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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 29 October 1992 to 16 July 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD Guideline and EU Method and according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Lithium bis(trifluoromethylsulfonyl)imide
EC Number:
415-300-0
EC Name:
Lithium bis(trifluoromethylsulfonyl)imide
Cas Number:
90076-65-6
Molecular formula:
C2F6NO4S2.Li
IUPAC Name:
Methanesulfonamide, 1,1,1-trifluoro-N- [(trifluoromethyl)sulfonyl]-,lithium
Test material form:
solid: particulate/powder
Details on test material:
Identity: T-4201
Chemical name: Lithium (bis) trifluoromethanesulfonimide
Purity: >99.5%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, England
- Age at study initiation: 28 ± 1 days old
- Weight at study initiation: 70 - 83g
- Fasting period before study: none
- Housing: The rats were housed in groups of five rats according to sex in metal cages with wire mesh floors.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The animals were acclimated for a period of 6 days before the beginning of the treatment period.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21.5°C
- Humidity (%): 31 - 57% (one incident of a slightly low humidity value (29% relative humidity) was recorded on Day 1 of the treatment period)
- Air changes (per hr): approximately 19 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)

IN-LIFE DATES: From: 3 November 1992 To: 18 December 1992

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared initially in distilled water (high dose level). A series of formulations were prepared by further direct dilution of the high dose level.
Formulations were prepared freshly each day.

VEHICLE
- Concentration in vehicle: 1.67, 10 and 60 mg/kg/day
- Amount of vehicle (if gavage): 10 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chemical stability and, for suspensions, homogeneity of test substance formulations were assessed prior to the start of treatment.

Concentration analyses of formulations prepared for administration on Days 1 and 22 were performed.

The absorption of the test substance was not determined in this study.

Data concerning the analytical purity and homogeneity of the test substance and its stability under the specified conditions of storage were available to the Sponsor.
Duration of treatment / exposure:
32 days
At the end of the treatment period, the remaining animals (groups 1 and 4) were retained for a post treatment observation period of
at least two weeks.
Frequency of treatment:
Once a day
Doses / concentrationsopen allclose all
Dose / conc.:
1.67 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Controls and 60 mg/kg bw/day: 10 animals per sex per dose
1.67 and 10 mg/kg bw/day: 5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dosage was selected on the basis of a preliminary oral toxicity investigation performed at this laboratory HRC Schedule No. MIN 79. The results of the preliminary study indicated that 60 mg/kg/day was a suitable choice for the high dose level.
- Post-exposure recovery period in satellite groups: Yes, 2 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked early in each working day and again in the late afternoon to look for dead or moribund animals. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for signs of ill health, behavioural changes or toxicosis. Any observed changes were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed prior to dosing and subsequently at weekly intervals throughout the study.

FOOD CONSUMPTION:
- 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 in each cage was measured at weekly intervals throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the water bottles were maintained throughout the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: These samples were collected shortly after 4 weeks of treatment (Day 30) and for rats of the recovery sub-groups shortly after their two-week recovery period (Day 44).
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, for an overnight period
- How many animals: five male and five female rats selected from groups 1 and 4 and all rats of groups 2 and 3
- Parameters indicated in the attached document were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: These samples were collected shortly after 4 weeks of treatment (Day 30) and for rats of the recovery sub-groups shortly after their two-week recovery period (Day 44).
- Animals fasted: Yes, for an overnight period
- How many animals: five male and five female rats selected from groups 1 and 4 and all rats of groups 2 and 3
- Parameters indicated in the attached document were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: These samples were collected shortly after 4 weeks of treatment (Day 30) and for rats of the recovery sub-groups shortly after their two-week recovery period (Day 44).
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, for an overnight period
- Parameters indicated in the attached document were examined.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1)
Samples of the following tissues from all rats were preserved in 10% buffered formalin.

ORGAN WEIGHT: Yes
The following organs from each animal killed after four week or six weeks were dissected free of fat and weighed:
* adrenals
* brain
* kidneys
* liver
* ovaries
* spleen
* testes

HISTOPATHOLOGY: Yes
Microscopic examinations were carried out for all rats of Group 1 (Control group) and Group 4 (high dosage group) killed after 4 weeks of treatment.
Examinations were extended, following documented approval from the Sponsor, to include the livers of all male and female rats of the intermediate and low dosage groups and recovery animals (Groups 1 and 4).

