Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Endpoint summary

Description of key information

Oral exposure to the reaction mass of TFSK/TFAK (OECD 422, GLP) did not result in any treatment related findings and the No Observed Adverse Effect Level (NOAEL) for oral subacute toxicity was considered to be 1000 mg/kg/day of active ingredient.

Oral exposure to the reaction mass of TFSK/TFAK (OECD 408, GLP) resulted in increased relative and absolute liver weights in male and female animals and treatment-related changes in blood biochemistry parameters (ALP and T-Bil). The NOAEL for subchronic toxicity was considered to be 100 and 300 mg/kg/day of active ingredient for males and females, respectively.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

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: 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

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.

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 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).

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

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.

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

Key result

Critical effects observed:

no

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.

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 May 2018 to xx November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For the Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate a new chemical substance notification in China has been prepared under the Regulation ‘Measures on the Environmental Management of New Chemical Substances' (Decree No. 7 of the Ministry of Environmental Protection of the P.R. China, also known as ‘China REACH’). Under this regulation a 90-day toxicity study according to OECD 408 is part of the data requirements for substances that are produced or imported in volumes >100 t/y. For this reason an OECD 408 study was performed in 2020 at Shenyang Research Institute of Chemical Industry. The results of the study are included in the dossier and serve as the valid test related to this endpoint.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Remarks:

2019-02-11

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF (Beijing) Biotechnology Co., Ltd, China
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 to 7 weeks on arrival
- Weight at study initiation: 201 to 240 g (males) and 171 to 203 g (females). At the grouping of the experiment, the weight variation in the same group used was ± 20% of the mean body weight for either sex.
- Fasting period before study: no
- Assigned to test groups randomly: yes
- Housing: animals were housed in suspended, stainless steel cages (L32.0xW28.0xH20.0cm). Animals were housed in groups of max. 2.
- Diet (e.g. ad libitum): SPF rodent growth and breeding feed supplied by Shenyang Mao Hua biological science and Technology Co., Ltd, ad libitum
- Water (e.g. ad libitum): Purified drinking water, ad libitum
- Acclimation period: 6 days. Clinical observations were performed daily until grouping.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: May 23, 2019 (animal arrival) To: August 29, 2019 (final sacrifice)

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions of the test item in the vehicle (drinking water) were prepared daily before the administration and were used within 4 hours after preparation. The test item concentrations were based on the active ingredient of the reaction mass (Potassium triflinate, TFSK: 14.4%, Potassium trifluoroacetate, TFAK: 14.1%). A dosing volume of 10 mL/kg was applied for all animals, which was adjusted based on the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CONCENTRATION VERIFICATION: Before the initiation of the main toxicity study, the stability of test item solutions was determined by a validated analytical method (Study No. G1999B0040). The formulations were found to be stable for at least 4h at room temperature (15-25°C). The concentration of the formulations was determined by analysis on the first dosing day, the 7th week and the 13th week of the dosing. Formulation samples of 100 µl were collected for analysis, at least three parallel samples each time. 100 µl of the vehicle was collected for analysis, at least two parallel samples each time. Analysis was performed with Ion Chromatography (IC) with a validated analytical method for the test item. To be considered acceptable, the actual results for the analysis of the dosing formulations were to be within ±10% of the nominal concentration.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Active ingredient

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Active ingredient

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Active ingredient

No. of animals per sex per dose:

- 10/sex/group for low and mid dose
- 16/sex/group for high dose and control

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The study doses selected were based on the pre-study results (Study No. G1857B0001A). The pre-study results indicated that the body weight gains were not affected by the test item at the dose of 1000 mg/kg a.i. during the 22-day dosing period. There were no deaths and the animals showed normal during the 22-day dosing period. Based on these results the dose level of 1000 mg/kg was choosen as the highest dose level in the main study.
- Rationale for animal assignment: Healthy animals were used for randomization grouping based on the body weight by the Provantis 9.4.3 Animal Arrangement Module. Animals were allocated to 4 groups: 16 animals of each sex in the control group and high-dose group, 10 animals of each sex in the other dose groups. At the grouping of the experiment, the weight variation in the same group used must not exceed ±20% of the mean body weight for either sex.
- Recovery group: The reversibility of effects was assessed after a recovery period of 28-days without exposure (6/sex/group for control and high dose).

