Registration Dossier

Administrative data

Description of key information

One oral repeated dose study is available: MHLW 2001, resulting in a NOAEL of 5 mg/kg bw/d.
Two dermal repeated dose studies are available: IITRI2000, 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. IBM1999 (reliability 4): a systemic NOAEL of 2.5 mg/kg bw for females and 5.5 mg/kg bw for males was established for TMAH.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Results based on OECD SIDS on tetramethylammoniumhydroxide (April 2006), which is considered to be peer-reviewed.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5 weeks
No further details are given on animals or environmental conditions.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqueous solution
Details on oral exposure:
Volume: 10 ml/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
5, 10, 20 mg/kg bw/ day
Basis:
actual ingested
No. of animals per sex per dose:
5 (5 and 10 mg/kg bw), 10 (0 and 20 mg/kg bw)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Not reported
- Rationale for selecting satellite groups: Not reported
- Post-exposure recovery period in satellite groups: 14 days (0 and 20 mg/kg bw)
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: not reported

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: not reported

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to dosing, on Day 5, and twice a week thereafter.

FOOD CONSUMPTION: YES
- Food consumption was monitored

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: right before autopsy
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: not reported
- Parameters examined: no details reported

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: right before autopsy
- Animals fasted: No
- How many animals: not reported
- Parameters examined: no details reported

URINALYSIS: Yes
- Time schedule for collection of urine: Collected for 4 and 24 hours in the 4th week of the administration period and the 2nd week of the recovery period.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: no details reported

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, organ weights of heart liver and kidney were determined. No data on other organs.
HISTOPATHOLOGY: Yes
Histopathological examinations by hematoxylin eosin staining were carried out on heart, liver, kidneys, spleen, adrenals, stomach, testes, epididymis and ovary of all the animals in the control and high dose groups, and on all gross lesions of all animals. Because treatment-related changes were observed in the kidney of male rats, histopathological examination was further performed on the kidney of all the male animals in the low and middle dose groups.
Statistics:
Dunnett's test, Student's t-test, Aspin-Welch t-test,Mann-Whitney U-test, Fisher test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality was observed. On the 6th day of administration and later, incidences of salivation were found in males and females at 10 and 20 mg/kg bw/day.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
A significant decrease in food consumption was observed in the 1st week of administration in male animals at 10 mg/kg bw/day, and male and female animals at 20 mg/kg bw/day. A significant increase in food consumption was observed in the 1st week of the recovery period, however, in the male animals at 20 mg/kg bw/day. The decrease in food consumption was considered to be related to the alkalinity of the substance.

URINALYSIS
There were no treatment-related changes observed in urinalysis, but a significant increase in urinary electrolyte excretion was noted in the 2nd week of the recovery period in the male animals at 20 mg/kg bw/day.

ORGAN WEIGHTS
Decreases were observed in the heart weights in male animals: a significant decrease in the absolute weights at 5 mg/kg bw/day and higher and that in the relative heart to body weights at 10 mg/kg bw/day and higher. The effect was not clearly dose-dependent. On the other hand, no treatment-related changes in organ weights were observed in female animals at any doses at the end of administration or male and female animals at the end of the recovery period. As no histopathology was observed, for now the decrease in heart weight is considered to be not toxicologically relevant, awaiting further data.

Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on decreases in food consumption seen at 10 mg/kg bw.
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on decreases in food consumption seen at 20mg/kg bw/day in females.
Critical effects observed:
not specified
Conclusions:
In a 28-days oral repeated dose exposure assay, the NOAEL was established to be 5 (males) and 10 (females) mg/kg bw/d.
Executive summary:

A 28-days oral repeated dose experiment was conducted according to OECD guideline 407 and GLP principles. No deaths were observed at the concentrations tested (5, 10 and 20 mg/ kg bw/d). A significant decrease in food consumption was observed in the first week of administration in male animals at 10 mg/kg bw/day, and male and female animals at 20 mg/kg bw/day. A decreased absolute and relative heart weight without dose-response and no correlated histopathological findings was observed at 10 mg/kg bw/day and higher in males only. This effect was not seen at the end of the recovery period. Therefore, this effect was not considered to be toxicologically relevant for the time being, awaiting further data.

