Lithium bis(trifluoromethylsulfonyl)imide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 May 2014 to 12 Dec 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD 421 compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

10/01/2013

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Number: 82 rats (41 males and 41 females) were received at CiToxLAB France on 03 June 2014.
- Strain and sanitary status: Sprague-Dawley, RjHan: SD (Rats CD®).
- Breeder: Janvier, Le Genest-Saint-Isle, France.
- Age/Weight: At the beginning of the treatment period, the males were 10 weeks old and had a mean body weight of 454 g (range: 406 g to 475 g). The females were 9 weeks old and had a mean body weight of 213 g (range: 196 g to 242 g). The animals were sexually mature and were not siblings. The females were virgin.

- Housing: The animals were individually housed, except during mating and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France).
Individual housing was chosen since it is preferable for pregnant animals and to avoid any aggressive behavior around mating.
Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litters as nesting material.
Each cage contained an object for the environmental enrichment of the animals (rat hut).

- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 6730736 (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 to the study conditions for a period of 7 days before the beginning of the treatment period. One supplementary animal per sex than necessary were allocated to the study and acclimated, in order to permit the selection and/or replacement of individuals.

ENVIRONMENTAL CONDITIONS
- temperature: 22 ± 2°C,
- relative humidity: 50 ± 20%,
- light/dark cycle: 12h/12h,
- ventilation: About 12 cycles/hour of filtered, non-recycled air.


IN-LIFE DATES: From: 03 June 2014 To: 01 August 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The weighing of the test item was performed using de humidified bag system (glove box under nitrogen/argon), then the test item was mixed with the required quantity of vehicle.
The test item dose formulations were prepared with a frequency (8 days) depending on their stability according to an homogeneity and stability study performed at CiToxLAB France (CiToxLAB France/Study No. 41182 AHS), stored at +2-8°C protected from light prior to use and delivered to the study room at room temperature and protected from light.


VEHICLE
The vehicle was prepared by CiToxLAB France and was used for preparation of test item dose formulations and administered to control group (control dose formulation).
The vehicle was drinking water treated by reverse osmosis using ELIX 5 (Millipore SA).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diluted samples of dose formulations were analyzed in single by Ion Chromatography (IC) with conductivity detection.
The test item peak area was determined for each sample and the corresponding concentration was calculated using the equation obtained from the calibration data.
All the results are expressed as mg/mL of LiTFSI PURE.
The test item concentrations in the administered dose formulations analyzed in Weeks 01, 03 and 05 remained within an acceptable range of variation (+0.9% to +4.8%) when compared to the nominal values (± 10% of the nominal concentrations).
LiTFSI PURE was not detected in control samples.

Details on mating procedure:

The females were paired with males from the same dose-level group. One female was placed with one male, in the latter's cage, during the night.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
The day of confirmed mating was designated Day 0 p.c..
Each female was placed with the same male until mating occurs or 7 days had elapsed. Pairs with no evidence of mating after 7 days were separated and the female was placed for a further 7 days with a different male from the same dose-level group who had already mated.
The pre-coital time was calculated for each female.
Group 3 female C27975, with no evidence of mating after 7 days with the second male was sacrificed 25 days after the end of the mating period.

Duration of treatment / exposure:

In the males:
 2 weeks before mating,
 during the mating period (up to 2 weeks),
 until sacrifice (at least 4 weeks in total).

In the females:
 2 weeks before mating,
 during the mating period (up to 2 weeks),
 during gestation,
 during lactation until Day 4 p.p. inclusive,
 until sacrifice for females with no evidence of mating or no delivery.

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

Frequency of treatment:

Daily

Duration of test:

Males: at least 28 days
Females: ca. 53 days

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
The dose-levels were selected, following the results of a previous 4-week toxicity study by oral route (gavage) in rats followed by a 2-week treatment-free period. In this previous study, the test item was administered at 15, 45 and 90 mg/kg/day.
The No Observable Effect Level (NOEL) was established at 15 mg/kg/day and the Lowest Observable Adverse Effect Level (LOAEL) was established at 45 mg/kg/day.
Therefore, 60 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose were selected using a ratio representing a 2-fold interval (i.e. 15 and 30 mg/kg/day).

Examinations

Maternal examinations:

Morbidity and mortality
Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.
Animal showing signs of poor condition, especially when death appeared imminent, was humanely sacrificed. Animals found dead or prematurely sacrificed were subjected to a macroscopic post-mortem examination.

Clinical signs
From arrival, the animals were observed once a day as part of routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

Body weight
The body weight of each male was recorded on the first day of treatment (Day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated (or until sacrifice for females with no evidence of mating), on Days 0, 7, 14 and 20 post-coitum and on Days 1 and 5 p.p.

Food consumption
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 mating 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 the mating period, during gestation for the intervals Days 0-7, 7 14 and 14-20 p.c. and during lactation for the interval Days 1-5 p.p..
During the mating period, food consumption was not measured for males or 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

The numbers of corpora lutea and implantation sites were recorded for female sacrificed as scheduled on Day 5 p.p. and for female sacrificed 25 days after the end of the mating period with no evidence of mating and for females sacrificed on Day 25 p.c. 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 (Salewski, 1964).

Fetal examinations:

A detailed soft tissue examination of thoracic (including a detailed examination of the heart and great vessels) and abdominal organs was performed on all pups sacrificed on Day 5 p.p..
Macroscopic lesions were preserved without any further investigations in the first instance.
The findings were described according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformations or variations (Chahoud et al., 1999, Makris et al., 2009):
 malformation refers to a permanent structural change that is likely to adversely affect the survival or health,
 variation refers to a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health (this might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development).

