Registration Dossier

Diss Factsheets

Administrative data

Description of key information

In a 90-day oral repeated dose toxicity study with rats, conducted according to OECD/EC guidelines and GLP principles, the NOAEL (oral route) was found to be exceed 100 mg/kg bw/day, based on absence of adverse effects at the highest dose level tested. A repeated dose toxicity was conducted with substance analogue TMAH comparable to OECD guideline 410 (1981) and according to GLP. Due to significant effects on the skin a NOAEL for local effects was determined to be 5,5 mg/kg/day for males and 2.5 mg/kg/day for females. The NOAEL for systemic toxicity was found to be 10 mg/kg/day for both sexes. This result is read across to TEAH, the justification is attached in Section 13.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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.
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
Specific details on test material used for the study:
Adjustment was made for specific gravity of the test item.
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
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
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)
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.
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.
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.
Key result
Critical effects observed:
no

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Study was performed according to OECD guidelines and GLP principles (Klimisch 1 study).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Limit test:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Early clinical signs of the animals that died due to TMAH dose were hypoactivity, ptosis, ataxia, tremors, dyspnea and convulsions. No clinical signs at 2.5, 5.5 and 10 mg/kg bw/day.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related gross necropsy findings included various dermal irritation observations at the application skin site (ie thickened, pigmentation, scab/scaly) which increased in incidence with dose level.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats treated with 50 mg/kg/day died during the study and mortality was observed in 8 of 10 (males and females) in the rats treated with 30 mg/kg/day. Nine females and two male rats in the 50 mg/kg dose group died on day 1. All but one remaining male rats in the 50 mg/kg dose group died on days 2-3, one male and one female rat in this group died on Day 8. Two female rats in the 30 mg/kg dose group died on day 1, all other rats died between day 1 and 14, apart from two males and two females that survived treatment with 30 mg/kg TMAH. No other deaths were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weight were observed during the study apart from a significant decrease in mean body weight gain for the two surviving male rats in the group treated with 30 mg/kg/day in week 2. Their body weight had recovered at the end of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in food consumption was observed during week 2 for the two surviving male rats in the group treated with 30 mg/kg/day. No significant differences were noted in any other test group.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in mean corpuscular hemoglobin concentration was observed in females treated with 10 mg TMAH/kg/day. However, this decrease was not considered toxicologically significant since the decrease was very small and no other parameters were affected.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the 30mg/kg/day dose level, the two surviving male rats exhibited a significant increase in cholesterol (152 % of control) while the two surviving females exhibited a significant increase in total bile acids (212 % of control) .
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Significant decreases in potassium levels were observed in the male exposed to 2.5 mg/kg/day and in both males and females in the group treated with 30 mg/kg/day. In addition, significant decreases in urine creatinine values were observed in the females treated with 10 and 30 mg/kg/day. No other significant differences were noted in the urine parameters.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant differences in absolute or relative organ weights for any tissue weighed from male TMAH-treated rats. For female rats, a statistically significant increase in absolute organ weight was seen in the adrenals of the rats exposed to 30 mg/kg/day. However, this difference is based on a group size of the two surviving rats and was not considered treatment-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Red or dark pigmented lungs were generally early deaths and thus correlated with congestion. Pulmonary congestion was noted in the early deaths of the rats treated with 30 mg/kg/day. No other significant differences to the control group were found.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necrosis at the application site was observed in 9 of 10 male rats and 8 of 10 females in the 30 mg/kg/day dose group. Necrosis was also noted in one rat/sex in the 10 mg/kg/day dose group, and in one female rat in the 5.5 mg/kg/day dose group. Other microscopic skin findings were exudative scab, squamous epithelial hyperplasia and inflammation (acute, subacute, or chronic active) at all dose levels, including controls. The magnitude of these findings increased in incidence, severity and distribution with increasing dose concentration of TMAH. Thymic lymphoid necrosis was observed in the group exposed to 30 mg/kg/day. The incidence of vacuolation in the liver was elevated in the males exposed to 30 mg/kg/day. The overall incidence of vacuolation was 1/10, 0/10 and 7/9 males and 6/10, 6/10 and 3/8 females in the control, 10 and 30 mg/kg/day dose groups resp. Vacuolation was accompanied with membrane bound homogenous, eosinophilic intracytoplasmic material. Hepatocellular eosinophilic intracytoplasmic material was present in 7/9 male rats and 2/8 female rats in the 30 mg/kg/day dose group. Individual hepatocyte necrosis was limited to only 3/9 male rats in the 30 mg/kg/day dose group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Acetylcholinesterase (AChE) activity in plasma and red blood cells was not inhibited in TMAH-treated rats compared to the vehicle controls on days 2 or 24.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
10 mg/kg bw/day
Based on:
other: TMAH
Sex:
male/female
Basis for effect level:
other: Thymic lymphoid necrosis, hepatocyte vacuolation with eosinophilic , intracytoplasmic material, hepatocyte necrosis pulmonary congestion at 30 mg/ kg bw/ day.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
10 mg/kg bw/day
Based on:
other: TMAH
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
other: Other microscopic skin findings were exudative scab, squamous epithelial hyperplasia and inflammation (acute, subacute, or chronic active) at all dose levels, including controls.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
5.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
other: Other microscopic skin findings were exudative scab, squamous epithelial hyperplasia and inflammation (acute, subacute, or chronic active) at all dose levels, including controls.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
liver
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