The NOAEL for repeated dose oral toxicity was considered to be 5 mg/kg bw/day for males and 10 mg/kg bw/day for females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study was performed according to OECD guidelines and GLP principles.

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:
experimental study
Adequacy of study:
key study
Study period:
May 21, 1999 - June 23, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Experiment was conducted comparable to OECD guidelines and with GLP principles. No details were given on test substance (purity).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Principles of method if other than guideline:
Application of the substance was done with an Hill top Chamber® (Hill Top Biolabs, Inc., Cincinnati, OH, USA), exact size of exposure area is not given. No recovery group was included in this study to assess reversibility of the symptoms.
GLP compliance:
yes
Remarks:
US EPA GLP Standards (40CFR Part 792)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Age at study initiation: appr. 5 weeks
- Weight at study initiation:76 - 139 g (random 15% of rats was weighed)
- Fasting period before study: yes, overnight prior to terminal sacrifice
- Housing: individually, in suspended stainless steel cages
- Diet: ad libitum, certified Purina rodent chow 5002 (PMI Feeds, Inc, St. Louis, MO, USA)
- Water: ad libitum, City of Chicago municipal water
- Acclimation period: ≥ 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 - 25.5
- Humidity (%): 27.3 - 69.0
- Air changes (per hr): not described
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
water
Remarks:
distilled
Details on exposure:
TEST SITE
- Area of exposure: Hill top Chamber® (Hill Top Biolabs, Inc., Cincinnati, OH, USA), occlusive
- Type of wrap if used: Chamber was secured with an elastic band secured with Velcro®
- Time intervals for shavings or clippings: 24 hours prior to treatment, three times/ week during the application period

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

TEST MATERIAL
- Amount applied (volume with unit): 1 ml/kg bw
- Concentration (if solution): 0%, 0.25%, 0.55%, 1%, 3%, 5%
- Constant volume used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, plastic neck collars
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of TMAH in water formulations was determined by titration using HCL. All dosing formulation concentrations were confirmed in first two weeks of experiment and were within 3% of the theoretical concentration. The stability of the highest and lowest concentrations of the test dosing formulations was determined over 8 and 13-day periods. Both concentrations were still stable after 13 days.
Duration of treatment / exposure:
6 hours/ day; 4 weeks
Frequency of treatment:
5 days/ week
Remarks:
Doses / Concentrations:
0, 2.5, 5.5, 10, 30 and 50 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
50 mg/kg NaOH
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on week days, once daily in weekends and holidays

DETAILED CLINICAL OBSERVATIONS: yes, at least weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: one day pre-dosing, weekly during treatment and terminal body weight

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of sacrifice
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, overnight (appr. 18 hours)
- How many animals: all surviving animals
- Parameters according to guideline (OECD 410, 1981) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:on day of sacrifice
- Animals fasted: Yes, overnight (appr. 18 hours)
- How many animals: all surviving animals
- Parameters according to guideline (OECD 410, 1981) were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight prior to scheduled sacrifice
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: urine volume, levels of sodium and potassium, color, appearance, refractive index, specific gravity, microscopic evaluation, and qualitative measurement of glucose, urobilinogen, pH, protein, ketones, blood, nitrites, leukocytes, creatinine and bilirubin.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Complete necropsy was performed on all rats, gross necropsy was performed on the 5% TMAH rats or rats not surviving at least two days of TMAH exposure.
HISTOPATHOLOGY: Yes.
Tissues according to guideline were collected from rats that were designated for complete necropsy. A complete set of tissues from all rats in the vehicle control group and in the two highest surviving dose groups (3% and 1% TMAH) were examined microscopically.
In addition, the skins (application site) in the 0.55% and 0.25% TMAH groups were also examined.
Adrenals, brain, heart, kidneys, liver, ovaries, spleen and testes were weighed, and the organ to fasted body weight ratios were calculated.
Other examinations:
On study days 2 and 24, blood samples for determination of acetylcholinesterase (AChE) activity were collected via the retroorbital sinus from anesthetized (70% CO2/30% air) rats at least two hours after application of dosing formulations.
Statistics:
Statistical analysis of continuous data was performed using analysis of variance followed, where appropriate, by Dunnett's test. All comparisons were performed using the vehicle control exposed animals as the control group. A minimum significance level of p≤0.05 was used for all comparisons.
Clinical signs:
effects observed, treatment-related
Dermal irritation:
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
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
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:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
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. 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.
BODY WEIGHT AND WEIGHT GAIN
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
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.
HAEMATOLOGY
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 CHEMISTRY
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
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 tretaed 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 WEIGHTS
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 PATHOLOGY
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. 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.
HISTOPATHOLOGY
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.
OTHER FINDINGS
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:
test mat.
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:
2.5 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Squamous epithelial hyperplasia, inflammation (acute, subacute or chronic active) and necrosis seen at 5.5 mg/kg bw/ day at application site.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
5.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Squamous epithelial hyperplasia and inflammation (acute, subacute or chronic active) and necrosis seen at 10 mg/kg bw/ day at application site.
Critical effects observed:
not specified

