Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22JUL2015 - 11DEC2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(September 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
(May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
(August 1998)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): TMAC – tetramethylammonium chloride
- Substance type: Organic
- Physical state: White crystalline powder
- Storage condition of test material: At room temperature protected from light
- pH: 6-8

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approx. 7 weeks old
- Weight at study initiation: 145-170 g (males); 122-149 g (females)
- Housing: Group housing of 5 animals per sex in Macrolon cages except during locomotor activity monitoring, when animals were housed individually in a Hi-temp polycarbonate cage
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) except overnight (for a maximum of 24 hours) before blood samples were collected
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70 (deviations from the daily mean relative humidity occurred, however laboratory historical data did not indicate an effect of the deviations)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 10SEP2015 to 11 December 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The formulations (w/w) were prepared daily within 5 hours prior to dosing. After confirmation of stability, formulations (w/w) were prepared weekly and were used within 8 days after preparation. The formulations were homogenized to a visually acceptable level. No correction was made for purity/composition of the test substance.


DOSE VOLUME: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). During pretest stability in vehicle over 5 hours at room temperature under normal laboratory light conditions and over 8 days in the refrigerator in the dark was also determined (highest and lowest concentration). Long term stability in the freezer was determined over 34 days. The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrationsopen allclose all
Dose / conc.:
3 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels are based on results of a 14-day oral range finding study with TMAC – tetramethylammonium chloride by daily gavage in the rat. Three females were exposed to either 30 or 100 mg/ kg bw/ day (5mL/kg bw/ day). Clinical signs were recorded daily, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Mortality was recorded at least twice daily. Body weighhts and food consumption were recorded weekly. All animals were subjected to an external, thoracic and abdominal examination after sacrifice.
No mortality occurred at 30 mg/kg bw/ day, at 100 mg/kg bw/ day two animals were found dead and one animal was sacrificed on day 1. The peak effect of occurrence of clinical signs occurred at 1 hour after dosing. This time point was taken into account for conduct of clinical observations and functional observation tests in the main study. No findings were recorded for clinical appearance, body weight and food consumption (only recorded for 30 mg/ kg bw/ day). No abnormalities were noted for all animals at macroscopic examination at sacrifice.

- Selection of animals for selected measurements:
Functional observations were performed on 5 animals/sex/group. Ophthalmoscopic examination were examined in all animals at pre-test, at week 13 in control and high dose group animals. Histopathology was performed on all tissues collected at the scheduled sacrifice from all animals in control and high dose groups, furthermore on all tissues from two males which died spontaneously, and on all gross lesions.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily: immediately after dosing and 1 hour (+/- 15 min) after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption was recorded weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-test and week 13
- Dose groups that were examined: all (at pre-test), control and high dose in week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: all
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (functional observation battery)
- Time schedule for examinations: week 13
- Dose groups that were examined: all
- Battery of functions tested: grip strength/ motor activity /total movements and ambulations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
Organs checked according to test guidelines