An extra acute study addressing the cause of mortality was included in this report. In both male and female rats treated with a single dose of 250 mg/kg TMAH a significant decrease in pH and an increase in pCO2 in the blood was observed. The males also had a decreased HCO3 levels, while male and female rats treated with a single dose of 50 mg/kg bw TMAH only had decreased PO2 levels. Corresponding hematology profiles indicated an increase in hemoglobin in the 25% TMAH-treated males and an increase in mean corpuscular volume in the 25% TMAH-treated females as well as increases in mean corpuscular hemoglobin in the 5% and 25% TMAH-treated females. Alterations in erythrocyte morphology were noted in both sexes in each of the test substance treatment groups, but not in all animals and not in a consistently dose-related fashion. No other differences to control group were found.

Conclusions:
A repeated dose toxicity was conducted comparable to OECD guideline 410 (1981) and according to GLP. Due to significant effects on the skin a NOAEL for local effects was determined to be 5,5 mg/kg/day for males and 2.5 mg/kg/day for females. The NOAEL for systemic toxicity was found to be 10 mg/kg/day for both sexes. This result is read across to TEAH.
Executive summary:

A repeated dose toxicity was conducted comparable to OECD guideline 410 (1981) and according to GLP. The aqueous substance was applied at doses of 0, 2.5, 5.5, 10, 30 and 50 mg/kg bw/day. All rats died at the highest concentration, and 8/10 (males and females) at 30 mg/kg/day. No changes in body weight gain or absolute and relative organ weight were noted for the surviving animals. At the 30 mg/kg/day dose level, the two surviving male rats exhibited a significant increase in cholesterol while the two surviving females exhibited a significant increase in total bile acids. A significant decrease in potassium levels at 2.5 mg/kg/day (males) and 30 mg/kg/day (males and females) and significant decreases in urine creatinine values at 10 and 30 mg/kg/day (male and female) were found. Histopathology revealed liver vacuolation (males) with eosinophilic intracytoplasmic material, thymic lymphoid necrosis and pulmonary congestion (males and females) at 30 mg/kg/day TMAH. Inflammation, necrosis and squamous epithelial hyperplasia of the skin was found at 10 mg/kg in males and 5.5 mg/kg in females. Due to the significant effects on the skin a NOAEL for local effects was determined to be 5.5 mg/kg/day for males and 2.5 mg/kg/day for females. The NOAEL for systemic toxicity was established to be 10 mg/kg/day for both sexes. This result is read across to TEAH.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliable study for substance analogue TMAH is available, conducted according to OECD guideline and GLP principles.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Limit test:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Early clinical signs of the animals that died due to TMAH dose were hypoactivity, ptosis, ataxia, tremors, dyspnea and convulsions. No clinical signs at 2.5, 5.5 and 10 mg/kg bw/day.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related gross necropsy findings included various dermal irritation observations at the application skin site (ie thickened, pigmentation, scab/scaly) which increased in incidence with dose level.
Mortality:
mortality observed, treatment-related
Description (incidence):
All rats treated with 50 mg/kg/day died during the study and mortality was observed in 8 of 10 (males and females) in the rats treated with 30 mg/kg/day. Nine females and two male rats in the 50 mg/kg dose group died on day 1. All but one remaining male rats in the 50 mg/kg dose group died on days 2-3, one male and one female rat in this group died on Day 8. Two female rats in the 30 mg/kg dose group died on day 1, all other rats died between day 1 and 14, apart from two males and two females that survived treatment with 30 mg/kg TMAH. No other deaths were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in body weight were observed during the study apart from a significant decrease in mean body weight gain for the two surviving male rats in the group treated with 30 mg/kg/day in week 2. Their body weight had recovered at the end of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in food consumption was observed during week 2 for the two surviving male rats in the group treated with 30 mg/kg/day. No significant differences were noted in any other test group.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in mean corpuscular hemoglobin concentration was observed in females treated with 10 mg TMAH/kg/day. However, this decrease was not considered toxicologically significant since the decrease was very small and no other parameters were affected.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the 30mg/kg/day dose level, the two surviving male rats exhibited a significant increase in cholesterol (152 % of control) while the two surviving females exhibited a significant increase in total bile acids (212 % of control) .
