Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route in rats is available for the reaction mass of TFAK/TFSK.

No effects were observed on reproductive organs, reproductive performances and progeny at the maximum dose tested : 1000 mg/kg bw/day.

Link to relevant study records

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP complaint OECD guideline study, klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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 active ingredient / kg bw / day,

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

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

Effects on developmental toxicity

Description of key information

The No-Observed-Adverse-Effect-Level (NOAEL) for maternal and embryo-fetal development toxicity in rats was considered to be 1000 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 Sep 2019 to 24 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP Guideline study

Justification for type of information:

For the Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate a new chemical substance notification in China has been prepared under the Regulation ‘Measures on the Environmental Management of New Chemical Substances' (Decree No. 7 of the Ministry of Environmental Protection of the P.R. China, also known as ‘China REACH’). Under this regulation an prenatal developmental toxicity study according to OECD 414 is part of the data requirements for substances that are produced or imported in volumes > 100 t/y. For this reason an OECD 414 study was performed in 2020 at Shenyang Research Institute of Chemical Industry. The results of the study are included in the dossier and serve as the valid test related to this endpoint.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2019-02-11

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF (Beijing) Biotechnology Co., Ltd
- Age at study initiation: 11 weeks of age at arrival at the test facility
- Weight at study initiation: 267.07 - 404.02 g
- Fasting period before study: No
- Housing: animals were housed in plastic cages (L46.0xW31.5xH20.0 cm). During the acclimatization period, the animals were housed in groups of max. 2. Mated females were housed individually in cages.
- Diet (e.g. ad libitum): SPF rodent growth and breeding feed supplied by Shenyang Mao Hua biological science and Technology Co., Ltd, ad libitum
- Water (e.g. ad libitum): Purified drinking water, ad libitum
- Acclimation period: 33 days. Clinical observations were performed daily during acclimatization period, and were continued until the start of the test.

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

IN-LIFE DATES: From: 13 Nov 2019 To: 5 Dec 2019

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item in the vehicle (drinking water) were prepared daily before the administration. The test item concentrations were based on the active ingredient of the reaction mass (Potassium triflinate, TFSK: 14.2%, Potassium trifluoroacetate, TFAK: 14.8%). A dosing volume of 10 mL/kg was applied for all animals, which was adjusted based on the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

CONCENTRATION VERIFICATION: The concentration of the test item was analyzed in the first and last week of the test item preparation with a validation analytical method (G1999B0040).

- Separation method: Ionic Chromatography (IC) (ICS-5000+ EG)
- Detection method: Conductivity detector.
- Conditions:
Analytical column: Dionex IonPac AS11-HC RFIC, 4 mm x 250 mm, 13 µm
Pre-column: Dionex IonPac AS11-HC RFIC, 4 mm x 50 mm, 13 µm
Column temperature: 30°C
    Flow rate: 1.0 mL/min
    Injection volume: 50 μL
    Mobile phase: 10mM potassium hydroxide aqueous solution
Retention time: About 11.2 and 13.7 min.
Run time: 17.0 minutes
- Data processing: the analytical data obtained were processed using the software of Chromeleion (c) Dionex. Since the test item is a reaction mass of two materials, the two main chromatography peaks in the chromatogram were considered as the target peak during sample analysis. The quantitative analysis was based on the sum of the two main peaks. The RSD of retention time was obtained from the first chromatography peak and the RSD of peak area was obtained from the sum of the two mean peaks in the system suitability test.
- External calibration and linearity range: A stock solution of 1.0 g active ingredient/L was diluted with ultra-pure water to obtain a series of standard solutions of 70.00, 60.00, 50.00, 40.00 and 30.00 mg/L. The accuracy of calibration of 5 concentrations should be within 90-110% by regression analysis and the coefficient of correlation (R) should be more than 0.99.
- System suitability/Method validation: To demonstrate the validity of the method, each analytical sequence consisted of at least:
+ A blank control (drinking water only), two repeats at a time.
+ Low dose (10 mg/ml), five repeats at a time.
+ Middle dose (30 mg/ml), five repeats at a time.
+ High dose (100 mg/ml), five repeats at a time.
Acceptance criteria: At the retention time of the target peak, the peak area of the blank matrix should be less than 10% of that of the lowest concentration standard solution. The determined concentrations of the low-, mid- and high-dose formulations should be 85-115% of the nominal concentration, and the RSD should not exceed 10%.
- Sample analysis: The dose formulations (nominal concentrations: 10, 30 and 100 mg/mL) were sampled and diluted within the linear range (30-70 mg/L), and determined by Ion Chromatography. The results of the determind concentration were in accordance with the target concentrations (accuracy 94.8 - 101.2%).

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 2 (or one) females : one male
- Length of cohabitation: until a sperm positive smear was detected.
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy

Duration of treatment / exposure:

Females were dosed daily in the morning from GD5 to GD19 by oral gavage.

