Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-11-21 to 2012-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was GLP, standardized guidelines compliant and well described.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Screening test (reproduction/developmental toxicity) combined with repeated dose toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-11-21 to 2012-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study, OECD 422 compliant

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-07-19

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories France, L’Arbresle, France. Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Age at study initiation: the males were approximately 10 weeks old, the females were approximately 9 weeks old
- Weight at study initiation: males: a mean body weight of 383 g (range 359 g to 421 g), and female a mean body weight of 211 g (range: 186 g to 236 g).
- Fasting period before study:
- Housing: The animals were individually housed, except during pairing, in polycarbonate cages (UAR, 43.0 cm x 21.5 cm x 18 cm) with stainless steel lids, and containing autoclaved sawdust (SICSA, Alfortville, France).
Toward the end of gestation and during lactation with their litter, autoclaved wood shavings (SICSA Alfortville, France) was provided as nesting material, a few days before delivery and during the lactation period.
Nylabone was given as enrichment of the environment of the rats.
The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): The animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 7055973 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated for a period of 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES: From:2011-11-24 To: 2012-01-24

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PPREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle.
The dosage formulations were prepared for 2 to 8 days (frequency based on the results of the stability study CIT/Study No. 38264 AHS).
The dosage forms were stored and delivered at room temperature and protected from light (see § Study plan adherence).

VEHICLE
drinking water treated by reverse osmosis using an ELIX 5 apparatus
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg of active ingredient
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:

- M/F ratio per cage:1/1
- Length of cohabitation: until mating occurs or 14 days
- Proof of pregnancy: vaginal plug or sperm in a vaginal lavage. referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Alone
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The High Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method for the determination of Reaction mass of TFSK/TFAK in dosage form samples was validated at CIT prior to dosage form analysis.
The analytical method was validated for concentration ranging from 2 to 200 mg/mL.

The concentration of the test item in samples of each control and test item dosage form prepared for use in weeks 1, 3 and 5 was determined. In addition, the concentration of the test item in a sample of the high-dose test item form prepared for use in week 2 was determined.

Acceptance criterion: measured concentration = nominal concentration ± 10%

The validation of the analytical method was conducted in CIT/Study No. 38263 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results were documented in the corresponding validation report.

Each analytical sequence consisted of at least:
+ A blank sample (diluent only),
+ Ten standard samples at nominal concentration, prepared from two independent standard solutions,
+ Study samples prepared from aliquots of the dosage forms.
The standard samples bracketed the dosage form samples.
The blank sample was checked for the absence of chromatographic interference.

Duration of treatment / exposure:

The dosage forms were administered daily according to the following schedule:
in the males:
- 2 weeks before pairing (from study day 1 to 15),
- during the pairing period (3 weeks)(from study day 16 until study day 17 to 31),
- until sacrifice (at least 5 weeks in total)(from study day 18 to 32 until study day 39).

in the females:
- 2 weeks before pairing (from study day 1 to 15),
- during the pairing period (3 weeks)(from study day 16 until study day 17 to 31),
- during gestation (from study day 18 to 32 until study day 38 to 52),
- during lactation until day 5 post-partum inclusive (from study day 39 to 51 until study day 43 to 56),
- until sacrifice for the non-pregnant female (from study day 16 to Day 42).

Study day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

Once a day at approximately the same time

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex and per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose-levels were selected in agreement with the Sponsor.
In a previous study (CIT/Study No. 38258 TSR), Reaction mass of TFSK/TFAK (batch No. SSK1278645) was given to rats (three/sex/group), by daily oral administration (gavage) for 2 weeks at 0, 150, 550 or 1000 mg/kg/day active ingredient. Ptyalism was observed in 2/3 males and in 1/3 females given 1000 mg/kg/day for 2 to 4 days during the second week of treatment. This clinical sign was attributed to the test item. Reflux at dosing was observed in 1/3 females given 500 mg/kg/day active ingredient on day 15. Soft feces, alopecia and scabs were noted in some animals; as these findings were isolated and/or without any dose relationship, they were considered as incidental.
Body weight and food consumption were considered to be unaffected by the treatment with the test item. On necropsy, there were no test item-related macroscopic post-mortem findings.
Therefore 1000 mg/kg/day, which is also the maximum recommended dose to be tested according to OECD Guideline No. 422, was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing a three-fold interval (i.e. 100 and 300 mg/kg/day active ingredient).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations:
+ males: on the first day of treatment (day 1), then once a week until sacrifice.
+ femelles: on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 p.c. and days 1 and 5 p.p..
The body weight of female sacrificed on day 25 p.c. for no delivery was recorded the day of sacrifice, presented in the report but not including in the statistical evaluation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1 5 p.p..
During the pairing period, the food consumption was measured neither for males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

