 to 2022-03-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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 was 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 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 observations 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. In addition, analysis of plasma samples taken in the dose range finding study (Study No. 8437241) demonstrated very high plasma concentrations up to 485 mg/L in F0 high-dose females and up to 394 mg/L in F1 offspring at PND 21. Evaluation of the plasma analysis data for the different time points indicated a sub-proportional increase of plasma concentrations with increasing dose levels of Sodium trifluoroacetate. Based on calculated AUC values for the timepoint Gestation Day 17 using a statistical data driven approach sub-proportionality started between 1500 to 2100 ppm. For Lactation Day 11 sub-proportionality started between 1200 to 3000 ppm. 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.

- 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.

Test material

Test material information

Constituent 1

Reference substance name:

Sodium trifluoroacetate

EC Number:

220-879-6

EC Name:

Sodium trifluoroacetate

Cas Number:

2923-18-4

Molecular formula:

C2HF3O2.Na

IUPAC Name:

sodium trifluoroacetate

Test material form:

solid

Details on test material:

- Batch number: 2019052304
- Appearance: White solid
- Storage conditions: At ambient temperature (15 to 25°C)
- Expiry date: 23 May 2021 (two years after date of manufacture)
- Purity: 99.9%
- Impurity: water, 0.0635%

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. The dietary dose levels are expressed as nominal NaTFA concentrations (ppm). The conversion factor from TFA-sodium salt to TFA is 0.84.

Test animals

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: RccHan™; WIST rat
- Age of F0 animnals at the start of the study (GD0): 28 to 34 days old.
- Weight at study initiation: 66-87 g (F0 females) and 70-107 g (F0 males),
- 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, blood chemistry and thyroid hormones and during the period of 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: 6 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Maintained within the range of 20-24ºC.The temperature was occasionally outside the indicted ranges but these deviations wer minor and/or of short duration.
- Humidity (%): Maintained within the range of 40-70%. The humidity was occasionally outside the indicted ranges but these deviations wer minor and/or of short duration.
- 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: 20 October 2020 To: 7 May 2021

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Once weekly
- Mixing appropriate amounts with (Type of food): The test substance was incorporated into the diet (SDS VRF1 Certified, powdered 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: At ambient temperature (15 to 25°C). 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). It was demonstrated stable for 22 days at ambient temperature (15 to 25°C).

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 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:

CONCENTRATION VERIFICATION:
Samples of each formulation prepared for administration in Week 1 (F0 generation), last week of lactation (F0/F1 generation and last week (F1 generation) of treatment were analyzed for achieved concentration of the test item. 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.
The mean achieved concentrations for formulation samples taken during the course of this study were within – 15%/+10% of nominal concentrations confirming the accuracy of formulation. Mean recovery results obtained at each concentration on each analytical occasion were within ±10% of nominal (except for 300 ppm at lactation with a mean 88.9% of nominal), demonstrating the continued accuracy of the method.

HOMOGENEITY:
The homogeneity of sodium trifluoroacetate in diet formulations was assessed at nominal concentrations of 50 ppm and 10000 ppm as part of another study (Covance Study no. LK56VW). A set specimen batch of diet formulation was prepared and sampled (200 g) in duplicate from the top, middle and bottom of the Turbula® drum for homogeneity assessment. The homogeneity samples were analyzed singly. The mean concentrations of the top, middle and bottom samples were within 3.8% of nominal values (CV < 4.66%set) and therefore met the acceptance criteria (analysed concentration within -15% and +10% of nominal, CV ≤ 5%).

STABILITY:
The stability of sodium trifluoroacetate in diet formulations was assessed at nominal concentrations of 50 ppm and 10000 ppm during ambient temperature and frozen storage. Samples (5 x 200 g) were taken from the middle of the drum for stability determination. The stability samples were analyzed in duplicate following 4, 7, 15 and 22 days ambient storage and following 22 days freezer storage. The initial mean analyzed concentration of the homogeneity analysis was taken as the initial stability timepoint. Stability was confirmed after ambient storage for 22 days and frozen storage for up to 22 days (acceptance criteria: Mean concentration within ± 10% of initial concentration).

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, for 10 weeks during premating, gestation and lactation. To compensate for the higher food intake during lactation, dietary concentrations in females 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

120 ppm (nominal)

Remarks:

Main phase

Doses / concentrations 2

Dose / conc.:

600 ppm (nominal)

Remarks:

Main phase

Doses / concentrations 3

Dose / conc.:

3 000 ppm (nominal)

Remarks:

Main phase

Doses / concentrations 4

Dose / conc.:

60 ppm (nominal)

Remarks:

Lactation Phase

Doses / concentrations 5

Dose / conc.:

300 ppm (nominal)

Remarks:

Lactation Phase

Doses / concentrations 6

Dose / conc.:

1 500 ppm (nominal)

Remarks:

Lactation Phase

No. of animals per sex per dose:

Parent generation: 25/sex/group
Cohort 1A: One male and/or one female per litter were selected (20/sex/group)
Cohort 1B: One male and/or one female pup per litter were 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 observations 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.
In addition, analysis of plasma samples taken in the dose range finding study (Study No. 8437241) demonstrated very high plasma concentrations up to 485 mg/L in F0 high-dose females and up to 394 mg/L in F1 offspring at PND 21. Evaluation of the plasma analysis data for the different time points indicated a sub-proportional increase of plasma concentrations with increasing dose levels of Sodium trifluoroacetate. Based on calculated AUC values for the timepoint Gestation Day 17 using a statistical data driven approach sub-proportionality started between 1500 to 2100 ppm. For Lactation Day 11 sub-proportionality started between 1200 to 3000 ppm.
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.

Examinations

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. 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. From GD 20 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were performed once per week on each animal to monitor general health for all F0 and selected F1 generation animals. During gestation and lactation period, detailed observations were conducted at least once on Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.

BODY WEIGHT: Yes
- F0 males: Day on which treatment commenced, weekly thereafter and before necropsy.
- F0 females: Day on which treatment commenced, weekly thereafter until mating detected, on Days 0, 7, 14 and 20 after mating; On days 1, 4, 7, 14, 21 and 28 of lactation, and before necropsy.
- F1 selected animals: Day 21 and 25 of age and then weekly from nominal four weeks of age (at the formal start of the F1 generation) and before necropsy.

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; Days 0-6, 7-13, and 14-19 after mating; Days 1-3, 4-6, 7-13, and 14-20 of lactation
Selected F1: Weekly from nominal four weeks of age.
- Calculation of food consumption: Yes, from these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

CLINICAL PATHOLOGY
- Time schedule for collection of blood: Blood samples were collected at test termination after overnight withdrawal of food from F0 adults (10/sex/dose) and F1 Cohort 1A animals (10/sex/dose). Blood samples (2 x 0.5 mL for hematology; 0.7 mL for clinical chemistry) were withdrawn from the sublingual vein
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: yes, food deprivation for blood samples for haematology and clinical chemistry.
- Haematology and clinical chemistry: Haematocrit (Hct), Hemoglobin (Hb), Erythrocyte count (RBC), Mean cell hemoglobin (MCH), Mean cell hemoglobin concentration (MCHC), Mean cell volume (MCV), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils,
Basophils, Monocytes, Large unstained cells), Platelet count, Prothrombin time (PT) and activated partial thromboplastin time (APTT).
- Clinical chemistry: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Non esterified fatty acids (NEFA), Triglycerides
(Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb). Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

OTHER: T4 AND TSH HORMONE DETERMINATIONS: See Table 2 ('any other information on materials and methods') for the occasions of blood sampling.

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 and for four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter or mate were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females.

Sperm parameters (parental animals):

Immediately after scheduled sacrifice of each F0 and F1 Cohort 1A male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed. The following parameters were examined:
- Sperm motility (all groups): Sperm motility was evaluated immediately after sacrifice. A sample of sperm was expressed from the left vas deferens. The percentages of motile and progressively motile sperm and sperm motion parameters were reported.
- Sperm morphology (F0 Group 1 and 4; F1 Cohort 1A all groups): At least 200 spermatozoa per sample were observed and classified as either normal (both head and midpiece/tail appear normal) or abnormal. The number and percentages of normal and abnormal sperm were reported.
- Sperm count (Groups 1 and 4): Groups 1 and 4): The cauda epididymis (left) was weighed and homogenized and the number of sperm was counted. The testis (left) was homogenized and the number of homogenization-resistant spermatids was counted. The sperm concentration (Million/g) and total number were reported.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.