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were inspected for signs of morbidity and mortality at the beginning and the end of work at least once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical signs were recorded at least once daily at about 1.5h after dosing for all animals during the study. During the recovery period, the observation time was performed at the same time each day. The healthy conditions and toxicity signs wase recorded.
Detailed physical examinations were performed for all animals prior to the first exposure (at grouping) and once a week during the treatment period and during the recovery period. The animals were taken out of the home cage for observation, and all the findings will be recorded in detail. Observation includes changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviors (e.g. self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed at grouping, once a week during the treatment period and recovery period, and in a moribund state or at death. Animals were fasted overnight (16-18h) prior to necropsy, and empty-stomach body weights were collected before necropsy.

FOOD CONSUMPTION: yes
- Time schedule for examinations: The weight of food supplied and of that remaining at the end of the food consumption period was recorded weekly for all animals during the treatment period. From these records the mean daily consumption was calculated.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were conducted in all animals after grouping and the day before the end of treatment.

CLINICAL PATHOLOGY INVESTIGATIONS: Yes
- Time schedule for collection of blood: All surviving animals at termination of dosing and after the recovery period were anesthetized by CO2 inhalation, and blood was collected via abdominal aorta for hematology, coagulation and serum biochemistry.
- Animals fasted: yes, all animals were fasted overnight before blood collection.
- Anaesthetic used for blood collection: Yes, CO2 anaesthesia
- Haematology: Parameters checked in Table 1 (see 'Any other information on materials methods') were examined.
- Coagulation: Prothrombin time, activated partial thromboplastin time and fibrinogen were investigated.
- Clinical chemistry: Parameters checked in Table 2 (see 'Any other information on materials methods') were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Before necropsy after termination of dosing and the recovery period, urine samples were collected from all surviving animals by abdominal extrusion.
- Metabolism cages used for collection of urine: No
- Animals fasted: Animals were fasted overnight prior to necropsy, but water was available.
- The following measurements were performed on urine: Appearance (visual observation), pH, urine specific gravity, occult blood, ketones, glucose, total protein, bilirubin, urobilinogen, nitrites, leukocytes, vitamin

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 11 of treatment
- Dose groups that were examined: all surviving animals in control and high dose group were assessed for general behavior, sensory reactivity by different types of stimulation, grip strength and motor activity.
- Battery of functions tested:
+ General behaviour assessment: Responses to the following manipulations was assessed in the open-field observations, home-cage observations, handling and manipulative observations. The following parameters were checked: Spontaneous Activity, Gait and Posture, Reactivity and Alertness, Abnormal Motor Movements (involuntary motor movements/abnormal/stereotypic behavior), Observations of Autonomic Nerve (Lacrimation/Salivation/Piloerection/Palpebral closure/Eye abnormal/Urination/Defecation/ Breath/Muscle tension), Reflexes (Pupil/Palpebral/Pinna/Extensor Thrust Reflex)
+ Motor activity Rat: Activity Record Instrument was used to assess motor activity. The evaluation period was three minutes for each animal.
+ Forelimb and Hindlimb Grip Strength: A tensile strength tester was used. Each animal was allowed to grip the board of the meter with its forepaws and hindpaws. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal and the average readings were used.
+ Sensory reactivity: Each animal was individually assessed for sensory reactivity to visual, auditory, and proprioceptive stimuli. Then the following parameters were observed: approach response, click response, tail pinch, touch response.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The necropsy included careful visual examinations of the external features of the carcass, external body orifices, the abdominal, thoracic, and cranial cavities and their contents. The location, size, hardness and the color of the abnormal findings were recorded. The following organ weights were recorded from all animals on the scheduled day of necropsy: brain, thyroid, heart, lung, liver, spleen, adrenal glands, kidney, thymus, uterus (female), testis (male), epididymis (male), prostate (male)

HISTOPATHOLOGY: Yes.
The samples of the tissues indicated in Table 3 (see 'Any other information on materials methods') were collected and preserved. The tissues from each animal were preserved in 10% neutral-buffered formalin (eyes were preserved in Davidson’s solution, and testis in Improved Davidson’s solution). Lungs were infused with fixative prior to immersion in 10% neutral-buffered formalin. Stomach, intestine and bladder were infused with formalin prior to immersion in 10% neutral-buffered formalin. Tissues and organs collected from the control and high dose group of animals and gross lesions found in the other dose group animals were sectioned, fixed and dehydrated with alcohol, embedded in paraffin, sliced using microtomy, and stained with hematoxylin and eosin, extracted/washed with dimethylbenzene, sealed with rosin and examined microscopically.
Histopathology was examined on the preserved organs and tissues of all animals in the control and high-dose group during the study.