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

OPHTHALMOSCOPIC EXAMINATION: Yes pupillary reflex during the FOB test
- Dose groups that were examined: 5 males and 5 female per group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females to deliver from each group
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- TTime schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females to deliver from each group
- Parameters checked in table were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period
- Dose groups that were examined: The first five males and the first five females to deliver
- Battery of functions tested:
+"touch escape" or ease of removal from the cage
+In the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis)
+ In the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia
+ The following parameter measurements, reflexes and responses were recorded:
 touch response,
 forelimb grip strength,
 pupillary reflex,
 visual stimulus response,
 auditory startle reflex,
 tail pinch response,
 righting reflex,
 landing foot splay,
 at the end of observation: rectal temperature.

OTHER:
+ at the end of observation: rectal temperature.
+Motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the females are mated.

Sperm parameters (parental animals):

No data

Litter observations:

Each live pup was identified individually on day 1 p.p., by subcutaneous injection of Indian ink.

Litter size
The total litter size and numbers of pups of each sex were recorded as soon as possible after birth. Any gross malformations in pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.

Clinical signs
The pups were observed daily for clinical signs or abnormal behavior.

Body weight
The body weight of each pup was recorded on days 1 and 5 p.p..

Postmortem examinations (parental animals):

SACRIFICE
GROSS PATHOLOGY: Yes
Parent animals:
A complete macroscopic post-mortem examination was performed on all parent animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 6 post partum and for female sacrificed on day 25 post-coitum due to no delivery. For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
Pup examinations:
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups showing relevant external abnormalities. No tissues were preserved.

HISTOPATHOLOGY: Yes (see table)

Postmortem examinations (offspring):

A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead pups. Special attention was paid to whether the pup had fed (e.g. presence of milk in the stomach). No tissues were preserved.

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups showing relevant external abnormalities. No tissues were preserved.

Statistics:

see attached documents statistique.doc

Reproductive indices:

- pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

- post-implantation loss:
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantations

- mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

- fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

- gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females

Offspring viability indices:

- live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

- viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males at 1000 mg/kg/day there was a minimal decrease in hemoglobin content (considered as minor toxicological significance).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males at 1000 mg/kg/day there was a minimal increase in inorganic phosphorus content and a light decrease in calcium content (considered as minor toxicological significance).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test item administration induced minimal centrilobular hypertrophy of hepatocytes in males and females at 1000 mg/kg/day, and in males at 300 mg/kg/day.
This microscopic change correlated with increased liver weights.
This change was not considered to be adverse in view of the absence of associated degenerative microscopic findings or clinical pathology changes in liver enzyme activities.

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths and no clinical signs related to the treatment with the test item.

BODY WEIGHT AND WEIGHT GAIN
There were no effects on mean body weight or mean body weight change, either in males or females.

FOOD CONSUMPTION
There were no effects on food consumption.

HAEMATOLOGY
In males and at 1000 mg/kg/day active ingredient there was a minimal decrease in hemoglobin content (-10.2% vs. controls, p <0.01).
Taking into account the amplitude of the changes and in absence of relevant effect in other parameters, a relationship with the test item treatment was considered unlikely up to 300 mg/kg/day active ingredient and of minor toxicological significance at 1000 mg/kg/day active ingredient (males only)

CLINICAL CHEMISTRY
In males and at 1000 mg/kg/day active ingredient there was a minimal increase in inorganic phosphorus content (+13.4% vs. controls, p <0.05) and a light decrease in calcium content (-4.9% vs. controls, p<0.05).
Taking into account the amplitude of the changes and in absence of relevant effect in other parameters, a relationship with the test item treatment was considered unlikely up to 300 mg/kg/day active ingredient and of minor toxicological significance at 1000 mg/kg/day active ingredient (males only).
Other statistically significant differences (chloride, glucose and albumin) and were minimal, without any dose-level relationship and/or in one sex only. Therefore a treatment-related effect was considered unlikely.


NEUROBEHAVIOUR
There were no relevant differences in treated groups when compared with control group.