PARAMETERS EXAMINED
- Litter evaluation: Litter size was recorded daily from Days 1-21 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
- Mortality and clinical signs: Observations were made approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to treatment. On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling
- Body weight: Individual offspring body weights were recorded on Days 1, 4 (before culling), 7, 14 and 21 of age. For unselected F1 offspring on Day 22 of age. For selected F1 generation on Days 25 of age and then weekly until termination.
- Food consumption: For selected F1 animals, each week from nominal four weeks of age.
- Anogenital distance: The anogenital distance (AGD) of each pup was measured Day 1 of age.
- Nipple retention in male pups: The presence of nipples/areolae was checked on Day 13 of age.
- Sexual maturation: Males were examined daily from Day 38 of age of age until balano-preputial separation occurred. Body weight was recorded on the day of completion of separation. Females were examined daily from Day 25 of age until vaginal opening occurred. Body weight was recorded on day of vaginal opening.
- Estrous cycle monitoring: For Cohort 1A females wet smears were taken following onset of vaginal patency until the first cornified (estrus) smear was 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 approximately Day 75 of age. For Cohort 1B females wet smears were taken for at least 3 days prior to the start of the necropsy phase and on the day of termination.
- T4 and TSH hormone determinations: See Table 2 for the occasions of blood sampling. Samples from F1-adults and F1 offspring at Day 22 were analysed for T4 by LC-MS/MS. Samples from F1-offspring at Day 4 were stored frozen, but analyses was not required. Serum samples for TSH analysis were stored frozen until analysis. TSH analysis was done by using Millipore Luminex multiplex kits.
- Clinical pathology: Blood sampling for F1 Cohort 1A was performed on the morning after overnight collection of urine. See section on observations and examinations for parental animals.
- Urinalysis: Urine samples were collected at test termination from F1 Cohort 1A animals (10/sex/dose). Urine samples were collected after overnight withdrawal of food and water. Animals were deprived of water overnight but had access to water for a minimum period of one hour prior to blood sampling. The following parameters were assessed: Volume, Appearance (clarity and colour by visual assessment), Specific gravity, pH, Glucose (Gluc), Ketones (Keto), Bile pigments (Bili), Blood pigments (UBld), Protein-total and concentration, Sodium-total and concentration, Potassium-total and concentration, Chloride-total and concentration. The sediment, obtained after centrifugation, was examined microscopically for epithelial cells (Epi), leucocytes (WBC), erythrocytes (RBC), casts, crystals, spermatozoa and other abnormal components.
- Sperm Analysis: Sperm analysis was done for selected F1 animals at scheduled termination.
- Immunox analysis: Ten males and ten females per group from F1Cohort 1A were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter. The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portions of the spleen was then weighed, placed into a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis. In Table 1 ('any other information on materials and methods') an overview of the cell markers assessed in immunophenotyping is provided.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: F0 males were sacrificed after weaning of the F1 animals, after confirmation that no further mating was required.
- Female animals :Surviving F0-females were sacrificed on Day 28 post partum). Females that failed to litter or mate were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females. All animals were weighed before necropsy.

GROSS NECROPSY
All F0 animals were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative and processed for histopathological examination (see Table 3). For F0 females the implantation site count was recorded.

HISTOPATHOLOGY/ORGAN WEIGHTS
The tissues indicated in Table 3 in ‘Any other information on materials and methods incl. tables’ were prepared for microscopic examination and weighed, respectively. Tissues were routinely preserved iin 10% neutral buffered formalin (NBF) with the following exceptions: eyes and testes were fixed in Modified Davidson’s fluid. Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with hematoxylin and eosin. Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). For the lower dose groups from F0 animals, slides were prepared and examined microscopically only for 1) abnormalities, 2) reproductive organs from F0 animals that showed reduced fertility (males that failed to sire a pregancy and females that were not pregnant and failed to litter), and 3) stomach (F0 females)

Postmortem examinations (offspring):

SACRIFICE
Unselected F1 offspring was sacrificed at culling on Day 4 and Day 22 of age. Selected Cohort 1A animals were sacrificed at approximately 13 weeks of age and Cohort 1B animals were sacrificed at approximately 14 weeks of age.

GROSS NECROPSY
All F1 animals, including surplus offspring culled on Day 4 of age and Day 22 of age unselected offspring were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative and processed for histopathological examination (see Table 3, 4 and 5). Decedents offspring ≤ 21 days of age, (found dead or welfare kill), where possible, were examined and carcass retained.
For females of Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.

HISTOPATHOLOGY/ORGAN WEIGHTS
Selected tissues and any abnormalities of selected F1of cohort 1A and 1B and unselected F1 offspring were weighed, fixed and examined as indicated in Table 3, 4 and 5, respectively. For unselected F1 offspring on Day 22 of age, organs were weighed from ten males and ten females per sex per group from as many litters as possible. Tissues were routinely preserved iin 10% neutral buffered formalin (NBF) with the following exceptions: eyes and testes were fixed in Modified Davidson’s fluid. For Cohort 1A animals, tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Sections were stained with hematoxylin and eosin. Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). For the lower dose groups from F1 animals, slides were prepared and examined microscopically only for 1) abnormalities and 3) stomach (F1 Cohort 1A females)
For Cohort 1B animals, tissue samples were dehydrated and embedded in parafin wax.

Statistics:

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis (except during gestation and lactation). For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated 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 data types were 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.
• Hematology
• Blood chemistry
• Urinalysis
• F0 Estrous cycles
• F0 Gestation length
• F1 Cohort 1A estrus before termination
• Litter (implantations, litter size, sex ratio – percentage male, post implantation survival index, llive birth index and viability index), for before cull study periods
• Ano-genital distance, adjusted for Day 1 pup body weight
• Sexual maturation, age and body weight at completion
• Sperm analysis motility, morphology and counts
• Organ weights, absolute and relative to body weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4
Group 1 vs 4 (as applicable)

Methods
For categorical data, the proportion of animals was analyzed for each treated group (as appropriate) versus the control group. For continuous data, Bartlett’s test was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups were then compared with the control group, incorporating adjustment for multiple comparisons where necessary. Further information on the statistical methods used is provided in the attached background information.