Statistics:

Data of hematology, coagulation, serum biochemistry and urinalysis was collected by Clinical Test Data Administration 1.0. The other data was collected by the Provantis 9.4.3 except nerve function observation and ophthalmological examinations. Where appropriate, quantitative data was analysed by the Provantis 9.4.3 Tables and Statistics Module.
- If > 2 groups, the data were analyzed by Bartlett test for variance homogeneity first. In case of heterogeneity of variance (p<0.05), the Kruskal-Wallis non-parametric analysis of variance for pair wise comparison was used to evaluate the difference between each treated group and the control group. If the test was significant (p<0.05), non-parametric Dunnett's test was then used for pairwise comparison between each treated group and the control group. Statistical analysis was finished when the Kruskal-Wallis test was non-significant (p≥0.05). In the case of homogeneity of variance (p≥0.05), one-way analysis of variance (ANOVA) was used to analyze above parameters. If the test was significant (p<0.05) and the number of animals of each group was the same, Dunnett's test was conducted for pairwise comparison between each treated group and the control group. If the test was significant (p<0.05) and the number of animals of each group was different, Duncan’s test was conducted. Statistical analysis was finished when the test will be non-significant (p≥0.05).
- If 2 groups, pair-wise tests was used by T-test (parametric) or Mann-Whitney U test (non-parametric). The incidences of pathological findings (gross pathological findings, non-graded histopathological findings) was analyzed by the Fisher’s exact test (one-tailed probability level).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected throughout the study. Animals showed hair loss which occurred in both the treatment and control groups and appeared sporadically. So the symptoms noted were considered unrelated to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant reduction in mean body weight on Day 91 and 98 were noted for the females in 1000 mg/kg dose group only during the recovery period. But no adverse effect was evident in the overall body weight (Day 91-119) and the reduction was considered not to be of toxicological importance. No test item-related body changes were noted. No significant differences were noted in mean body weight or body weight gains during the treatment period for either sex.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effect in food consumption was detected in treated animals when compared to controls. Throughout the whole test, including the treatment and recovery phase, animals in the treatment groups showed normal variation in the mean food consumption, incidentally including significant increases or decreases as compared with the control group, but without any toxicological meaning.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmic examinations were conducted on the high-dose (1000 mg/kg) and control group animals (0 mg/kg) prior to the dosing and at the end of treatment. The results indicated that there were no treatment-related changes in the periocular area of eyes, conjunctiva, iris, cornea, optic disk and blood vessel of the eye fundus.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- At the end of the treatment period: Among males, the mean Total Leukocyte counts (WBC) value was statistically significantly increased at 1000 mg/kg (P<0.05, +29.9% vs.controls), but not at 300 mg/kg and only observed in one sex only. Therefore, this was casual without toxicological meaning. Other statistically significant differences were minimal, without any dose relationship.
- At the end of the recovery period: For the males, the high-dose group of 1000 mg/kg showed statistically significant increases in PDW, P-LCR (P <0.05) and RDW-CV, RDW-SD (P<0.01) compared with the untreated control group. For the females, the high-dose group of 1000 mg/kg showed statistically significant increases in RDW-CV and RDW-SD (P<0.05) compared with the control group. These above changes only occurred in the recovery period and were not associated with other indices. No test item related changes were found during the recovery period.
There were no toxicologically significant effects detected in the hematology and coagulation parameters examined.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period: The males at the dose of 300 and 1000 mg/kg showed a significant increase in ALP (+31.3% at P<0.001, +63.6% at P<0.05) compared with the control group. Males in the 100, 300 and 1000 mg/kg dose groups showed a significant decrease in T-Bil, -100%, -95.8% and -129.2% respectively. The females at the dose of 1000 and 300 mg/kg also showed a significant decrease in T-Bil (-90.6% at P<0.001 and -57.6% at P<0.05). The above changes returned to normal after four weeks' withdrawal.
The males in 1000 mg/kg dose group showed a significant decrease in TP and GLB compared with the control group, and in all treated groups showed a significant increase in Urea and A/G, but without a dose-response relationship in the above findings, it was considered there was no toxicological significance.
Females in the 1000 mg/kg dose group showed a significant decrease in GLB (- 8.5% at P<0.05) and a significant increase in A/G (+8.6% at P<0.01). The above changes were minimal and returned to normal after four weeks' withdrawal and it was considered to be a normal biological variation.
At the end of the recovery period: The males at the dose of 1000 mg/kg showed a significant increase in TP and GLB (P<0.05). The males at the dose of 1000 mg/kg showed a significant decrease in A/G (P<0.05). The difference was very small in the males at the end of the recovery period and the results were opposite to the treatment period, it was considered unrelated to the test item. No abnormal findings were observed in the female animals.
(see Table 5 in 'any information on results including tables' for an overview of the results).