ORGAN WEIGHTS
The mean absolute and relative liver weights were increased in males treated at 300 or 1000 mg/kg/day active ingredient. These variations were statistically significant. Minimal, not statistically significant increases were also seen in females at 1000 mg/kg/day active ingredient. This increase correlated with microscopic changes observed in the liver.
There were statistically significant increases of the mean absolute and relative kidney weights in females treated at 1000 mg/kg/day active ingredient. In view of the low amplitude of the changes and the absence of microscopic correlates, these variations were considered to be of minor toxicological significance
Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude

GROSS PATHOLOGY
There were no macroscopic changes attributed to the test item administration.
The few macroscopic findings noted had no histologic correlates or correlated with common histologic findings in control Sprague-Dawley rats, and were considered to be incidental.


HISTOPATHOLOGY: NON-NEOPLASTIC
The test item administration induced minimal centrilobular hypertrophy of hepatocytes in 4/5 males and 2/5 females at 1000 mg/kg/day active ingredient, and in 1/5 males at 300 mg/kg/day active ingredient. This microscopic change correlated with increased liver weights. This change was not considered to be adverse in view of the absence of associated degenerative microscopic findings or clinical pathology changes in liver enzyme activities.
In males given the test item at all dose-levels, the incidence and severity of hematopoiesis in the spleen were minimally increased compared to controls. This increased hematopoiesis may have been compensatory to the changes in red blood cell parameters.
There were no microscopic changes attributed to the test item administration in the male and female genital organs.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.

Key result

Dose descriptor:

NOAEL

Remarks:

Parental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Effects considered to be of minor toxicological significance

Remarks on result:

other: Histopathological observation: Centrilobular hepatocellular hypertrophy but not judged adverse (in view of the absence of associated degenerative microscopic findings or clinical pathology changes in liver enzyme activities).

Key result

Dose descriptor:

NOEL

Remarks:

Parental reproductive performance

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: mating and fertility

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
There were no treatment-related effects on pup mortality

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs in pups considered to be treatment-related

BODY WEIGHT (OFFSPRING)
There were no effects on mean body weight neither in male nor in female pups during the lactation period (day 1 or day 5 p.p.).

GROSS PATHOLOGY (OFFSPRING)
There were no treatment-related findings in found dead or pups sacrificed on day 5 p.p..

Key result

Dose descriptor:

NOEL

Remarks:

Toxic effects on progeny

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Any treatment-related effect on pups at 1000 mg/kg/day

Key result

Reproductive effects observed:

no

The summary of mating datais presented in the following table:

 

Dose-level (mg/kg/dayactive ingredient)	0	100	300	1000
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	9	10	10	10
Number of females mated	10	10	10	10
Mean number of days taken to mate	3.6	3.0	2.0	3.4
Number of pregnant females	10	10	9	10
Male fertility index	90%	100%	100%	100%
Female fertility index	100%	100%	90%	100%

M: males; F: females.

 

The summary of delivery datais presented in the following table:

 

Dose-level (mg/kg/dayactive ingredient)	0	100	300	1000
Number of pregnant females	10	10	9	10
Number of females which delivered	10	10	9	10
Mean duration of gestation (days)	21.3	21.6	21.4	21.4
Mean number ofcorpora luteaper female	18.1	16.9	17.3	18.9
Mean number of implantation sites per female	17.2	15.7	15.9	15.8
Mean pre-implantation loss (%) per female	4.6	6.6	8.8	14.6
Mean number of pups delivered per female	15.0	13.7	15.0	14.1
Mean post-implantation loss (%)^aper female	11.5	12.4	4.5	11.1
Viability index on day 4p.p.(%) per female	90.7	99.3	96.3	96.5

a: manually calculated, no statistics performed.