Reproductive indices:

Mating performance and fertility - F0 generation
♦ Percentage mating (%): (No. of animals mating/Animals paired) x 100
♦ Conception rate (%): (No. of animals achieving pregnancy/Animals mated) x 100
♦ Fertility index (%): (No. of animals achieving pregnacy/Animals paired) x 100

Gestation length and Gestation Index - F0 generation
♦ Gestation index (%): (No. of live litters born/animals paired) x 100

Offspring viability indices:

Survival Indices – F0 Generation
♦ Post-implantation survival index (%): (Total no. of offspring born/Total no. of uterine implantation sites) x 100
♦ Live birth index (%): (No. of offspring on Day 1 after littering/Total no. of offspring born) x 100
♦ Viability index (%): (No. of live offspring on Day 4 before culling/No. of live offspring on Day 1 after littering) x 100
♦ Lactation index (%): (No. of live offspring on Day 21 after littering/No. of live offspring on Day 4 after culling) x 100
♦ Sex ratio (% males): (No. of males in litter/Total no. of offspring in litter) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During Week one of treatment body weight gain for males and females receiving 3000 ppm and males receiving 600 ppm was low when compared with Controls (p<0.01). Overall body weight gain for males at 3000 ppm was slightly low at approximately 92% of Controls (p<0.01); overall weight gain for males at 120 or 600 ppm or females at all dose levels were similar to Controls.
During gestation body weight gain at 3000 ppm was slightly but statistically significantly low from GD7 to GD14 (p<0.01) and the mean body weight on GD14 at this dietary concentration was slightly low when compared with Controls (p<0.05). On Day 1 of lactation group mean body weight was slightly low at 3000/1500 ppm when compared with Controls (p<0.05). Overall body weight gain during gestation and lactation was unaffected by administration of Sodium Trifluoroacetate at dietary concentrations up to and including 3000/1500 ppm.
Detailed results are provided in Table 1-3 (see attached document).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Overall food consumption during the ten-week pre-pairing treatment period showed no clear dose-related adverse effects of Sodium Trifluoroacetate administration. There was no clear effect on food consumption during gestation; however, at 3000 ppm mean food consumption values from GD7-19 were slightly but statistically significantly low when compared with Controls (p<0.01) and during GD7-13 mean consumption was also slightly low at 120 and 600 ppm (p<0.05). During lactation food consumption was low for females receiving 3000/1500 ppm, with statistical significance attained from LD4 to LD20 (p<0.05/0.01); consumption during lactation for females at 120/60 ppm or 600/300 ppm was similar to Controls.
Detailed results are provided in Table 4 and 5 (see attached document).
The mean achieved dose levels over the ten-week pre-pairing treatment period at 120, 600 and 3000 ppm were 9.71, 49.2 and 248 mg/kg/day for males and 10.26, 53.9 and 265 mg/kg/day for females, respectively. During gestation mean achieved dose levels were 8.65, 44.3 and 223 mg/kg/day for females receiving 120, 600 and 3000 ppm and during lactation the mean achieved dose levels were 9.85, 47.5 and 233 mg/kg/day for females receiving 60, 300 and 1500 ppm, respectively.
The mean achieved dose levels for the individual study periods are summarized in Table 6 (see attached document).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males at 3000 ppm and females at 3000/1500 ppm showed slightly but statistically significant low hemoglobin (p<0.05/0.01) and low mean cell hemoglobin (p<0.05). Females at 3000/1500 ppm also showed low hematocrit (p<0.05) and low mean cell hemoglobin concentration (p<0.01). At 3000 ppm males showed low monocyte counts (p<0.05) and short activated partial thromboplastin time (p<0.01) and at 3000/1500 ppm females showed high platelet counts (p<0.01).
Detailed results are provided in Table 7 (see attached document).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Detailed results are provided in Table 8 (see attached document).
Alkaline phosphatase activity for males at 600 or 3000 ppm was elevated when compared to Controls (p<0.01). Males and females at all dose levels showed low plasma glucose levels (p<0.05/0.01) and low levels of non-esterified fatty acids (p<0.01); males at all dose levels also showed low triglyceride concentrations (p<0.01) when compared to Controls.
Other statistically significant differences were as follows:
- bilirubin concentrations at all dose levels were slightly low for females only (p<0.05 at 120/60 ppm; p<0.01 at 600/300 or 3000/1500 ppm)
- sodium concentrations at 3000 ppm were high for males (p<0.05)
- potassium concentrations were high at 3000/1500 ppm for females (p<0.05)
- calcium concentrations were low at 3000 ppm in males (p<0.01)
- A/G ratio was high in males at 600 or 3000 ppm and females at 3000/1500 ppm (p<0.01)

Thyroid hormone analyses:
Assessment of TSH serum levels in F0 males and females at scheduled termination did not reveal any differences that could be attributed to administration of Sodium Trifluoroacetate at dietary concentrations up to and including 3000 ppm. Detailed results are provided in Table 21 (see attached document).
Mean serum T4 concentrations in F0 males at 3000 ppm and females at 3000/1500 ppm were low when compared with Controls (p<0.01). Detailed results are provided in Table 22 (see attached document).