Statistics:
All statistical analyses were carried out separately for males and females.

Standard deviations (sample standard deviations) were likewise calculated separately.

Data relating to food consumption were analysed on a cage basis. For all other parameters the analyses were carried out using the individual animal as the basic experimental unit.

Food consumption data were analysed using cumulative values for each cage of animals. Bodyweight data were analysed using weight gains.

A sequence of statisctical tests was used for bodyweight, food consumption, organ weight and clinical pathology data, see below "Any other information on materials and methods incl. tables" for explanations.

Organ weight analyses were initially carried out using analysis of variance as outlined, below, on absolute organ weights. Covariate analysis of organ weight data (with final bodyweight as covariate) was also be performed using adjusted weights for organs where a correlation between organ weight and bodyweight was established at the 10% level of significance.

Additional or alternative statistical methods were used when considered appropriate.

Significant differences between control animals and those treated with the test substance are expressed at the 5% (*P<0.05) or 1% (**P<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY (see table 2 in "Any other information on results incl. tables")
There were no mortalities.

Hypersensitivity, pilo-erection and abnormal gait (walking on toes) were seen intermittently among male and female rats treated with TFSILi at 60 mg/kg/day, mainly during the first half of the treatment period. Body tremors and lethargy were noted for four male rats at this dosage level on Day 22.
Slight fur loss was noted for three male rats treated at 60 mg/kg/day from Day 4 to 15.
For rats treated at 10 mg/kg/day, pilo-erection was noted for one male rat on Days 7 and 11 and for one female on Days 11, 21 and 22. Pilo-erection was accompanied, for the female rats, on Days 21 and 22 by hypersensitivity and abnormal gait. No clinical signs were seen for the remaining rats in
this group throughout the treatment period.
No clinical signs were seen during the treatment period amongst rats treated at 1.67 mg/kg/day or for control rats.
No clinical signs were seen for control rats or rats previously treated at 60 mg/kg/day during the recovery period.

BODY WEIGHT
No significant differences in group mean bodyweight or cumulative bodyweight gain were recorded between treated and control rats of either sex during the treatment period.
During the recovery period, females previously treated at 60 mg/kg/day showed a statistically significantly lower (P < 0.05) cumulative bodyweight gain relative to controls. However, group mean bodyweights for these rats were comparable to those of controls and the variation was considered to
be coincidental.
Group mean bodyweights and cumulative gains for male rats previously treated with TFSILi were similar to those of controls during the recovery period.

FOOD CONSUMPTION
For females receiving 60 mg/kg/day cumulative food consumption for the treatment period was statistically significantly higher (P < 0.01) relative to controls. However, the percentage increase was only 5% and the variation was not considered to be treatment-related.
Food consumption values for females during the recovery period and for males throughout the study were comparable to controls.

CLINICAL PATHOLOGY
Blood was withdrawn during Week 5 from all animals scheduled for sacrifice on Day 32 (terminal kill) and during Week 7 from all animals scheduled for sacrifice on Day 46 (recovery kill) for haematological and biochemical investigations. Urine samples were similarly collected. The parameters examined at the end of the recovery period were those for which significant differences from control had been seen on Week 5.

HAEMATOLOGY
- Week 5
The thrombotest times were statistically significantly higher (P <0.05, P < 0.01) for male rats treated with TFSILi at 60 and 10 mg/kg/day in comparison with controls. The change at 60 mg/kg/day was considered to be treatment-related and possibly so at 10 mg/kg/day.
There were no other statistically significant differences between control and treated rats.
Analysis of blood slides revealed the occurrence of slight polychromasia and/or slight anisocytosis among rats from treated and control groups. This finding is not uncommon among young laboratory rats and was not considered to be important.
- Week 7
There were no significant differences in thrombotest times for control rats (male) and male rats previously treated at 60 mg/kg/day.

