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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April 2017 - 31 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was initiated after final ECHA decison TPE-D-2114346827-38-01/F was received.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraethylammonium hydroxide
EC Number:
201-073-3
EC Name:
Tetraethylammonium hydroxide
Cas Number:
77-98-5
Molecular formula:
C8H20N.HO
IUPAC Name:
tetraethylammonium hydroxide
Test material form:
liquid
Remarks:
Clear colourless to light yellow liquid
Details on test material:
- Name of test material (as cited in study report): Tetraethylammoniumhydroxide 35% in water
- Purity/composition correction factor required: Yes, correction factor is 2.83 (correction of water content, adjust to 100% TEAH)
- Storage condition of test material: At room temperature
- pH: >13
- Specific Gravity / Density: 1.02
Specific details on test material used for the study:
Adjustment was made for specific gravity of the test item.

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 142 - 175g (males), 118 - 144g (females) (main study)
- Fasting period before study: Animals were only deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm), except during locomotor activity monitoring, when animals were housed individually in a Hi-temp polycarbonate cage
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum (except during locomotor activity monitoring, when no food was available and overnight before sacrifice (maximum 24 hours))
- Water: tap water, ad libitum (except during locomotor activity monitoring, when no water was available)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS (actual daily mean)
- Temperature (°C): 22
- Humidity (%): 41 - 59
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 April 2017 To: 31 Aug 2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of exposure was selected because this is the intended route of human exposure.
Vehicle:
water
Remarks:
(Elix)
Details on oral exposure:
- Dose volume: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy and homogeneity were determined for formulations prepared for in week 1, week 6 and week 13 and stability was determined for formulations prepared for week 1.
For determination of accuracy, duplicate samples were taken at middle position (50% height) (vehicle control and 30 mg/kg bw/day groups) or at top, middle and bottom position (90%, 50% and 10% height) (10 and 100 mg/kg bw/day groups). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.
For determination of stability, additional samples were taken for the 10 and 100 mg/kg bw/day groups at 50% height and stored at room temperature under normal laboratory light conditions for 5 hours.
The samples were diluted to obtain concentrations within the calibration range and analyses were conducted according to a validated method using LC-MS/MS.

The accuracy of preparation was considered acceptable if the mean measured concentrations are 90-110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation is ≤ 10%. Formulations were considered stable if the relative difference before and after storage is maximally 10%.
Duration of treatment / exposure:
at least 90 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 females (dose range finding study);
10 (main study)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a 14-day range finding study (for details see below)

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 once daily from start of treatment onwards. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena.

FUNCTIONAL OBSERVATIONS:
During week 12-13 of treatment, the following tests were performed on the first 5 animals/sex/group after dosing at no specific time point, but within a similar time period after dosing for the respective animals:
- hearing ability, pupillary reflex (L/R), static righting reflex;
- fore- and hind-limb grip strength (recorded as the mean of three measurements);
- locomotor activity (recording period: 1 hour under normal laboratory light conditions, using a computerized monitoring system, total movements and ambulations are reported).

BODY WEIGHT: Yes
- Weekly

FOOD CONSUMPTION :
- Weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during Pretreatment in all study and spare animals, and at the end of the Dosing Period in Week 13 in all animals of the control and high dose group.

ESTROUS CYCLE DETERMINATION
- Daily vaginal lavage was performed for all females from Week 11 (day 71) up to and including Week 13 (day 91). Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment (day of necropsy between 7.00 and 10.30 AM)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: all animals


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment (day of necropsy between 7.00 and 10.30 AM)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: all animals

URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were necropsied and descriptions of all macroscopic abnormalities recorded.