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:

effects observed, treatment-related

Description (incidence and severity):

There was a test item-related increased incidence of minimal gland dilatation in the stomachs of the females given 600/300 ppm and 3000/1500 ppm. This finding was present in the fundic portion of the glandular stomach. This finding was considered non-adverse based on the minimal severity. Detailed results are provided in Table 11 (see attached document).

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles were unaffected by administration of Sodium Trifluoroacetate at dietary concentrations up to and including 3000 ppm. The majority of females showed an estrus smear prior to termination.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistical effects observed on sperm motility/motion or testis parameters. There were some changes of the parameters related to the cauda epididymis at 3000 ppm Sodium Trifluoroacetate. The slight non-statistically significant increase of progressive motile sperm (%) was within HCD, while the control value was outside the HCD range. In addition, the motion parameters Rapid was slightly decreased. The value at 3000 ppm was higher than the HCD range and is considered as non-adverse. There were correlating increases in slow and static sperm which were however inside the HCD range.
At 3000 ppm there were also statistically significant decreases in cauda epididymis weights and total sperm along with a non-statistically significant decrease in cauda epididymal sperm number per gram when compared with Controls. However, it has to be noted that the values of the concurrent control were all outside the HCD range, while the values of the high-dose group were within the HCD-range. In addition, mean epididymal weight at termination showed no significant difference to Controls at termination.
Morphologically there was a slight but statistically significant decrease in normal % and an increase in total abnormal % sperm with no particular abnormality, when compared with Controls. But all the values were within the HCD range. The abnormality head misshapen was highlighted as statistically significant but the incidence was lower than in the control group.
Detailed results are provided in Table 10-12 (see attached document).

Reproductive performance:

no effects observed

Description (incidence and severity):

- Fertility data: There were no test item-related changes in fertility data, including male mating index, male fertility index, female mating index, female fertility index, and fecundity index in this study. Detailed results are provided in Table 14 (see attached document).
- Cohabitation Duration: There were no test item-related cohabitation duration changes in the study. All the data in test item treated groups were comparable to the control.
- Gestation duration: There was no adverse effects on either gestation length or gestation index at all dose levels.
- Litter Size, Survival Indices and Sex Ratio: There were no treatment-related effects on Live birth, viability and lactation indices at dose level of Sodium Trifluoroacetate up to and including 3000 / 1500 ppm. Post-implantation loss was slightly high at 3000/1500 ppm (p<0.05), however the mean value was within the historical control range and the litter size was unaffected by treatment. Sex ratio was unaffected by treatment. Detailed results on litter data are provided in Table 13 (see attached document).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed in reproductive and developmental parameters and general toxicity at any of the dose levels

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The general condition of F1 offspring was unaffected by parental treatment.

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):

Detailed results on the F1-offspring bodyweight and body weight gain during lactation are provided in Table 15 (see attached document).
Offspring body weight on Day 1 of age and subsequent weight gain up to Day 4 of age showed no effects of parental treatment. From Day 4 to Day 14 of age male and female offspring at 3000/1500 ppm showed statistically significantly low body weight gain when compared with Controls (p<0.05/0.01) and overall body weight gain from Day 1 of age up to weaning on Day 21 of age at 3000/1500 ppm was low when compared with Controls (p<0.05); however as the weight gain was only 7% lower than Controls, this affect was considered minor and was considered non adverse. Body weight gain for offspring at 120/60 ppm or 600/300 ppm was unaffected by administration of Sodium Trifluoroacetate.
Detailed results on the F1-males and females bodyweight and body weight gain are provided in Table 16 and 17 (see attached document).
At weaning on Day 21 of age mean bodyweights for selected F1 animals receiving 3000/1500 ppm were low when compared with Controls (p<0.01) and for females at these dietary concentrations the gain up to Day 25 of age was also low (p<0.01); this was not apparent for selected males. The mean absolute body weights for selected F1 animals at 3000/1500 ppm remained low from Day 1 to Day 64 of the F1 generation, with males at these dietary concentrations also showing low overall weight gain during this period (p<0.01); overall weight gain for high dose females was similar to Controls over the same period. Body weight and body weight gain at 120/60 or 600/300 ppm were unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Overall, there was no adverse effect on food consumption during treatment of the F1 generation. Females at 3000/1500 ppm showed slightly low food consumption (p<0.05) during Week 1, 8 and 10 of the F1 generation and males at 3000/1500 ppm also showed slightly low food consumption (p<0.05) during Week 4. Females at 600/300 showed slightly but statistically significantly high food consumption (p<0.01) during Week 5 of the F1 generation when compared with Controls; this was not evident for males at this dietary concentration.
Detailed results are provided in Table 18 (see attached document).
The mean achieved dose levels were 9.37, 47.3 and 242 mg/kg/day for males, and were 9.83, 49.4 and 248 mg/kg/day for females, at 120/60, 600/300 and 3000/1500 ppm respectively. The mean achieved dose levels for the individual study periods of the F1 animals are summarized in Table 6 (see attached document).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males at 3000/1500 ppm and females at 600/300 or 3000/1500 ppm showed low hematocrit and hemoglobin count. Lymphocyte and monocyte counts were slightly low in males at 3000/1500 ppm (p<0.05); this was not evident for females at this dose level. Females at 600/300 or 3000/1500 ppm had slightly high platelet counts (p<0.05); there was no evidence of a dose response, and it was not evident in males.
Detailed results are provided in Table 19 (see attached document).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry evaluation showed some changes that were considered to be test-substance related. Alkaline phosphatase activity for males at 600 or 3000 ppm was elevated when compared to Controls (p<0.01).
Males and females at all dose levels showed low plasma glucose levels (p<0.05/0.01) and low levels of non-esterified fatty acids (p<0.01); males at all dose levels also showed low triglyceride concentrations (p<0.01) when compared to Controls.
Other statistically significant differences were as follows:
• bilirubin concentrations at all dose levels were slightly low for females only (p<0.05 at 120/60 ppm; p<0.01 at 600/300 or 3000/1500 ppm)
• sodium concentrations at 3000 ppm were high for males (p<0.05)
• potassium concentrations were high at 3000/1500 ppm for females (p<0.05)
• calcium concentrations were low at 3000 ppm in males (p<0.01)
• A/G ratio was high in males at 600 or 3000 ppm and females at 3000/1500 ppm (p<0.01).
Detailed results are provided in Table 20 (see attached document).