Frequency of treatment:

Once daily

Duration of test:

15 treatment days

Dose / conc.:

100 mg/kg bw/day

Remarks:

Active ingredient

Dose / conc.:

300 mg/kg bw/day

Remarks:

Active ingredient

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Active ingredient

No. of animals per sex per dose:

27 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route in rats is available for the Reaction mass of TFSK/TFAK. No effects were observed on reproductive organs, reproductive performances and progeny at the maximum dose level tested (1000 mg active ingredient/kg bw/day). The No Observed Adverse Effect Level (NOAEL) for parental toxicity, reproductive performance (mating and fertility) and effects on progeny was therefore considered to be 1000 mg/kg/day. For this reason the maximum dose level of 1000 mg a.i./kg bw/day was selected as the highest dose level in the OECD 414 study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed daily by cage-side observations until the termination. After dosage, dams were observed for morbidity and mortality twice daily (once daily on non-working days)

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

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All rats were killed on GD20, and the uterus was removed for examination immediately.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included: the whole gravid uteri and total placentas were weighed. The number of corpora lutea, absorbed fetuses (early and late separately), dead fetuses (early and late separately) and viable fetuses were counted.

Fetal examinations:

- External examinations: Yes, the sex and body weight of each viable fetus were determined. Each fetus was examined for external alterations, including head, trunk, limbs and tail.
- Soft tissue examinations: Yes, one-half live fetuses of each litter were immersed in modified Davidson's fixative (the recipe for 100 ml of fixative: 14 ml ethanol, 6.25 ml glacial acetic acid, 37.5 ml saturated 37% formaldehyde and 37 ml distilled water) for one week, rinsed twice with tap water and stored in 70% isopropyl alcohol for soft tissue examination. Limbs and tail of the fetus were cut down first, then four chips were cut to examine the structural alterations of the head. Finally, thorax and abdomen of the fetus were opened to examine the size, shape and position of the organs.
- Skeletal examinations: Yes, the other half of each litter was prepared and examined for skeletal alterations. After removal of the skin and soft tissue, the remainder of the fetus was stained by using Alizarin red staining method, and stored in 70% glycerol for examination. This procedure include the skull, vertebra, sternum, ribs, limb bones and pelvis. At the same time the number of sternal ossification points, namely the number of ossified sternal segments, was recorded.

Statistics:

Average and standard deviation of the data for each group were calculated including body weight, food consumption, body weight change corrected for gravid uterine weight, number of corpora lutea and implantations, number of viable fetuses and body weight of fetuses, number of absorptive and dead fetuses.
Data statistics: If variance homogeneity test was P >0.05, Anova was used. If variance homogeneity tests P ≤0.05, Kruskal Wallis test was used. The data about ratio of viable fetuses, dead fetus and absorptive fetus, and incidence of anomalies were evaluated by the chi-square test. Anova and non-parametric analysis was done with Provantis 9.4.3.0 software, the chi-square test was done with SPSS 17.0 software.

Indices:

Pre-implantation loss (%): (No. of corpora lutea - No. of implantations) / No. of corpora lutea ×100 %
Post-implantation loss (%): (No. of implantations - No. of live fetuses) / No. of implantations ×100 %
Sex ratio distribution (%): Number of male fetuses / Number of fetuses x 100%
External abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%
Visceral abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%
Skeletal abnormalities/litter (%): Number of fetuses with abnormality / Number of fetuses x 100%

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed in the course of the study.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No deaths were observed in the course of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

During the administration period, the body weight and body weight change of pregnant rats in dose groups had no significant difference compared with the control group (P>0.05). The mean corrected body weight gain on GD20 (body weight minus gravid uterine weight) and net body weight change (body weight on GD20 minus body weight on GD5 minus gravid uterine weight) during the gestation period in the pregnant rats in all dose groups had no significant difference compared with the control group (P>0.05). Detailed results are provided in Table 1 see 'Any other information on results'.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the administration period, the food consumption of the pregnant rats in all dose groups had no statistically significant difference compared with the control group (P>0.05). Detailed results are provided in Table 2 see 'Any other information on results'.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The percentage of absorbed fetuses and the pre-implantation loss of high-dose group had a significant decrease compared with the control group (P≤0.05 or P≤0.01), but without toxicological significance and that had no statistically difference in the low- and mid- dose group (P>0.05). Detailed results are provided in Table 3 see 'Any other information on results'.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

not examined

Other effects:

not specified

Details on maternal toxic effects:

In all dose groups, no statistically significant difference was observed in the mean numbers of corpora lutea, implantation sites and total placental weights, and the gravid-uterine weights compared with the control group (P>0.05). Detailed results are provided in Table 3 see 'Any other information on results'.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Basis for effect level:

other: No effects observed at the highest dose level tested.

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference in the body weight of live fetuses was observed in all dose groups compared with the control group (P>0.05).
Detailed results are provided in Table 4 see 'Any other information on results'.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

In the high-dose group there was a statistically significant increase in the percent of live fetuses (P≤ 0.05).

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No statistically significant difference in the sex distribution of live fetuses was observed in all dose groups compared with the control group (P>0.05).