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Significant decreases in potassium levels were observed in the male exposed to 2.5 mg/kg/day and in both males and females in the group treated with 30 mg/kg/day. In addition, significant decreases in urine creatinine values were observed in the females treated with 10 and 30 mg/kg/day. No other significant differences were noted in the urine parameters.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant differences in absolute or relative organ weights for any tissue weighed from male TMAH-treated rats. For female rats, a statistically significant increase in absolute organ weight was seen in the adrenals of the rats exposed to 30 mg/kg/day. However, this difference is based on a group size of the two surviving rats and was not considered treatment-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Red or dark pigmented lungs were generally early deaths and thus correlated with congestion. Pulmonary congestion was noted in the early deaths of the rats treated with 30 mg/kg/day. No other significant differences to the control group were found.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necrosis at the application site was observed in 9 of 10 male rats and 8 of 10 females in the 30 mg/kg/day dose group. Necrosis was also noted in one rat/sex in the 10 mg/kg/day dose group, and in one female rat in the 5.5 mg/kg/day dose group. Other microscopic skin findings were exudative scab, squamous epithelial hyperplasia and inflammation (acute, subacute, or chronic active) at all dose levels, including controls. The magnitude of these findings increased in incidence, severity and distribution with increasing dose concentration of TMAH. Thymic lymphoid necrosis was observed in the group exposed to 30 mg/kg/day. The incidence of vacuolation in the liver was elevated in the males exposed to 30 mg/kg/day. The overall incidence of vacuolation was 1/10, 0/10 and 7/9 males and 6/10, 6/10 and 3/8 females in the control, 10 and 30 mg/kg/day dose groups resp. Vacuolation was accompanied with membrane bound homogenous, eosinophilic intracytoplasmic material. Hepatocellular eosinophilic intracytoplasmic material was present in 7/9 male rats and 2/8 female rats in the 30 mg/kg/day dose group. Individual hepatocyte necrosis was limited to only 3/9 male rats in the 30 mg/kg/day dose group.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Acetylcholinesterase (AChE) activity in plasma and red blood cells was not inhibited in TMAH-treated rats compared to the vehicle controls on days 2 or 24.
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
10 mg/kg bw/day
Based on:
other: TMAH
Sex:
male/female
Basis for effect level:
other: Thymic lymphoid necrosis, hepatocyte vacuolation with eosinophilic , intracytoplasmic material, hepatocyte necrosis pulmonary congestion at 30 mg/ kg bw/ day.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
10 mg/kg bw/day
Based on:
other: TMAH
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
other: Other microscopic skin findings were exudative scab, squamous epithelial hyperplasia and inflammation (acute, subacute, or chronic active) at all dose levels, including controls.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
5.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
other: Other microscopic skin findings were exudative scab, squamous epithelial hyperplasia and inflammation (acute, subacute, or chronic active) at all dose levels, including controls.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
liver
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

An extra acute study addressing the cause of mortality was included in this report. In both male and female rats treated with a single dose of 250 mg/kg TMAH a significant decrease in pH and an increase in pCO2 in the blood was observed. The males also had a decreased HCO3 levels, while male and female rats treated with a single dose of 50 mg/kg bw TMAH only had decreased PO2 levels. Corresponding hematology profiles indicated an increase in hemoglobin in the 25% TMAH-treated males and an increase in mean corpuscular volume in the 25% TMAH-treated females as well as increases in mean corpuscular hemoglobin in the 5% and 25% TMAH-treated females. Alterations in erythrocyte morphology were noted in both sexes in each of the test substance treatment groups, but not in all animals and not in a consistently dose-related fashion. No other differences to control group were found.