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all control and high dose animals (according to guideline)
- adrenal glands of all males of low and mid dose groups, based on (possible) treatment-related changes in these organs in high dose group animals,
- all gross lesions.
- For the testes of all males of the control and 100 mg/kg bw/day groups detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. Any cell- or stage-specificity of testicular findings were noted.
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. Whenever, the overall test was significant, the Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
All statistical tests was conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs (including during arena observations) were noted. At 100 mg/kg bw/day, one male displayed rales for a duration of three days during week 7 of treatment, however this was considered incidental based on the lack of incidence, severity and its transient nature.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item related mortality occurred. One high dose female was killed in extremis on day 57 of the study due to the poor health condition of the animal. This animal displayed abnormal/hunched posture and uncoordinated movements immediately after dosing and prior to sacrifice. Macroscopic findings included thickened and gelatinous, dark red axillary region (right side), with the microscopic correlate of edema. No cause of death could be determined from the slides examined, however a gavage related error may be a possible cause. This death was considered incidental and not test item-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A slight trend towards lower body weight and body weight gain was observed in the 100 mg/kg males. This effect was not statistical significant when compared to controls over the study period and not considered toxicological significant.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Higher creatinine and potassium levels were noted in 100 mg/kg females when compared to controls (statistically significant). These findings were small in their extent, within historical control ranges, and lacking any histopathological correlate, and therefore not considered toxicologically significant.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Foregrip strength was statistically significantly reduced in 30 and 100 mg/kg males in a dose dependent manner. There were no supporting behavioural or histopathological findings, therefore this effect is not considered toxicologically significant.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations. Al 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:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous Cycle Determination
Three females at 10 mg/kg bw/day, two females at 100 mg/kg bw/day showed an irregular estrous cycle length, and one female at 30 mg/kg bw/day showed acyclic cycle. All other females showed a normal (regular) estrous cycle of 4 days. The incidence of irregular/acyclic estrous cycle length showed no relationship to the dose, and was therefore considered unrelated to treatment.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose level tested. Higher doses were not tested due to test item related toxicity in the dose range finding study.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Analysis of formulations:

No test item was detected in the control group formulation.

The mean accuracies for the concentrations in the formulations of the 10, 30 and 100 mg/kg bw/day groups (Week 1, Week 6 and Week 13 formulations) were between 98.0 and 103.3% and therefore in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).

The coefficient of variation for the formulations of the 10 and 100 mg/kg bw/day groups were between 0.8 and 1.4% and therefore the formulations were considered homogeneous (i.e. coefficient of variation ≤ 10%).

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤10%, actual differences found were -0.4 and 0.3% for the 10 and 100 mg/kg bw/day groups, respectively). In addition the test item was stable in water over a storage period of at least 20 days at a temperature of ≤ -70°C.

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 was found to be >100 mg/kg bw/day, based on absence of adverse effects at the highest dose level tested.
Executive summary:

A 90-day oral repeated dose toxicity study was conducted with tetraethylammoniumhydroxide (in water) according to OECD/EC guidelines and in accordance with GLP principles. Male and female rats were exposed to 10, 30 and 100 mg TEAH/kg bw/day via oral gavage. Chemical analysis of the formulations confirmed correct concentrations and stability. No mortality occurred during the study. No treatment-related changes were noted in clinical appearance of the animals, food consumption, estrous cycle, motor activity and opthalmic examinations. No treatment-related effects were observed on haematology parameters, coagulatory parameters and organ weights and during macroscopic and microscopic examinations including detailed qualitative examination of the testis.

A slight trend towards a lower body weight and body weight gain was observed in 100 mg/kg bw/day males. The effects were minimal and not statistically significant and therefore not considered toxicologically significant.

Functional observation tests revealed a statistically significant reduction in foregrip strength in 30 and 100 mg/kg bw/day males, however there were no supporting behavioural or histopathological findings, therefore this effect is not considered toxicologically significant.

Clinical chemistry analysis revealed increased creatinine and potassium levels in 100 mg/kg bw/day females, however these findings were small in their extent, within historical control ranges, and lacking any histopathological correlate, and therefore not considered toxicologically significant.

Taking all data together, the NOAEL of Tetraethylammoniumhydroxide was determined to be >100 mg/kg bw/day based on the absence of adverse effects at the highest dose level tested. Higher dose levels were not tested due to test item related mortality, related clinical signs and macroscopic findings observed in the dose range finding study.