CLINICAL CHEMISTRY (see table 3 in "Any other information on results incl. tables")
- Week 5
Statistically significantly (P < 0.01) higher albumin and lower globulin levels were recorded for male and female rats treated at 60 mg/kg/day. The A/G ratio was consequently increased for these animals, achieving statistical significance (P < 0.01) in comparison with controls. The total protein
remained unaffected.
Lower globulin levels (achieving statistical significance for females, P < 0.05) were also seen for male and female rats treated at 10 and 1.67 mg/kg/day, with an associated higher A/G ratio. The change in A/G ratio achieved statistical significance (P < 0.05) for both sexes in comparison with controls. However, the magnitude of the difference from control was small and a strict dosage relationship was not observed. Thus a continuation of the possible effect seen on the high dosage was considered unlikely and statistically significant differences in globulin and A/G ratios for rats dosed at 10 or
1.67 mg/kg/day were considered to be chance occurrences.
Cholesterol and triglyceride levels were lower than controls for male rats treated at 60 mg/kg/day, this change was statistically significant (P < 0.01).
Significantly (P < 0.05) lower than control cholesterol levels were also recorded for female rats at all dosage levels. Although these differences were considered of minor importance the possibility of a treatment relationship could not be excluded.
Blood urea nitrogen (BUN) levels were statistically significantly (P < 0.01) higher for male and female rats treated at 60 mg/kg/day than for controls. The magnitude of the difference in female rats was small and there was overlap of individual values between the groups. A treatment-related effect on
BUN was not considered likely for female rats.
Statistically significantly (P < 0.01) higher alkaline phosphatase (AP) levels were recorded for male rats treated at 60 mg/kg/day in comparison with controls.
Slightly lower potassium ion concentration was recorded for female rats treated at 60 mg/kg/day, achieving statistical significance in comparison with controls (P < 0.01).
Chloride ion concentration was statistically significantly lower (P < 0.01, P < 0.05) for male rats treated at 60 and 10 mg/kg/day than for controls. There was no dosage relationship and the change was minor. This difference was considered to have arisen by chance.
Statistically significantly (P < 0.01) higher glucose levels were recorded for male rats treated at 60 mg/kg/day in comparison with controls. The individual values showed considerable variation and a treatment-related effect was not suspected.
- Week 7
The changes seen in Week 5 were not seen following the recovery period.
Higher globulin levels (with consequently higher total protein levels) were recorded for female rats previously treated at 60 mg/kg/day, achieving statistical significance (P < 0.01). However, this minor change was not thought to be related to treatment with TFSILi.

URINALYSIS
- Week 5
Statistically significantly (P < 0.01) lower urinary pH was recorded for male and female rats treated at 60 and 10 mg/kg/day in comparison with controls.
The volume of urine collected was statistically significantly (P < 0.05) higher for male rats treated at 60 mg/kg/day than for controls. However, intergroup differences in urine volume were not strictly dosage-related and there was considerable variation in individual values and overlaps between the
groups. This apparent change may have arisen by chance.
- Week 7
Slightly lower urinary pH was recorded for male and female rats previously treated with 60 mg/kg/day following the recovery period. This change achieved statistical significance (P < 0.01) in comparison with the controls.
There was no significant difference in the volume of urine collected between control and treated male rats.

ORGAN WEIGHTS
- Week 5
Increased liver weights (absolute and adjusted) were recorded for male and female rats treated at 60 mg/kg/day, achieving statistical significance (P < 0.01) in comparison with controls.
Statistically significantly (P < 0.05) higher kidney (adjusted) weights were also recorded for male rats treated at 60 mg/kg/day in comparison with controls.
Higher spleen weights were also recorded for male rats treated at 60 mg/kg/day, achieving statistical significance (P < 0.05) in comparison with controls. This apparent change was minor and was not dosage-related. There was no histopathological finding in this tissue and a treatment-related effect
on the spleen was considered unlikely.
- Week 7
Higher absolute brain weights were recorded for male rats previously treated at 60 mg/kg/day. This difference was statistically significant (P < 0.05) when compared to controls.
For female rats previously treated with TFSILi at 60 mg/kg/day, statistically significantly (P < 0.05) lower absolute spleen weights were recorded in comparison with controls.
These minor differences from control were considered to be chance occurrences.