 

The number of pups found dead and the number of litters affected

Dose-level (mg/kg /dayactive ingredient)	0	100	300	1000
Number of pups found dead (litter)	5 (2)	1 (1)	5 (3)	2 (2)
Number of pups cannibalized (litter)	10 (3)	0	1 (1)	3 (2)
Number of pups prematurely sacrificed	0	0	0	0

 

 

The live birth, viability and lactation indexes

 

Dose-level (mg/kg/dayactive ingredient)	0	100	300	1000
Live Birth Index (%)	100	100	100	100
Viability Index (day 4p.p., %).	90.7	99.3	96.3	96.5
Lactation Index (day 5p.p., %)	99.3	100.	99.2	100

 

 

Macroscopicpost-mortemexamination of pups

Dose-level (mg/kg/dayactive ingredient)	0	100	300	1000
Dead pups
Number of pup examined (litter)	5 (2)	1 (1)	5 (3)	2 (2)
Autolysis	1 (1)	0	0	0
Stomach: absence of milk	1 (1)	1 (1)	3 (2)	2 (2)
Total pup dead observations	2 (1)	1 (1)	3 (2)	2 (2)
Schedule sacrificed pups
Number of pup examined (litter)	0	0	1 (1)	1 (1)
Cutaneous lesion	0	0	0	1 (1)
Total pup scheduled sacrifice observations	0	0	0	1 (1)

 

 

Conclusions:

The test item, Reaction mass of TFSK/TFAK, was administered daily by gavage to male and female Sprague Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until day 5 post partum, at dose-levels of 100, 300 or 1000 mg/kg/day active ingredient (approximately 38 days for males and at maximum 57 days for females).
Slight but non adverse effects were observed in the liver from parent animals..
No effect on reproductive performance or reproductive organs was observed.
In pups, there were no treatment-related findings.
Based on the experimental conditions of this study:
- The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day active ingredient,
- The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day active ingredient,
- The No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 1000 mg/kg/day active.

Executive summary:

The purpose of this study (OECD 422 , 2012), was to generate information concerning the effects of TFSK/ TFAK on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

TFSK/ TFAK was administered to male rats for at least 38 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The following dose levels were applied:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

A dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (distilled water).

The following results were obtained:

In parent animals, in males and females, body weight and body weight gain were not considered to be affected by the treatment with the test item. At 300 and 1000 mg/kg/day a centrilobular hepatocellular hypertrophy was observed in some animals. This microscopic change correlated with increased liver weights. In view of the absence of associated degenerative microscopic findings or clinical pathology changes in liver enzyme activities this change was considered to be non adverse.

 

No effect on reproductive performance or parental reproductive organs was observed.

In pups, the mean number of pups at first litter check and on day 4 post partum was not affected by the treatment with the test item. The sex ratio was also not affected, and weight development was not affected by the treatment with the test item. At necropsy of pups, no test item-related findings were noted.

 

Based on the experimental conditions of this study:

-         The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 1000 mg/kg/day active ingredient,

-         the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day active ingredient,

-         the No Observed Effect Level (NOEL) for toxic effects on progeny was considered to be 1000 mg/kg/day active.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-07-19

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

EC Number:

911-467-3

Molecular formula:

C2O2F3K and CO2F3KS

IUPAC Name:

Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Remarks:

aqueous solution

Details on test material:

Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate / 911-467-3

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Charles River Laboratories France, L’Arbresle, France. Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Age at study initiation: the males were approximately 10 weeks old, the females were approximately 9 weeks old
- Weight at study initiation: males: a mean body weight of 383 g (range 359 g to 421 g), and female a mean body weight of 211 g (range: 186 g to 236 g).
- Fasting period before study: fasting overnight during the night prior the blood collection
- Housing: The animals were individually housed, except during pairing, in polycarbonate cages (UAR, 43.0 cm x 21.5 cm x 18 cm) with stainless steel lids, and containing autoclaved sawdust (SICSA, Alfortville, France).
Toward the end of gestation and during lactation with their litter, autoclaved wood shavings (SICSA Alfortville, France) was provided as nesting material, a few days before delivery and during the lactation period.
Nylabone was given as enrichment of the environment of the rats.
The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): The animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 7055973 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated for a period of 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES: From:2011-11-24 To: 2012-01-24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle.
The dosage formulations were prepared for 2 to 8 days (frequency based on the results of the stability study CIT/Study No. 38264 AHS).
The dosage forms were stored and delivered at room temperature and protected from light (see § Study plan adherence).


VEHICLE
drinking water treated by reverse osmosis using an ELIX 5 apparatus
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg of active ingredient
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The High Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method for the determination of Reaction mass of TFSK/TFAK in dosage form samples was validated at CIT prior to dosage form analysis.
The analytical method was validated for concentration ranging from 2 to 200 mg/mL.

The concentration of the test item in samples of each control and test item dosage form prepared for use in weeks 1, 3 and 5 was determined. In addition, the concentration of the test item in a sample of the high-dose test item form prepared for use in week 2 was determined.