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no statistically significant or treatment-related changes in urinalysis parameters in male or female animals.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The results of the behavioural observations during the 11th week of the treatment period indicated that no neurotoxicity was found related to the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and relative liver weights were statistically significantly higher in male rats at 300 and 1000 mg/kg and in female rats at 1000 mg/kg when compared to controls (see Table 4 in 'any information on results including tables'). The liver weights in both male and female animals of the 1000 mg/kg dose groups showed statistically significant in mean absolute increases (+26.3% for the males and +19.2% for the females both at p<0.01) and relative increases (organ-to-body weight ratios: +41.2% for the males at and +26.2% for the females, both at p<0.001; organ-to-brain weight ratios: +30.5% for the males at p<0.001 and +19.2% for the females at p<0.01). The liver weights in male animals of the 300 mg/kg dose groups showed statistically significant in mean absolute increases (+17.1% at p<0.05) and relative increases (organ-to-body weight ratios: +22.7% at p<0.001; organ-to-brain weight ratios: +16.3% at p<0.05). In the male and female test groups of the recovery phase, liver weights were normal without significant deviation from the respective controls.
The liver weights were increased at the highest dose level and the clearly treatment-related effect occurred in both sexes. Although in the absence of any associated histology correlates, the intergroup differences were considered to be of toxicological significance taking into account of the changes of ALP and T-Bil in blood biochemistry.
All other statistically significant organ weight differences were judged to be incidental in view of their individual variation and in the absence of any correlated histopathological finding.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy and macroscopic observation did not provide evidence of any signs of toxicity related to the test item during the whole test.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was no evidence of treatment-related microscopic abnormalities.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

Formulation analysis: The concentration of the formulations was determined by analysis on the first dosing day, the 7th week and the 13th week of the dosing, and the actual results for the analysis of the dosing formulations were within ±10% of the nominal concentration.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Table 4. Liver weight changes at terminal sacrifice (% change when compared to controls)

Liver weight changes at terminal sacrifice (% change when compared to controls
Sex		Male				Female
Dose level TFSK/TFAK (mg/kg)		0	100	300	1000	0	100	300	1000
Mean absolute liver weight (g) [a]	Mean SD N	13.0354 1.8718 10	13.8423 1.5024 10	15.2701^* 1.4764 10	16.4643^** 2.7111 10	7.6480 0.6255 10	8.5629 1.2161 10	8.3204 1.0144 10	9.1165^** 0.6580 10
Mean liver to body weight ratio (%) [a]	Mean SD N	2.474 0.195 10	2.726 0.210 10	3.034^*** 0.278 10	3.492^*** 0.264 10	2.569 0.159 10	2.892^** 0.155 10	2.951^*** 0.258 10	3.242^*** 0.217 10
Mean liver to brain weight ratio (%) [a]	Mean SD N	587.798 80.178 10	622.568 67.219 10	683.494^* 81.651 10	767.266^*** 111.444 10	372.627 24.254 10	411.884 55.083 10	402.721 48.395 10	444.302^** 38.637 10

  [a] - Anova & Dunnett: * = p < 0.05; ** = p < 0.01; *** = p < 0.001

Table 5. Clinical chemistry parameter changes in males and females (% change when compared to controls)