GROSS PATHOLOGY
- Terminal kill
Enlarged livers were seen for all five male and three of the five female rats treated at 60 mg/kg/day.
All other findings were considered to be incidental and not related to treatment.
- Recovery kill
An enlarged liver was seen for one male rat previously treated at 60 mg/kg/day following a 2-week recovery period.
No other findings were considered to be related to treatment with TFSILi.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histological examination was confined to the heart, spleen, kidneys and adrenals of terminal rats in Groups 1 and 4 only and the livers of all rats on study. The following comments are made in summary:
Liver: Generalised hepatocyte enlargement was recorded in all male rats and 4/5 female rats given 60 mg/kg/day TFSILi (Group 4). A single female rat given 60 mg/kg/day TFSILi showed centrilobular hepatocyte enlargement.

These changes were not recorded in any rats in any other treated group and there was no evidence of hepatocyte enlargement in rats given 60 mg/kg/day following a 2-week recovery period.
All other changes seen are considered to be spontaneous in origin and therefore of no toxicological importance.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOEL
Effect level:
1.67 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

DISCUSSION

In this subacute study with TFSILi the liver was identified as a primary target organ at the high dosage of 60 mg/kg/day particularly in males. The livers were enlarged, increased in weight and showed generalised hepatocyte enlargement. Amongst the biochemical parameters, significantly

increased albumin and AP levels were observed and significantly decreased cholesterol and triglyceride levels were observed. The thrombotest times were also higher for male rats and since clotting factors are proteins this could also be linked to a treatment-related effect in the liver. Also at 60 mg/kg/day clinical signs of toxicity were seen (e.g. pilo-erection, lethargy) increased kidney weights for males, increased blood urea nitrogen levels and a possible disturbance of urinary pH. No histopathoiogical effect on the kidney was seen, however.

Following the two-week recovery period for rats treated at 60 mg/kg/day there was no effect on liver weight and hepatocyte enlargement was no longer apparent histopathologically.

At the dosage of 10 mg/kg/day a slight increase in thrombotest time in males was statistically significant. There was a minor decrease in plasma cholesterol level among females; occasional other statistically significant differences among blood biochemistry parameters were considered incidental. The incidence of clinical signs was low both in terms of number of affected animals and frequency of occurrence, and the signs were non-specific in nature. Nevertheless they may have represented a continuation of the signs seen at 60 mg/kg/day. Also at 10 mg/kg/day there was a possible minor disturbance of urinary pH, but no effect on kidney weight and no histopathological effect for the liver. Overall it was considered that possible effects of treatment at 10 mg/kg/day were minor in nature.

At the low dosage of 1.67 mg/kg/day there were no differences from controls considered to be of toxicological importance. Other than for a possible minor reduction in plasma cholesterol level for females only, 1.67 mg/kg/day can be regarded as a no effect level.

Table 2: Clinical signs

Sex

Male

Female

Dose-level (mg/kg/day)

0

1.67

10

60

0

1.67

10

60

Number of animals/group

10

5

5

10

10

5

5

10

Piloerection

0

0

1

10

0

0

1

10

Abnormal gait

0

0

0

9

0

0

1

10

Fur loss

0

0

0

3

0

0

0

0

Body tremors

0

0

0

4

0

0

0

0

Lethargy

0

0

0

4

0

0

0

0

Hypersensitivity

0

0

0

10

0

0

1

10

Swollen right eye

1

0

0

0

0

0

0

0

Table 3: Blood chemistry parameters (Week 5)

Sex

Male

Female

Dose-level (mg/kg/day)

0

1.67

10

60

0

1.67

10

60

Glucose (mg/dL)

109

112

112

122*

122

114

121

117

Total protein (g/dL)

6.1

6.1

6.0

6.0

6.5

6.2

6.2

6.4

Albumin (g/dL)

2.7

2.8

2.8

3.1**

2.9

2.9

3.0

3.3**

Globulin (g/dL)

3.4

3.2

3.2

2.9**

3.5

3.2*

3.3*

3.1**

A/G ratio

0.78

0.87*

0.89*

1.05**

0.83

0.91*

0.92*

1.05**

Urea Nitr (mg/dL)

12

12

11

16**

11

14

12

14**

AP (mU/mL)

376

373

350

498**

209

270

290

268

K (mEq/L)