Acceptance criterion: measured concentration = nominal concentration ± 10%

The validation of the analytical method was conducted in CIT/Study No. 38263 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results were documented in the corresponding validation report.

Each analytical sequence consisted of at least:
+ A blank sample (diluent only),
+ Ten standard samples at nominal concentration, prepared from two independent standard solutions,
+ Study samples prepared from aliquots of the dosage forms.
The standard samples bracketed the dosage form samples.
The blank sample was checked for the absence of chromatographic interference.

Duration of treatment / exposure:

The dosage forms were administered daily according to the following schedule:
in the males:
- 2 weeks before pairing (from study day 1 to 15),
- during the pairing period (3 weeks)(from study day 16 until study day 17 to 31),
- until sacrifice (at least 5 weeks in total)(from study day 18 to 32 until study day 39).

in the females:
- 2 weeks before pairing (from study day 1 to 15),
- during the pairing period (3 weeks)(from study day 16 until study day 17 to 31),
- during gestation (from study day 18 to 32 until study day 38 to 52),
- during lactation until day 5 post-partum inclusive (from study day 39 to 51 until study day 43 to 56),
- until sacrifice for the non-pregnant female (from study day 16 to Day 42).

Study day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

once a day, at approximately the same time.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor.
In a previous study (CIT/Study No. 38258 TSR), Reaction mass of TFSK/TFAK (batch No. SSK1278645) was given to rats (three/sex/group), by daily oral administration (gavage) for 2 weeks at 0, 150, 550 or 1000 mg/kg/day active ingredient. Ptyalism was observed in 2/3 males and in 1/3 females given 1000 mg/kg/day for 2 to 4 days during the second week of treatment. This clinical sign was attributed to the test item. Reflux at dosing was observed in 1/3 females given 500 mg/kg/day active ingredient on day 15. Soft feces, alopecia and scabs were noted in some animals; as these findings were isolated and/or without any dose relationship, they were considered as incidental.
Body weight and food consumption were considered to be unaffected by the treatment with the test item. On necropsy, there were no test item-related macroscopic post-mortem findings.
Therefore 1000 mg/kg/day, which is also the maximum recommended dose to be tested according to OECD Guideline No. 422, was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing a three-fold interval (i.e. 100 and 300 mg/kg/day active ingredient).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations:
+ males: on the first day of treatment (day 1), then once a week until sacrifice.
+ femelles: on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 p.c. and days 1 and 5 p.p..
The body weight of female sacrificed on day 25 p.c. for no delivery was recorded the day of sacrifice, presented in the report but not including in the statistical evaluation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1 5 p.p..
During the pairing period, the food consumption was measured neither for males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

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

OPHTHALMOSCOPIC EXAMINATION: Yes pupillary reflex during the FOB test
- Dose groups that were examined: 5 males and 5 female per group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females to deliver from each group
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- TTime schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females to deliver from each group
- Parameters checked in table were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period
- Dose groups that were examined: The first five males and the first five females to deliver
- Battery of functions tested:
+"touch escape" or ease of removal from the cage
+In the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis)
+ In the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia
+ The following parameter measurements, reflexes and responses were recorded:
 touch response,
 forelimb grip strength,
 pupillary reflex,
 visual stimulus response,
 auditory startle reflex,
 tail pinch response,
 righting reflex,
 landing foot splay,
 at the end of observation: rectal temperature.

OTHER:
+ at the end of observation: rectal temperature.
+Motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Parent animals:
A complete macroscopic post-mortem examination was performed on all parent animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 6 post partum and for female sacrificed on day 25 post-coitum due to no delivery. For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
Pup examinations:
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups showing relevant external abnormalities. No tissues were preserved.

HISTOPATHOLOGY: Yes (see table)

Other examinations:

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure

Statistics:

See attached document: statistiques.doc

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males and at 1000 mg/kg/day, a minimal decrease in hemoglobin content (-10.2% vs. controls, p <0.01).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males and at 1000 mg/kg/day, there was a minimal increase in inorganic phosphorus content (+13.4% vs. controls, p <0.05) and a light decrease in calcium content (-4.9% vs. controls, p<0.05).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The mean absolute and relative liver weights were increased in males treated at 300 or 1000 mg/kg/day. These variations were statistically significant. Minimal, not statistically significant increases were also seen in females at 1000 mg/kg/day active ingredient. This increase correlated with microscopic changes observed in the liver.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths and no clinical signs related to the treatment with the test item.