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 th 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.
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 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.

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 is available, conducted according to OECD guideline and GLP principles. A second study with reliability 4 presented no clear data on local effects and lacked exminations on organ weights and microscopy.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 21, 1999 - June 23, 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Experiment was conducted comparable to OECD guidelines and with GLP principles. No details were given on test substance (purity).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Principles of method if other than guideline:
Application of the substance was done with an Hill top Chamber® (Hill Top Biolabs, Inc., Cincinnati, OH, USA), exact size of exposure area is not given. No recovery group was included in this study to assess reversibility of the symptoms.
GLP compliance:
yes
Remarks:
US EPA GLP Standards (40CFR Part 792)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI, USA
- Age at study initiation: appr. 5 weeks
- Weight at study initiation:76 - 139 g (random 15% of rats was weighed)
- Fasting period before study: yes, overnight prior to terminal sacrifice
- Housing: individually, in suspended stainless steel cages
- Diet: ad libitum, certified Purina rodent chow 5002 (PMI Feeds, Inc, St. Louis, MO, USA)
- Water: ad libitum, City of Chicago municipal water
- Acclimation period: ≥ 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.0 - 25.5
- Humidity (%): 27.3 - 69.0
- Air changes (per hr): not described
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
occlusive
Vehicle:
water
Remarks:
distilled
Details on exposure:
TEST SITE
- Area of exposure: Hill top Chamber® (Hill Top Biolabs, Inc., Cincinnati, OH, USA), occlusive
- Type of wrap if used: Chamber was secured with an elastic band secured with Velcro®
- Time intervals for shavings or clippings: 24 hours prior to treatment, three times/ week during the application period

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

TEST MATERIAL
- Amount applied (volume with unit): 1 ml/kg bw
- Concentration (if solution): 0%, 0.25%, 0.55%, 1%, 3%, 5%
- Constant volume used: yes

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, plastic neck collars
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of TMAH in water formulations was determined by titration using HCL. All dosing formulation concentrations were confirmed in first two weeks of experiment and were within 3% of the theoretical concentration. The stability of the highest and lowest concentrations of the test dosing formulations was determined over 8 and 13-day periods. Both concentrations were still stable after 13 days.
Duration of treatment / exposure:
6 hours/ day; 4 weeks
Frequency of treatment:
5 days/ week
Remarks:
Doses / Concentrations:
0, 2.5, 5.5, 10, 30 and 50 mg/kg
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
50 mg/kg NaOH
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on week days, once daily in weekends and holidays

DETAILED CLINICAL OBSERVATIONS: yes, at least weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: one day pre-dosing, weekly during treatment and terminal body weight