Thyroid hormone analyses:
Assessment of TSH serum levels in F1 offspring on Day 22 of age and selected F1 Cohort 1A animals at scheduled termination did not reveal any differences that could be attributed to administration of Sodium Trifluoroacetate at dietary concentrations up to and including 3000 ppm. Detailed results are provided in Table 21 (see attached document).

At 1500 ppm mean serum T4 concentrations for F1 male and female offspring on Day 22 of age were low when compared with Controls (p<0.01), with most individual values below the lowest value in the concurrent Controls. The mean serum T4 concentration in male offspring only at 300 ppm was slightly low (p<0.05) and as the value was very close to the mean historical control data (HCD) value no effect of treatment was inferred.

Mean serum T4 concentrations in F1 males at 600 or 3000 ppm were low when compared with Controls (p<0.01), with most individual values below the lowest value in the concurrent Controls. At 120 ppm, the mean T4 level in F1 males was lower than the concurrent Controls (p<0.05) but the value was less than 1 SD away from the HCD mean and no effect of treatment was inferred. Considering that except for a slight effect on the growth of the animals, there were no effects on reproductive performance, parturition, offspring, survival, clinical condition or sexual maturation, and changes in the reproductive organs (all parameters/processes depending on normal thyroid function). Thus, the low T4 levels seen in F0 males and females, male and female F1 offspring on PND22 and F1A males are considered not to represent an adverse effect of treatment.
Detailed results are provided in Table 22 and 23 (see attached document).

Urinalysis findings:

no effects observed

Description (incidence and severity):

Specific gravity for females at 3000/1500 ppm was slightly low (p<0.05) when compared with Controls, but this was consistent with the slightly higher urinary volume; there was no similar finding in males. Total sodium and protein levels were slightly high in males at 600/300 or 3000/1500 ppm; but these differences did not attain statistical significance Total potassium levels were low and total sodium concentrations slightly high for females at 3000/1500 ppm (p<0.05).
Detailed results are provided in Table 24 (see attached document).

Sexual maturation:

no effects observed

Description (incidence and severity):

The age and body weight at attainment of balano preputial separation or vaginal opening was considered to be unaffected by administration of Sodium Trifluoroacetate at dietary levels up to and including 3000/1500 ppm. The mean age on completion of vaginal opening at 600/300 or 3000/1500 ppm was 34.0 or 33.7 days vs 32.6 days in the Control animals with the mean differences of 1.5 or 1.1 days attaining statistical significance (p<0.05). As the animals are only looked at once a day this difference is not considered of toxicological significance.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Ano-genital distance for both male and female offspring were similar across the groups and showed no effects of parental treatment. Detailed results are provided in Table 25 (see attached document).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

On Day 13 of age male offspring were assessed for the presence or absence of nipple/areolae and no nipples were observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Brain, spleen and thymus weights of unselected F1 offspring on Day 22 of age were unaffected by parental treatment with Sodium Trifluoroacetate.