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

One fetus in the mid-dose group showed sign of narrow tail and 1 fetus in the high-dose group showed sign of tail absent. Two fetuses in the control group showed sign of small brain, and 1, 0, 1 and 3 fetuses in the control group, low-, mid- and high-dose groups showed signs of uronephrosis or small kidney in unilateral or bilateral kidneys. Five, 0, 5 and 4 fetuses in the control group, low-, mid- and high-dose groups showed signs of parietal imcomplete ossification, and one fetus showed signs of wavy rib, but the frequency of these abnormalities in the dose groups had no significant difference compared with the control group (P>0.05), which were considered as incidental findings. No adverse effect attributable to treatment was observed across all groups with respect to external, soft-tissue and skeletal malformations or variations; some examined fetuses had less than six sternal ossification points,but the mean number of sternal ossification points in all dose groups had no statistically difference compared with the control group (P>0.05).

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested.

Abnormalities:

no effects observed

Developmental effects observed:

no

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

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

Day(s) relative to mating			0 mg/kg d	100 mg/kg d	300 mg/kg d	1000 mg/kg d
Body weight gain (g)	5-20 [a]	Mean SD N	121.5 20.1 27	130.1 24.4 27	124.0 23.5 27	122.6 22.7 27
Corrected body weight on GD20 (g)	[a]	Mean SD N	378.25 27.98 27	381.26 33.47 27	375.95 25.77 27	375.19 32.46 27
Corrected body weight gain (g)	[a]	Mean SD N	54.327 17.186 27	56.334 21.815 27	52.323 16.921 27	49.599 17.193 27
Net body weight change (g)	[a]	Mean SD N	25.02 12.77 27	28.44 13.52 27	24.71 20.83 27	22.37 12.81 27

[a] – Anova&Dunnett

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

		Day(s) Relative to mating
		7 → 8	10 → 11	13 → 14	16→ 17
0 mg/kg d	Mean SD N	28.15 4.60 27	27.77 4.76 27	29.77 4.98 27	32.31 4.67 27
100 mg/kg d	Mean SD N	26.71 3.96 27	28.65 4.82 27	28.70 4.12 27	32.38 5.32 27
300 mg/kg d	Mean SD N	25.99 4.22 27	27.77 4.26 27	29.61 4.75 27	32.12 5.38 27
1000 mg/kg d	Mean SD N	25.39 4.44 27	27.92 7.17 27	27.27 4.29 27	31.71 4.76 27

Table 3: Developmental endpoint results

		0 mg/kg d	100 mg/kg d	300 mg/kg d	1000 mg/kg d
Mated female	(n)	28	28	28	28
Pregnant female	(n)	27	27	27	27
Corpora Lutea [a]	Mean SD Sum	16.9 3.7 456	17.2 2.8 465	16.7 2.9 451	16.6 3.2 449
Implantation[a]	Mean SD Sum	16.1 3.5 436	16.4 2.8 442	16.1 2.7 434	16.1 3.1 436
Gravid Uterus [a] (g)	Mean SD	96.4771 22.7950	101.6460 19.9209	99.3060 16.2687	100.2396 17.2214
Total placental weight[a] (g)	Mean SD	14.1308 3.2056	14.7799 2.9829	14.6335 2.5385	14.7466 2.7125
Live fetus[a]	Mean SD Sum	15.1 3.7 409	15.5 3.0 419	15.4 2.7 415	15.7 3.0 424
Percent[c] (%)		93.8	94.8	95.6	97.2*
Absorbed fetus[a]	Mean SD Sum	0.9 1.2 25	0.8 1.1 23	0.7 1.0 19	0.4 0.5 10
Percent[c] (%)		5.7	5.2	4.4	2.3**
Dead fetus[k]	Mean SD Sum	0.1 0.3 2	0.0 0.0 0	0.0 0.0 0	0.1 0.4 2
Percent[c] (%)		0.5	0.0	0.0	0.5
Pre-implantation loss[c] (%)		4.4	4.9	3.8	2.9
Post-implantation loss[c] (%)		6.2	5.2	4.4	2.8*

[a] – Anova&Dunnett

[k] – Kruskal-Wallis&Wilcoxon

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

Table 4: Fetal examination results

		0 mg/kg d	100 mg/kg d	300 mg/kg d	1000 mg/kg d
Body weight of fetus[a](g)	Mean SD	3.974 0.308	4.068 0.273	4.019 0.264	3.965 0.322
Number of male fetuses		203	210	231	222
Number of female fetuses		206	209	184	202
Sex distribution (males/total) [c] (%)		49.63	50.12	55.66	52.35
[c] – Chi-square test

 

Conclusions:

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

Executive summary:

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

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

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

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP complaint OECD guideline study, klimisch 1

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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

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

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

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

Justification for classification or non-classification

Based on the overall evaluation of the available data for the Reaction mass of TFSK/TFAK on reproduction and developmental toxicity, no classification and labelling for reproduction is deemed to be justifiedaccording to regulation (EC) 1272/2008.