BODY WEIGHT AND WEIGHT GAIN
There were no effects on mean body weight or mean body weight change, either in males or females.

FOOD CONSUMPTION
There were no effects on food consumption.

HAEMATOLOGY
In males and at 1000 mg/kg/day active ingredient there was a minimal decrease in hemoglobin content (-10.2% vs. controls, p <0.01).
Taking into account the amplitude of the changes and in absence of relevant effect in other parameters, a relationship with the test item treatment was considered unlikely up to 300 mg/kg/day active ingredient and of minor toxicological significance at 1000 mg/kg/day active ingredient (males only)

CLINICAL CHEMISTRY
In males and at 1000 mg/kg/day active ingredient there was a minimal increase in inorganic phosphorus content (+13.4% vs. controls, p <0.05) and a light decrease in calcium content (-4.9% vs. controls, p<0.05).
Taking into account the amplitude of the changes and in absence of relevant effect in other parameters, a relationship with the test item treatment was considered unlikely up to 300 mg/kg/day active ingredient and of minor toxicological significance at 1000 mg/kg/day active ingredient (males only).
Other statistically significant differences (chloride, glucose and albumin) and were minimal, without any dose-level relationship and/or in one sex only. Therefore a treatment-related effect was considered unlikely.


NEUROBEHAVIOUR
There were no relevant differences in treated groups when compared with control group.

ORGAN WEIGHTS
The mean absolute and relative liver weights were increased in males treated at 300 or 1000 mg/kg/day active ingredient. These variations were statistically significant. Minimal, not statistically significant increases were also seen in females at 1000 mg/kg/day active ingredient. This increase correlated with microscopic changes observed in the liver.
There were statistically significant increases of the mean absolute and relative kidney weights in females treated at 1000 mg/kg/day active ingredient. In view of the low amplitude of the changes and the absence of microscopic correlates, these variations were considered to be of minor toxicological significance
Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude

GROSS PATHOLOGY
There were no macroscopic changes attributed to the test item administration.
The few macroscopic findings noted had no histologic correlates or correlated with common histologic findings in control Sprague-Dawley rats, and were considered to be incidental.


HISTOPATHOLOGY: NON-NEOPLASTIC
The test item administration induced minimal centrilobular hypertrophy of hepatocytes in 4/5 males and 2/5 females at 1000 mg/kg/day active ingredient, and in 1/5 males at 300 mg/kg/day active ingredient. This microscopic change correlated with increased liver weights. This change was not considered to be adverse in view of the absence of associated degenerative microscopic findings or clinical pathology changes in liver enzyme activities.
In males given the test item at all dose-levels, the incidence and severity of hematopoiesis in the spleen were minimally increased compared to controls. This increased hematopoiesis may have been compensatory to the changes in red blood cell parameters.
There were no microscopic changes attributed to the test item administration in the male and female genital organs.
Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Hematology

 

Sex	Male				Female
Dose-level (mg/kg/dayactive ingredient)	0	100	300	1000	0	100	300	1000
Mean corpuscular hemoglobin (pg)	18.1	18.2	17.7	18.0	19.4	18.2*	19.0	18.9
Hemoglobin (g/dL)	16.6	15.2* (-8.4)	15.5 (-6.6)	14.9** (-10.2)	14.4	13.9 (-3.5)	13.8 (-4.2)	13.6 (-5.6)
Pack cell volume (L/L)	0.48	0.44*	0.46	0.44*	0.43	0.41	0.41	0.40

(): in brackets, percent changesvs.controls.

Statistically significant: *: p<0.05, **: p <0.01.

 

 

Blood biochemistry

 

Sex	Male				Female
Dose-level (mg/kg/dayactive ingredient)	0	100	300	1000	0	100	300	1000
Chloride (mmol/L)	103.7	101.1*	100.6*	101.5	99.4	99.1	102.0	97.3
Calcium (mmol/L)	2.64	2.57 (-2.7)	2.56 (-3.0)	2.51* (-4.9)	2.79	2.64	2.69	2.64
Inorganic phosphorus (mmol/L)	2.09	2.08 (-0.5)	2.13 (+1.9)	2.37* (+13.4)	2.60	2.60	2.49	2.89
Glucose (mmol/L)	7.12	6.09	6.54	6.02	6.36	5.67	5.37**	5.76
Total bilirubin (µmol/L)	1	0*	0	1	1	1	0	0
Albumin (g/L)	39	38 (-2.6)	37 (-5.1)	36* (-7.7)	39	39	39	38

(): in brackets, percent changesvs.controls.