Cohort 1A
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.
Mean absolute testes weight was low (p<0.01) in the Cohort 1A males at 1500/3000 ppm when compared with Controls, but relative weights were comparable in all groups.
Mean absolute weight of the epididymides was low (p<0.05) in the Cohort 1A males at 1500/3000 ppm when compared with Controls. This change is considered to be incidental based on the lack of statistically significant low epididymides weights in Cohort 1B males, the lack of microscopic and sperm analysis correlation and the low magnitude of this change.
Mean absolute weight of the pituitary was low in the Cohort 1A females that received 1500/3000 ppm (p<0.05) when compared with Controls. This change is considered incidental as all individual weights are within the range of the control animals and this this difference was not apparent in Cohort 1A males or Cohort 1B females.
Terminal body weights were low for males (p<0.01) and females (p<0.05) at 1500/3000 ppm when compared with Controls. This caused statistically significant differences in organ weights of various organs including the thyroids and parathyroids, adrenals, kidneys and heart; these differences were not considered a direct effect of the test item.

Cohort Cohort 1B
Mean absolute testes weight was low in the males treated with 1500/3000 ppm (p<0.05) when compared to Controls. Relative testes weights were comparable in all dose groups.

Detailed results are provided in Table 26 and 27 (see attached document).

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring that either died prematurely, were culled on Day 4 of age or terminated on Day 22 of age did not reveal any findings that could be related to administration of Sodium Trifluoroacetate.
In Cohorts 1A and 1B all macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent Controls), and/or were as expected for rats of this age and strain. Therefore, they were considered not test item-related.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

There was a test substance-related increased incidence of minimal to slight glandular dilatation in the stomachs of females given 1500/3000 ppm. This finding was present in the fundic portion of the glandular stomachs. This finding was also present in the F0 females given 600/300 ppm and 3000/1500 ppm.
In the glandular stomachs of females given 1500/3000 ppm there was a high incidence of minimal to slight decreased secretion in the fundic mucous neck cells. In affected animals the mucous neck cells were attenuated and exhibited a small amount of pale eosinophilic cytoplasm with minimal or no mucus content. The gastric surface epithelium was intact. These finding were considered non-adverse based on their low severity grade.

Description (incidence and severity):

- Sperm assessment (Cohort 1A): There were no adverse effects on % motile sperm and % progressive sperm although there appeared to be a slight effect on testis weight. Morphologically: at 3000 ppm there was a statistically significant increase in abnormal sperm heads specifically flat head when compared with Controls and the incidence was outside of HCD range, however the overall % of normal sperm in the Control group exceeded the HCD range. At 3000/1500 ppm there were statistically significant decreases in testis weight, testis spermatid count and testis total million compared with Control; however only the testis weight was outside of HCD. The effect on testis weight is consistent with the mean combined weight testis recorded at necropsy of both Cohort 1A and 1B.
At 3000 ppm there was a statistically significant decrease in motion values VAP, VCL and BCF compared to concurrent control. At 600 ppm there was a statistical decrease in motion values VAP and VCL compared to concurrent control. At 120 ppm there was a statistical decrease in motion value VAP compared to concurrent control. All values were inside HCD range. The statistical significance of these parameters is considered to be due to the Control motion values being high (above the HCD range).
Detailed results are provided in Table 28-30 (see attached document).

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

A decreasing trend was observed in the cells/spleen immunophenotyping parameters in both male and female treated groups (Groups 2 to 4) when compared to the control group. A decrease in female CD4+ T cell percentage and an increase in female CD8+ T cell percentage was observed at dose 60/120 ppm compared to the Group 1 control. These differences were not related to the dietary administration of Sodium Trifluoroacetate. There were no observable effects on the remaining immunophenotyping parameters in male or female Han Wistar rat spleen leukocytes as a result of the dietary administration of Sodium Trifluoroacetate at doses 60/120, 300/600 and 1500/3000 ppm.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed in reproductive and developmental parameters and general toxicity at any of the dose levels

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Dietary administration of Sodium Trifluoroacetate to Han Wistar rats at concentrations of 120, 600 or 3000 ppm reduced to 60, 300 or 1500 pm for F0 females during lactation and F1 offspring up to nominal Day 28 of age was well tolerated with no adverse effects on general condition, body weight or food consumption, reproductive performance, fertility or offspring development/sexual maturation. Based on the available results it is concluded that 3000/1500 ppm (approximating 242 -265 mg NaTFA/kg/day) was the No Observed Adverse Effect Level (NOAEL) for both both reproductive performance/offspring development and for general systemic toxicity.