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of sacrifice
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes, overnight (appr. 18 hours)
- How many animals: all surviving animals
- Parameters according to guideline (OECD 410, 1981) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:on day of sacrifice
- Animals fasted: Yes, overnight (appr. 18 hours)
- How many animals: all surviving animals
- Parameters according to guideline (OECD 410, 1981) were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight prior to scheduled sacrifice
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: urine volume, levels of sodium and potassium, color, appearance, refractive index, specific gravity, microscopic evaluation, and qualitative measurement of glucose, urobilinogen, pH, protein, ketones, blood, nitrites, leukocytes, creatinine and bilirubin.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
Complete necropsy was performed on all rats, gross necropsy was performed on the 5% TMAH rats or rats not surviving at least two days of TMAH exposure.
HISTOPATHOLOGY: Yes.
Tissues according to guideline were collected from rats that were designated for complete necropsy. A complete set of tissues from all rats in the vehicle control group and in the two highest surviving dose groups (3% and 1% TMAH) were examined microscopically.
In addition, the skins (application site) in the 0.55% and 0.25% TMAH groups were also examined.
Adrenals, brain, heart, kidneys, liver, ovaries, spleen and testes were weighed, and the organ to fasted body weight ratios were calculated.
Other examinations:
On study days 2 and 24, blood samples for determination of acetylcholinesterase (AChE) activity were collected via the retroorbital sinus from anesthetized (70% CO2/30% air) rats at least two hours after application of dosing formulations.
Statistics:
Statistical analysis of continuous data was performed using analysis of variance followed, where appropriate, by Dunnett's test. All comparisons were performed using the vehicle control exposed animals as the control group. A minimum significance level of p≤0.05 was used for all comparisons.
Clinical signs:
effects observed, treatment-related
Dermal irritation:
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
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
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:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
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. 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.
BODY WEIGHT AND WEIGHT GAIN
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
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.
HAEMATOLOGY
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 CHEMISTRY
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
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 tretaed 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 WEIGHTS
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 PATHOLOGY
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. 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.
HISTOPATHOLOGY
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.
OTHER FINDINGS
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:
test mat.
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:
2.5 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Squamous epithelial hyperplasia, inflammation (acute, subacute or chronic active) and necrosis seen at 5.5 mg/kg bw/ day at application site.
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
5.5 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Squamous epithelial hyperplasia and inflammation (acute, subacute or chronic active) and necrosis seen at 10 mg/kg bw/ day at application site.
Critical effects observed:
not specified

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 th 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.
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 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.

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 is available, conducted according to OECD guideline and GLP principles. A second study with reliability 4 presented no clear data on local effects and lacked exminations on organ weights and microscopy.

Additional information

Oral exposure:

A 28-days oral repeated dose experiment was conducted according to OECD guideline 407 and GLP principles. No deaths were observed at the concentrations tested (5, 10 and 20 mg/ kg bw/d).A significant decrease in food consumption was observed in the first week of administration in male animals at 10 mg/kg bw/day, and male and female animals at 20 mg/kg bw/day. A decreased absolute and relative heart weight without dose-response and no correlated histopathological findings was observed at 10 mg/kg bw/day and higher in males only. This effect was not seen at the end of the recovery period. Therefore, this effect was not considered to be toxicologically relevant for the time being, awaiting further data.

The NOAEL for repeated dose oral toxicity was considered to be 5 mg/kg bw/day for males and 10 mg/kg bw/day for females.

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.

In another dermal repeated dose study (reliability 4) male rats were dosed with 0, 5.5, 50, 120 and 250 mg/kg bw/d and female rats with 0, 2.5, 5.5, 10 and 50 mg/kg bw/d TMAH (5d/w; 6h/d) during 28 days. All rats exposed to 50 mg/kg bw/d or more died. Skin irritation at the application site was seen in all groups treated with the test substance and included erythema, edema and/or scabbing. Clinical signs noted in a few of the rats at 120 and 250 mg/kg bw were lethargy followed by convulsions, tremors, and death beginning within one and a half to 2 hours after administration of the first dose of the test substance. No overt clinical signs of toxicity were observed at the lower doses (males; 5.5 mg/kg bw: females; 2.5, 5.5 or 10 mg/kg bw). No significant differences were seen in body weights, body weight gains or food consumption at any time during the study. No overt changes in blood hematology or chemistry were seen. Red ovaries were observed in all surviving rats at the 10 mg/kg bw (10/10), 4/10 at the 5.5 mg/kg bw and 2/10 at 50 mg/kg bw dose levels. Based on these data, a systemic NOAEL of 2.5 mg/kg bw for females and 5.5 mg/kg bw for males was established for TMAH.


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.

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

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

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 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, with thymic lymphoid necrosis, hepatocyte vacuolation with eosinophilic, intracytoplasmic material, and hepatocyte necrosis at 30 mg/ kg bw/ day. In another 28-day rep dose study all rats died at 50 mg/kg bw and higher. Therefore, based on the available data, TMAH is classified STOT-RE 1 for dermal exposure according to CLP Regulation (EC) No. 1272/2008. Relevant target organs are thymus, and liver.