Sex		Male				Female
Dose level TFSK/TFAK (mg/kg)		0	100	300	1000	0	100	300	1000
ALP [a] (U/L)	Mean SD N %Diff	104.9 18.9 10	126.6 20.4 10 20.7	137.7* 38.0 10 31.3	171.6*** 45.5 10 63.6	52.6 10.7 10	58.3 14.7 10 10.8	54.4 16.0 10 3.4	52.1 7.4 10 -1.0
ALT [a] (U/L)	Mean SD N %Diff	54.4 20.5 10	53.4 12.9 10 -1.8	55.4 14.1 10 1.8	57.7 16 10 6.1	49.1 18.6 10	48.0 13.6 10 -2.2	57.9 33.1 10 17.9	43.1 10.9 10 -12.2
AST [a] (U/L)	Mean SD N %Diff	117.8 55.4 10	124.7 42.6 10 5.9	112.4 19.3 10 -4.6	114.6 27.2 10 -2.7	109.5 29.8 10	106.8 27.2 10 -2.5	125.1 58.7 10 14.2	96.1 32.5 10 -12.2
Urea [a] (mmol/L)	Mean SD N %Diff	5.700 0.823 10	8.371** 4.448 10 46.9	6.950** 0.890 10 21.9	7.323*** 0.769 10 28.5	8.032 2.632 10	8.653 1.215 10 7.7	9.091 2.083 10 13.2	8.913 1.386 10 11.0
Creatinine [a] (µmol/L)	Mean SD N %Diff	29.5 3.6 10	40.3 26.5 10 36.6	32.7 3.0 10 10.8	31.9 4.4 10 8.1	40.1 6.3 10	38.3 5.7 10 -4.5	39.6 4.2 10 -1.2	38.4 6.1 10 -4.2
T-BIL [a] (µmol/L)	Mean SD N %Diff	0.48 0.40 10	0.00** 0.25 10 -100.0	0.02** 0.23 10 -95.8	-0.14*** 0.32 10 -129.2	0.66 0.39 10	0.38 0.21 10 -42.4	0.28* 0.31 10 -57.6	0.06*** 0.23 10 -90.9
GLU [a] (mmol/L)	Mean SD N %Diff	9.348 1.361 10	9.287 2.001 10 -0.7	10.589 1.421 10 13.3	12.281** 2.209 10 31.4	9.132 1.910 10	7.449 1.900 10 -18.4	7.412 1.431 10 -18.8	6.983 2.491 10 -23.5
Triglyceride [a] (mmol/L)	Mean SD N %Diff	0.492 0.190 10	0.510 0.148 10 3.7	0.437 0.095 10 -11.2	0.465 0.058 10 -5.5	0.504 0.191 10	0.456 0.117 10 -9.5	0.473 0.100 10 -6.2	0.477 0.140 10 -5.4
CHO [a] (mmol/L)	Mean SD N %Diff	1.702 0.496 10	1.728 0.243 10 1.5	1.674 0.304 10 -1.6	1.526 0.309 10 -10.3	2.469 0.532 10	2.246 0.296 10 -9.0	2.344 0.314 10 -5.1	2.522 0.349 10 2.1
TP [a] (mmol/L)	Mean SD N %Diff	66.01 2.55 10	64.89 2.21 10 -1.7	65.09 3.24 10 -1.4	61.17** 2.95 10 -7.3	73.61 4.09 10	73.53 4.47 10 -0.1	75.01 3.06 10 1.9	71.02 2.26 10 -3.5
ALB [a] (g/L)	Mean SD N %Diff	41.33 1.51 10	41.88 1.28 10 1.3	41.82 1.49 10 1.2	40.59 0.99 10 -1.8	46.46 2.44 10	46.78 2.67 10 0.7	47.97 2.47 10 3.3	46.17 1.59 10 -0.6
Potassium [a] (mmol/L)	Mean SD N %Diff	6.149 0.459 10	6.049 0.347 10 -1.6	6.184 0.419 10 0.6	6.155 0.396 10 0.1	5.670 0.771 10	6.143 0.832 10 8.3	5.877 0.595 10 3.7	5.554 0.452 10 -2.0
Sodium [a] (mmol/L)	Mean SD N %Diff	149.5 3.3 10	152.6 1.9 10 2.1	151.6 2.1 10 1.4	149.9 3.3 10 0.3	152.0 1.9 10	151.3 2.3 10 -0.5	152.0 1.9 10 0.0	151.9 2.5 10 -0.1
GLB [a] (g/L)	Mean SD N %Diff	24.68 1.33 10	23.01 1.82 10 -6.8	23.27 1.90 10 -5.7	20.58*** 2.21 10 -16.6	27.15 1.89 10	26.75 2.23 10 -1.5	27.04 1.31 10 -0.4	24.85* 1.31 10 -8.5
A/G [a]	Mean SD N %Diff	1.68 0.07 10	1.83** 0.17 10 9.2	1.80** 0.10 10 7.5	1.99*** 0.22 10 18.8	1.71 0.07 10	1.76 0.11 10 2.4	1.78 0.11 10 3.6	1.86** 0.11 10 8.6
Total bile acid [a] (µmol/L)	Mean SD N %Diff	28.18 17.36 10	30.94 35.03 10 9.8	32.61 24.46 10 15.7	37.66 27.57 10 33.6	38.04 17.82 10	38.27 23.57 10 0.6	32.53 17.19 10 -14.5	25.66 7.99 10 -32.5