Statistically significant: *: p<0.05, **: p <0.01.

 

 

Organ weights

 

Sex.	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/dayactive ingredient)	100	300	1000	100	300	1000
Exam. animals / Num. of animals	5/10	5/10	5/10	5/10	5/10	5/10
Body weight	+2	0	-3	-4	-3	-4
- Liver
. absolute	+12	+25*	+29**	-4	+4	+12
. relative	+8	+23**	+30**	-5	+1	+12
- Kidneys
. absolute	+3	+8	+9	+9	+12	+15*
. relative	0	+6	+10	+8	+8	+14**

Statistically significant from controls: *: p<0.05, **: p<0.01.

Statistical significance determined for organ weights values and not percent changes.

 

 

Incidence and severity of centrilobular hypertrophy in the liver

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/dayactive ingredient)	0	100	300	1000	0	100	300	1000
Exam. animals / Num. of animals	5/10	5/10	5/10	5/10	5/10	5/10	5/10	5/10
Liver
- hypertrophy; hepatocytes
Grade 1	-	-	1	4	-	-	-	2

-: no affected animals.

 

Incidence and severity of hematopoiesis in the spleen

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/dayactive ingredient)	0	100	300	1000	0	100	300	1000
Exam. animals / Num. of animals	5/10	5/10	5/10	5/10	5/10	0/10	0/10	5/10
Spleen
- hematopoiesis
Grade 1	4	2	3	3	-	NA	NA	1
Grade 2	-	3	2	2	4	NA	NA	4
Grade 3	-	-	-	-	1	NA	NA	-

-: no affected animals. NA: not applicable.

Applicant's summary and conclusion

Conclusions:

The test item, Reaction mass of TFSK/TFAK, was administered daily by gavage to male and female Sprague Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until day 5 post partum, at dose-levels of 100, 300 or 1000 mg/kg/day active ingredient (approximately 38 days for males and at maximum 57 days for females).
In parent animals, the test item administration at 300 and 1000 mg/kg/day active ingredient induced centrilobular hepatocellular hypertrophy. This microscopic change correlated with increased liver weights. This change was considered to be non adverse in view of the absence of associated degenerative microscopic findings or clinical pathology changes in liver enzyme activities.

In absence of observed adverse effects, the repeated oral toxicity NOAEL (No Adverse Observed Effect Level) was set at the maximum dose administered: 1000 mg/kg bw/day.

Executive summary:

The purpose of this study (OECD 422, 2012), was to generate information concerning the effects of TFSK/ TFAK on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time.

The test item was administered orally by gavage once daily at dose levels of 100, 300 and 1000 mg/kg/body weight/day, for approximately 38 days for males and at maximum 57 days for females. The following observations were made:

 

No effects were met on mortality, clinical observations FOB, food consumption and body weight.

 

Organ Weights

The mean absolute and relative liver weights were increased in males treated at 300 or 1000 mg/kg/day active ingredient. These variations were statistically significant. Minimal, not statistically significant increases were also seen in females at 1000 mg/kg/day active ingredient. This increase correlated with microscopic changes observed in the liver.

There were statistically significant increases of the mean absolute and relative kidney weights in females treated at 1000 mg/kg/day active ingredient. In view of the low amplitude of the changes and the absence of microscopic correlates, these variations were considered to be of minor toxicological significance

Other organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude

 

Histopathological Examinations

The test item administration induced minimal centrilobular hypertrophy of hepatocytes in 4/5 males and 2/5 females at 1000 mg/kg/day active ingredient, and in 1/5 males at 300 mg/kg/day active ingredient. This microscopic change correlated with increased liver weights. This change was not considered to be adverse in view of the absence of associated degenerative microscopic findings or clinical pathology changes in liver enzyme activities.

In males given the test item at all dose-levels, the incidence and severity of hematopoiesis in the spleen were minimally increased compared to controls. This increased hematopoiesis may have been compensatory to the changes in red blood cell parameters. There were no microscopic changes attributed to the test item administration in the male and female genital organs. Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.

 

In absence of observed adverse effects in the repeated oral toxicity, the NOAEL (No Adverse Observed Effect Level) was set at the maximum dose administered: 1000 mg/kg bw/day.