Executive summary:

In a GLP compliant Extended One Generation Reproduction Study in rat according to OECD 443, sodium trifluoroacetate was adminstered daily by oral gavage to Wistar rats. 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 ca. 10, 50 and 250 mg/kg bw/day, for 10 weeks premating, gestation and lactation. To compensate for the higher food intake during lactation, dietary concentrations were reduced in females to 60, 300 and 1500 ppm during lactation. A similarly constituted Control group received untreated basal diet. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. During the lactation period, the F1 animals were not administered with test item directly. The F1 animals consisted of 2 cohorts (Cohort 1A and Cohort 1B) from weaning and were treated with the test item directly from weaning (Day 21 of age) up to 13 and 14 weeks, respectively. Cohort 1A was allocated for primary assessment of effects upon reproductive systems and of general toxicity, and Cohort 1B for possible histopathology follow-up assessment of reproductive system. The Cohort 1A and 1B animals (20/sex/group) were treated from Day 21 to nominal Day 35 of age at dietary concentrations of 60, 300 or 1500 ppm and from Day 35 of age to their scheduled termination at dietary concentrations of 10, 600 or 3000 ppm. A similarly constituted Control group received untreated basal diet.

Criteria for evaluation included viability (morbidity/mortality), clinical observations, body weight, food consumption, estrus cycle, cohabitation duration, sperm analysis, fertility data, gestation duration, parturition observation, litter evaluation, litter data, development landmarks, clinical pathology (hematology, serum chemistry, urinalyses), thyroid hormone analysis (TSH and T4), spleen lymphocyte subpopulations, gross (necropsy) evaluation, organ weight, and histopathological evaluation (F1 Cohort 1B animals were not examined microscopically).

Results

No test item-related mortality was observed in both F0 and F1 animals during the study period. There were no test item-related changes on body weight or food consumption. There were no test item related changes in mating performance, fertility, gestation duration, litter evaluation, litter data (number of corpora lutea, implantation sites, and litter size, pre- and postimplantation loss, sex ratio, pup viability). There were no test item-related changes on developmental landmarks (including nipple retention, testes descent, and vaginal opening), anogenital distance, hematology, urinalysis, spleen lymphocyte subpopulations percentage (including Total T cells, Helper T cells, Cytotoxic T cells, B cells and NK cells) and TSH levels in this study.

Test item-related microscopic findings were observed in the fundic portion of the glandular stomachs of the F0 females and F1 Cohort 1A females with an increased incidence of minimal to slight gland dilatation in the F0 females at 600/300 ppm and 3000/1500 ppm and the F1 cohort 1A females at 1500/3000 ppm. The F1 cohort 1A females at 1500/3000 ppm also exhibited a high incidence of minimal to slight decreased secretion in the mucous neck cells located in the fundic region of the glandular stomach. As the severity of these findings were minimal to slight, the general condition of the females was unaffected and both bodyweight performance and food consumption of the females showed no adverse effects of treatment, these findings were considered non-adverse in this study. In the F0 and F1 generation body weight relative liver weight was high at 3000/1500ppm in both male and female animals. In the F0 and F1 generation males and females showed low plasma glucose levels and low levels of non-esterified fatty acids, males at all dose levels also showed low triglyceride concentrations. Bilirubin plasma concentrations were low for F0 females at all dose levels, for F1 males at 600/300 or 3000/1500 ppm and F1 females at 3000/1500 ppm. The increased liver weight and changes in the biochemistry of the plasma indicate some alteration in liver metabolism however in the absence of any macroscopic or microscopic liver pathology, these effects are considered to be non-adverse.

Kidney weights were high for F0 males and females at 3000/1500 ppm and for F0 males at 600 ppm. Plasma sodium levels in F0/F1 males and potassium levels in F0/F1 females were high, however in the absence of an effect on kidney weight in the F1 animals, with no adverse effects on the urinary composition in the F1 generation or any correlative microscopic changes in either the F0 or F1 generation these changes were considered non-adverse within the context of this study. In F1 generation Cohort 1A and 1B males had slightly low testes weights, however sperm assessment showed that the spermatid count in the Cohort 1A animals although slightly low was with in the historical control range. Histopathological examination of the Cohort 1A testes showed that the majority exhibited normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present. Tubular degeneration and atrophy occurred at a low incidence in both treated and control males but was considered unrelated to treatment as the incidence was similar across the groups.

Mean serum T4 concentrations in F0 males at 3000 ppm and females receiving 3000/1500 ppm were low when compared with Controls (p<0.01). At 1500 ppm mean serum T4 concentrations for F1 male and female offspring on Day 22 of age were low when compared with Controls (p<0.01), with most individual values below the lowest value in the concurrent controls. Mean serum T4 in F1A males at 600 or 3000 ppm were low when compared with Controls (p<0.01), with most individual values below the lowest value in the concurrent Controls. In the absence of any effects on reproductive performance, parturition, offspring, survival, clinical condition or sexual maturation, and changes in the reproductive organs (all parameters/processes depending on normal thyroid function), the low T4 levels seen in F0 males and females, male and female offspring on PND22 and F1A males are considered no to represent an adverse effect of treatment. 

Conclusion

Based on the available results it is concluded that 3000/1500 ppm (approximating 242 -265 mg NaTFA/kg/day) was the No Observed Adverse Effect Level (NOAEL) for both both reproductive performance/offspring development and for general systemic toxicity.