HISTOPATHOLOGY: Yes
According to test guidelines
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 5) was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were noted in control rats. In animals at 30 mg/kg bw/day, clinical signs consisted of lethargy, hunched and/or flat posture, piloerection and ptosis. In addition, abdominal swelling was noted at this dose level in females only. The animals at 10 mg/kg bw/day showed lethargy, hunched posture, piloerection and/or ptosis at a lower incidence and severity compared to animals at 30 mg/kg bw/day. At 3 mg/kg bw/day, hunched posture and piloerection were noted at low incidence in males only. Salivation seen after dosing among the animals at 10 and 30 mg/kg bw/day was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). No additional clinical signs during weekly arena observations were noted during the observation period that were considered to be related to treatment. No clinical signs were noted in the deceased rats.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two males treated at 30 mg/kg bw/ day were found dead at day 57 and day 80, respectively (with autolysis in several organs or missing organs (head region) due to cannibalism).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated at 30 mg/kg bw/day showed lower body weight (statistically significant from week 6 to end of study, appr. -12% for both absolute and relative values at study termination compared to controls) and body weight gain (statistically significant in week 2 and from week 7 to end of study). Body weights and body weight gain of female animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Absolute food consumption of males treated at 30 mg/kg bw/day was found to be lower compared to controls during the second half of the treatment period. No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for females.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No substance-related adverse effects were seen in heamatological parameters for exposed males compared to control group. A statistically significant decrease of prothrombin time in males dosed at 3 mg/kg bw/day was considered to be an incidental effect and not to be substance-related. In high dose females, the percentage lymphocytes was found to be decreased (-5.5%), with an increased percentage of monocytes (+29%), both effects were statistically significant. In absence of a clear dose-relationship of the effects, this was not found to be adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males exposed to 30 mg/kg bw/day showed statistically significant increase in alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels (respectively +67% and +20% compared to controls). ALAT levels were also increased in high dose females (+86%), and found together with increased alkaline phosphatase levels (+82%, both statistically significant).
Furthermore, concentration bilirubin was increased in high dose males (+23%) and females (+19%). Glucose levels were decreased in high dose males (-13%). A dose-related increase in bile acids was observed for all exposed males (+50%, +150% and +719% for males dosed at 3, 10 and 30 mg/kg bw/day, respectively, statistically significant only at highest dose). This increase was also seen in females with +38%, +83% and +1560% at respectively 3, 10 and 30 mg/kg bw/day (statistically significant only at highest dose). Furthermore, effects on ionic balance were noted in males (increased sodium levels at 3 and 10 mg/kg bw/day, dose related increased potassium (only statistically signficant at 30 mg/kg bw/day) and increased inorganic phophate levels at 30 mg/kg bw/ day) and females (increased potassium at highest dose). In high dose females, also effects were noted on total protein and albumin (both decreased with -11%, statistically significant effect). Cholesterol levels increased for all exposed females (+5%, +5% and +24% (statistically significant only at high dose).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Females exposed to 30 mg/kg bw/day showed statistically significant reduction in total movements (-73.5%) and ambulations (-83%), no such effect was seen in males. For males, grip strenth was decreased for all exposed rats compared to control group (appr. -23% and -21%, -27% and -17%, -29% and -20% for respectively fore and hind grip at 3, 10 and 30 mg/kg bw/day), however these effects were not statistically significant. Grip strenth was not affected in females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Females dosed at 30 mg/kg bw/day were found to have increased liver weights (appr. +23% and +25% for absolute and relative values respectively, both statistically significant). In parallel, relative liver weight was also slightly increased in males in the highest dose group (+7%, statistically significant). Lower thymus weights were noted in females (18% for absolute and 17% for relative weight, both statistically significant) at 30 mg/kg bw/day.
Other statistically significant changes in organ weights at the end of the treatment were considered to be of no toxicological significance as they remained within the range considered normal for rats of this age and strain or were in males considered to be the result of a test item-related effect on final body weight. These observations included effects on relative testes weights which were affected in a dose related manner compared to control males with +3%, +11% and +13% respectively for rats exposed to 3, 10 and 30 mg/kg bw/day (statistically significant only for two highest dose groups). Further observations included decreased weight of the brain in males only at 30 mg/kg bw/day (appr. -4% for absolute and +7.5% relative to body weight, both statistically significant). Relative kidneys weights were slightly increased for males in the highest dose group (+11%, statistically significant). At 10 mg/kg bw/ day, absolute and relative ovary weights were increased, but in absence of dose-relationship this was found to be not substance-related. No other findings were noted in males and females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Emaciation was noted in one female at 30 mg/kg bw/day at macroscopic examination. Swelling of the abdomen was noted for several rats. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination of the two animals deceased before scheduled sacrifice was only possible to a limited agree due to the status. Findings consisted of many dark red foci of the thymus (correlated with congestion/hemorrhage) and many dark red foci of the thymus in one of the two rats. Therefore conclusion on cause of pre-scheduled deaths was not possible.
In all exposed females microscopic findings were noted in the liver, consisting of hepatocellular hypertrophy at minimal degree. The effects were found in 4/10 females at 3 and 10 mg/kg bw/day and in 8/10 females exposed at 30 mg/kg bw/day (all grade 1, minimal effects). Due to the fact that this effect was present at low severity (minimal) and in absence of effects in other parameters this observation was considered to be non-adverse at 3 and 10 mg/kg bw/day. In the thymus of 2/10 females at 30 mg/kg bw/day atrophy at minimal degree was observed. Based on the low severity, this effect was found to be non-adverse. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

No test substance was detected in the control group formulations. The concentrations analyzed in the formulations with the test substance were in agreement with the target concentrations (i.e. mean accuracies between 95.1% and 96.5%). The formulations of high and low dose group were prepared homogeneous (i.e. coefficient of variation ≤ 1.8%). Formulations at the entire range were stable when stored at room temperature protected from light for at least 5 hours and for 8 days at room temperature (i.e. relative difference ≤ 3.2%). The long term storage samples were stable at ≤-70°C for 34 days.

Applicant's summary and conclusion

Conclusions:
In a 90-day oral repeated dose toxicity study with rats, conducted according to OECD/EC guidelines and GLP principles, the NOAEL for TMAC was determined to be 10 mg/kg bw/day based on adverse effects on liver at 30 mg/kg bw/ day.
Executive summary:

A 90-day oral repeated dose toxicity study was conducted according to OECD/EC guidelines and GLP principles. Male and female rats were exposed to 0, 3, 10 and 30 mg/kg bw/day via oral gavage. Two males exposed to 30 mg/kg bw/day died during the study, on exposure days 57 and 80, respectively. In absence of clinical signs, effects on body weight and/or food consumption and observations during necropsy, no cause of death could be established. In the surviving exposed rats clinical signs consisted of lethargy, hunched and/or flat posture, piloerection and ptosis, especially at the highest dose. Severity and incidence decreased with dose. Males treated at 30 mg/kg bw/day showed statistically significant lower body weight (gain). Hematological parameters were not affected by the test substance. Rats exposed to 30 mg/kg bw/day showed increase in ALAT (males and females), ASAT (males only) levels, increased ALP levels (females only), bilirubin and bile acid levels. Cholesterol levels decreased for high dose females only. Females exposed to 30 mg/kg bw/day showed statistically significant reduction in total movements and ambulations, no other functional effects were observed. Females dosed at 30 mg/kg bw/day were found to have statistically significantly increased relative and absolute liver weights and lower thymus weights. Histopathology revealed hepatocellular hypertrophy in 4/10 females at 3 and 10 mg/kg bw/day and in 8/10 females exposed at 30 mg/kg bw/day (all grade 1, minimal effects). Due to the fact that this effect was present at low severity at 3 and 10 mg/kg/day and in absence of effects in other parameters this observation was considered to be non-adverse at these dose levels. In the thymus of females at 30 mg/kg bw/day atrophy at minimal degree was observed in 2/10 females with low severity.

Taken all data together, the NOAEL of TMAC was determined to be 10 mg/kg bw/day based on adverse effects at 30 mg/kg bw/day.