[a] - Anova & Dunnett: * = p < 0.05; ** = p < 0.01***; = p < 0.001

 

Conclusions:

In the 90-day oral study with Sprague Dawley rats, the reaction mass of TFSK/TFAK was administered daily by oral gavage to male and female rats at 100, 300 and 1000 mg a.i./kg bw/day. The NOAEL for males was considered to be 100 mg/kg, based on marked increase in liver absolute and relative weights and ALP and also decrease in T-Bil at the high and mid dose level. The NOAEL for females was considered to be 300 mg/kg, based on marked increase in liver absolute and relative weights and decrease in T-Bil at the high dose level.

Executive summary:

In a GLP-compliant OECD Guideline 408 study, the reaction mass of TFSK/TFAK was administered daily by oral gavage to separate groups of Sprague Dawley rats (10/sex/group for low and mid dose and 16/sex/group for high dose) at concentrations of 100, 300 and 1000 mg active ingredient/kg bw/day for 90 days. A similarly constituted group of 16 males and 16 females received the vehicle (purified drinking water) and acted as a control. At the end of dosing, surviving animals were sacrificed and subjected to a full gross necropsy, while the residual animals (6 males and 6 females) in the control and high-dose were kept for at least four weeks (the recovery period) without treatment to detect delayed occurrence or persistence of, or recovery from toxic effects.

All animals survived until scheduled sacrifice. No treatment-related changes were noted in clinical appearance, functional observations, body weight, food consumption, haematology, urinalysis and ophthalmoscopy during the study. Dose-related increases were found in ALP for the males at 300 and 1000 mg/kg. Males in all treated groups showed a significant decrease in total bilirubin. Females in the 300 and 1000 mg/kg dose groups also showed a significant decrease in total bilirubin. These changes returned to normal after the four week recovery period.

Dose-related increases in absolute and relative liver weights were found for the 1000 and 300 mg/kg dose groups in males (mean absolute increase of +26.3% and +17.1%, respectively) and for the 1000 mg/kg dose group in females (mean absolute increase of +19.2%). Within the four weeks recovery period, the liver weights regressed to normal.

Based on the results of this study, the no-adverse-observed-effect-level (NOAEL) for the reaction mass of TFSK/TFAK was considered to be 100 mg a.i./kg/day for males, based on marked increase in relative and absolute liver weights and ALP and decrease in T-Bil at the high and mid dose level. The NOAEL for females was considered to be 300 mg a.i./kg/day for females, based on marked increase in relative and absolute liver weights and decrease in T-Bil at the high dose level.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP, OECD408 compliant study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For the reaction mass of TFSK/TFAK, a combined oral repeated dose toxicity study with reproduction/developmental toxicity screening test performed according to OECD 422 and a sub-chronic oral study according to OECD 408 are available for assessment.

In the OECD 422 study the test item, Reaction mass of TFSK/TFAK, was administered daily by gavage to male and female Sprague Dawley rats, 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. 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 not considered to be 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 in the repeated oral toxicity, the NOAEL (No Adverse Observed Effect Level) was set at the maximum dose administered: 1000 mg/kg bw/day.

In the 90-day oral study with Sprague Dawley rats, the reaction mass of TFSK/TFAK was administered daily by oral gavage to male and female rats at 100, 300 and 1000 mg a.i./kg bw/day. The NOAEL for males was considered to be 100 mg/kg, based on marked increase in liver absolute and relative weights and ALP and also decrease in T-Bil at the high and mid dose level. The NOAEL for females was considered to be 300 mg/kg, based on marked increase in liver absolute and relative weights and decrease in T-Bil at the high dose level.

Justification for classification or non-classification

Based on the available data, classification for repeated dose toxicity is not warrented in accordance with EU Directive 67/584/EEC andEU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.