3.7

3.9

3.9

3.4

3.8

3.7

3.6

3.2**

Cl (mEq/L)

98

97

96*

97*

98

99

99

99

Cholesterol (mg/dL)

75

75

66

30**

73

61*

60*

59*

Tri-glyc (mg/dL)

114

106

104

66**

59

71

59

53

Applicant's summary and conclusion

Conclusions:
Based on the results of this study with bis trifluoromethanesulfonimide lithium in the rat the liver was identified as a principal target organ at 60 mg/kg/day, the high dose. The effects of treatment at 10 mg/kg/day were considered minor in nature and for this reason this dose level is considered to be the NOAEL. Other than for a possible minor reduction in plasma cholesterol for females only, the low dosage of 1.67 mg/kg/day can be regarded as a NOEL.
Executive summary:

In this study the test material, bis trifluoromethanesulfonimide lithium (TFSILi) formulated in distilled water, was administered to groups of

rats at dosages of 1.67, 10 and 60 mg/kg/day for a minimum of 28 consecutive days. Control animals received the vehicle alone.

All rats of Groups 2 and 3 (1 .67 and 10 mg/kg/day) and five males and five females from each of Groups 1 and 4 (Controls and 60 mg/kg/day) were killed following the four-week treatment period (Day 32). The remaining animals (five males and five females from Groups 1 and 4) were retained

for a minimum two-week recovery period, following which they were also killed (Day 46).

Bodyweights, food consumption and clinical observations were recorded during the study. Blood samples were taken from all rats shortly prior to termination following the four-week treatment and two-week recovery periods. All animals were killed and subsequently examined macroscopically;

specified tissues were then prepared for histological examination.

All animals survived until scheduled sacrifice. No treatment-related changes in bodyweight development and food consumption were observed during the study. The following treat-ment related effects have been observed in the study:

Clinical signs - For rats receiving 60 mg/kg/day, hypersensitivity, piio-erection and abnormal gait (walking on toes) were seen intermittently mainly during the first half of the treatment period. Body tremors and lethargy were noted for four male rats at this dosage on Day 22.

For rats receiving 10 mg/kg/day, pilo-erection was noted infrequently for one male and one female and was accompanied on two occasions, for the female rat, by hypersensitivity and abnormal gait.

Haematology (Week 5) - The thrombotest times were higher for males rats treated with TFSILi at 60 and to a minor extent at 10 mg/kg/day.

Biochemistry (Week 5) - Higher albumin and lower globulin (with consequently higher A/G ratios) were recorded for male and female rats treated at 60 mg/kg/day.

Lower cholesterol and triglyceride levels and higher blood urea nitrogen and alkaline phosphatase levels were recorded for male rats from this high dosage group in comparison with controls. Slightly lower cholesterol levels were observed for females at all dosages of TFSILi.

Slightly lower potassium ion concentration was recorded for females treated at 60 mg/kg/day in comparison with controls.

Urinalysis (Week 5) - A minor decrease in urinary pH was observed at 60 mg/kg/day and at 10 mg/kg/day. For recovery animals at 60 mg/kg/day at Week 7 a similar difference from controls was seen as at Week 5.

Organ weights (Terminal kill) - Higher liver weights were recorded for male and female rats treated at 60 mg/kg/day than for controls. Higher kidney weights were also recorded for male rats of this group.

Macroscopic pathology (Terminal kill) - Enlarged livers were seen for all five male and 3/5 female rats treated at 60 mg/kg/day.

(Recovery kill) - An enlarged liver was seen for 1 male rat from the high dosage group.

Microscopic pathology (Terminal kill) - Generalised hepatocyte enlargement was seen in all male and 4/5 female rats treated at 60 mg/kg/day. The remaining female showed centrilobular hepatocyte enlargement.

Based on the results of this study with bis trifluoromethanesulfonimide lithium in the rat, the liver was identified as a principal target organ at 60 mg/kg/day, the high dose. The effects of treatment at 10 mg/kg/day were considered minor in nature and for this reason this dose level is considered to be the No-Observed-Adverse Effect-Level (NOAEL). Other than for a possible minor reduction in plasma cholesterol for females only, the low dosage of 1.67 mg/kg/day can be regarded as a No-Observed-Effect-Level (NOEL).

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