Statistics:

Body weight, food consumption and reproductive data
Data are compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).

Organ weights
PathData software was used to perform the statistical analysis of organ weight data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
At 30 mg/kg/day:
- one female was sacrificed for no delivery. This female was not pregnant,
- three females were sacrificed for dead litter.

Prior to sacrifice, piloerection was observed in one female sacrificed for dead litter. No necropsy findings were found at macroscopic post-mortem examinations.

At 60 mg/kg/day:
- two females were sacrificed for no delivery. These females were not pregnant,
- two females were sacrificed for humane reasons (moribunds) and one female was found dead,
- five females were sacrificed for dead litter.

Prior to sacrifice or death, signs of poor clinical condition were observed in 5/9 females as follows:
- ptyalism, piloerection, chromodacryorrhea, chromorhinorreha, round back, clonic contraction, tonic contraction, convulsions, hypersensitivity to the touch, reddish mouth area and/or lateral decubitus. No clinical signs were observed during the lactation period in four females sacrificed for dead litter,
- brownish discoloration of the wall stomach was observed in 2/9 females together with foamy brownish content in one of them or brownish discoloration in lungs in the other one.

All deaths recorded at 30 and 60 mg/kg/day were considered to be related to the test item treatment. There were no changes at microscopic examination that could explain the cause of deaths.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOEL

Effect level:

< 15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

mortality

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

< 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

changes in postnatal survival

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the experimental conditions of this study:
- the NOAEL for reproductive performance and embryo/fetal development was considered to be 30 mg/kg/day based on lower fertility and gestation indexes at 60 mg/kg/day,
- the No Observed Effect Level (NOEL) for developmental toxicity was considered to be lower than 15 mg/kg/day based on lower survival index on Day 4 p.p. at this dose-level.
Despite a context of severe to excessive maternal toxicity, there was no indication of any teratogenic potential.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, LiTFSI PURE, following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until Day 4post-partum (p.p.), according to the OECD guideline n°421 and under GLP conditions.

 

Three groups of ten male and ten female Sprague-Dawley rats received the test item,LiTFSI PURE (batch No. TFSILi-PU-1305001), daily, by oral administration (gavage), before mating, during mating and, for the females, throughout gestation until Day 5p.p., at dose-levels of 15, 30 or 60 mg/kg/day for at least 4 weeks in males and up to 6 weeks in females.An additional group of ten males and ten females received the vehicle control, drinking water treated by reverse osmosis, under the same experimental conditions. The dosing volume was 5 mL/kg/day.

Animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation.

The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 5p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on Days 1 and 5 p.p..

The males were sacrificed after completion of the mating period. Dams were sacrificed on Day 5 p.p.. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed, with particular attention paid to the reproductive organs. A microscopic examination was also conducted on selected organs from the animals in the control groups and the high‑dose groups. Microscopic examination was conducted on all macroscopic lesions from all groups. Pups, including those found dead before study termination, were also submitted for a macroscopic post‑mortem examination.

Mortality: Fifteen animals died during the study, nine females and one male in the high-dose group and four females and one male in the mid-dose group. In males and females, all deaths recorded at 30 and 60 mg/kg/day were considered to be related to the test item treatment. In females, these deaths were considered to represent excessive maternal toxicity at 30 and 60 mg/kg/day

 

Clinical signs: There were no test item treatment-related findings at 15 mg/kg/day. There were signs of neurological disorders (i.e. tremors, convulsions, and tonic contraction) from 30 mg/kg/day, in males and at 60 mg/kg/day in females.

 

Body weight and body weight change: In males, at 60 mg/kg/day, and when compared with controls, there was a minimal lower mean body weight on Day 8 (-4.6%, p < 0.01) resulting in a lower mean body weight gain over the first week of treatment (+24 gvs. +38 g, p < 0.05). In females, there were dose-related decreases in mean body weight and mean body weight change from 15 mg/kg/day.

   

Food consumption: In males, from 15 mg/kg/day, and when compared with controls, there were minimal reductions in mean food consumption during the first week of the treatment period (down to 32 g/animal/day at 60 mg/kg/day vs. 36 g/animal/day). In females during the lactation period, from 15 mg/kg/day, and when compared with controls, there were lower mean food consumptions (down to 35 g/animal/day at 30 mg/kg/day vs. 48 g/animal/day, p < 0.01).

 

Mating and fertility data: at 60 mg/kg/day, there were toxicologically significant lower fertility and gestation indexes.

 

Pup mortality: there were dose-related increased percentages of found dead and cannibalized pups from 15 mg/kg/day.

 

Pup clinical signs: when compared with controls, there was a dose-related increased number of litters with pups with absence of milk in the stomach from 15 mg/kg/day.

 

Pup body weight and body weight gain:on Days 1 and 5 p.p., and when compared with controls, there were lower mean body weights and mean body weight changes from 15 mg/kg/day.

 

Pups necropsy: at heart and cardiovascular examinations of the pups, there were no findings.

 

Pathology: there were no organs weight changes and macroscopic or microscopic findings considered to be test item treatment-related.

 

Based on the experimental conditions of this study:

- the No Observed Effect Level (NOEL) for developmental toxicity was considered to be lower than 15 mg/kg/day based on lower survival index on Day 4 p.p. at this dose-level.

 

Despite a context of severe to excessive maternal toxicity, there was no indication of any teratogenic potential.