Conclusions:
A repeated dose toxicity was conducted comparable to OECD guideline 410 (1981) and according to GLP. Due to significant effects on the skin a NOAEL for local effects was determined to be 5,5 mg/kg/day for males and 2.5 mg/kg/day for females. The NOAEL for systemic toxicity was found to be 10 mg/kg/day for both sexes. This result is read across to TEAH.
Executive summary:

A repeated dose toxicity was conducted comparable to OECD guideline 410 (1981) and according to GLP. The aqueous substance was applied at doses of 0, 2.5, 5.5, 10, 30 and 50 mg/kg bw/day. All rats died at the highest concentration, and 8/10 (males and females) at 30 mg/kg/day. No changes in body weight gain or absolute and relative organ weight were noted for the surviving animals. At the 30 mg/kg/day dose level, the two surviving male rats exhibited a significant increase in cholesterol while the two surviving females exhibited a significant increase in total bile acids. A significant decrease in potassium levels at 2.5 mg/kg/day (males) and 30 mg/kg/day (males and females) and significant decreases in urine creatinine values at 10 and 30 mg/kg/day (male and female) were found. Histopathology revealed liver vacuolation (males) with eosinophilic intracytoplasmic material, thymic lymphoid necrosis and pulmonary congestion (males and females) at 30 mg/kg/day TMAH. Inflammation, necrosis and squamous epithelial hyperplasia of the skin was found at 10 mg/kg in males and 5.5 mg/kg in females. Due to the significant effects on the skin a NOAEL for local effects was determined to be 5.5 mg/kg/day for males and 2.5 mg/kg/day for females. The NOAEL for systemic toxicity was established to be 10 mg/kg/day for both sexes. This result is read across to TEAH.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
18.75 µg/cm²
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliable study for substance analogue TMAH is available, conducted according to OECD guideline and GLP principles.

Additional information

Oral exposure:


Dermal exposure:

A repeated dose toxicity was conducted comparable to OECD guideline 410 (1981) and according to GLP. The aqueous substance was applied at doses of 0, 2.5, 5.5, 10, 30 and 50 mg/kg bw/day. All rats died at the highest concentration, and 8/10 (males and females) at 30 mg/kg/day. No changes in body weight gain or absolute and relative organ weight were noted for the surviving animals. At the 30mg/kg/day dose level, the two surviving male rats exhibited a significant increase in cholesterol while the two surviving females exhibited a significant increase in total bile acids. A significant decrease in potassium levels at 2.5 mg/kg/day (males) and 30 mg/kg/day (males and females) and significant decreases in urine creatinine values at 10 and 30 mg/kg/day (male and female) were found. Histopathology revealed liver vacuolation (males) with eosinophilic intracytoplasmic material, thymic lymphoid necrosis and pulmonary congestion (males and females) at 30 mg/kg/day TMAH. Inflammation, necrosis and squamous epithelial hyperplasia of the skin was found at 10 mg/kg in males and 5.5 mg/kg in females. Due to the significant effects on the skin a NOAEL for local effects was determined to be 5.5 mg/kg/day for males and 2.5 mg/kg/day for females. The NOAEL for systemic toxicity was established to be 10 mg/kg/day for both sexes.


Conversion dermal study:

Average rat body weight: 300 g (TNO report V98.390), in report males 220-361 g, females 161-250 g (from day 0 to week 4)

Average total body surface rat: 400 cm2 (TNO report V98.390), acc to OECD 410 at least 10% has to be exposed

2.5 mg/kg bw/d x 0.300 kg / 40 cm2 = 18.75 µg/cm2.

The rationale to read across these data to TEAH is included in section 13.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
One study available for substance analogue TMAH.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
One reliable study available for substance analogue TMAH.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Only one reliable study available.

Repeated dose toxicity: dermal - systemic effects (target organ) cardiovascular / hematological: thymus; digestive: liver

Justification for classification or non-classification

In an oral repeated dose study with TMAH, the nature of the observed effects was not found severe enough to justify classification. For dermal exposure, the NOAEL was found to be 10 mg/kg bw/day, with thymic lymphoid necrosis, hepatocyte vacuolation with eosinophilic, intracytoplasmic material, and hepatocyte necrosis at 30 mg/kg bw/day. As the data of TMAH are read across to TEAH (following the rationale attached in section 13), TEAH is also classified STOT-RE 1 for dermal exposure according to CLP Regulation (EC) No. 1272/2008. Relevant target organs are thymus, and liver.

As the generic concentration limit triggering classification of a mixture is ≥ 10%, a 35% aqueous solution of TEAH is also classified

STOT-RE cat. 1 for dermal exposure.

Categories Display