Tetramethylammonium hydroxide

Endpoint summary

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

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Rationale for read-across: see attached read-across document in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were noted in control rats. In animals at 30 mg/kg bw/day, clinical signs consisted of lethargy, hunched and/or flat posture, piloerection and ptosis. In addition, abdominal swelling was noted at this dose level in females only. The animals at 10 mg/kg bw/day showed lethargy, hunched posture, piloerection and/or ptosis at a lower incidence and severity compared to animals at 30 mg/kg bw/day. At 3 mg/kg bw/day, hunched posture and piloerection were noted at low incidence in males only. Salivation seen after dosing among the animals at 10 and 30 mg/kg bw/day was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). No additional clinical signs during weekly arena observations were noted during the observation period that were considered to be related to treatment. No clinical signs were noted in the deceased rats.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two males treated at 30 mg/kg bw/ day were found dead at day 57 and day 80, respectively (with autolysis in several organs or missing organs (head region) due to cannibalism).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated at 30 mg/kg bw/day showed lower body weight (statistically significant from week 6 to end of study, appr. -12% for both absolute and relative values at study termination compared to controls) and body weight gain (statistically significant in week 2 and from week 7 to end of study). Body weights and body weight gain of female animals remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Absolute food consumption of males treated at 30 mg/kg bw/day was found to be lower compared to controls during the second half of the treatment period. No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for females.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No substance-related adverse effects were seen in heamatological parameters for exposed males compared to control group. A statistically significant decrease of prothrombin time in males dosed at 3 mg/kg bw/day was considered to be an incidental effect and not to be substance-related. In high dose females, the percentage lymphocytes was found to be decreased (-5.5%), with an increased percentage of monocytes (+29%), both effects were statistically significant. In absence of a clear dose-relationship of the effects, this was not found to be adverse.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males exposed to 30 mg/kg bw/day showed statistically significant increase in alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels (respectively +67% and +20% compared to controls). ALAT levels were also increased in high dose females (+86%), and found together with increased alkaline phosphatase levels (+82%, both statistically significant).
Furthermore, concentration bilirubin was increased in high dose males (+23%) and females (+19%). Glucose levels were decreased in high dose males (-13%). A dose-related increase in bile acids was observed for all exposed males (+50%, +150% and +719% for males dosed at 3, 10 and 30 mg/kg bw/day, respectively, statistically significant only at highest dose). This increase was also seen in females with +38%, +83% and +1560% at respectively 3, 10 and 30 mg/kg bw/day (statistically significant only at highest dose). Furthermore, effects on ionic balance were noted in males (increased sodium levels at 3 and 10 mg/kg bw/day, dose related increased potassium (only statistically signficant at 30 mg/kg bw/day) and increased inorganic phophate levels at 30 mg/kg bw/ day) and females (increased potassium at highest dose). In high dose females, also effects were noted on total protein and albumin (both decreased with -11%, statistically significant effect). Cholesterol levels increased for all exposed females (+5%, +5% and +24% (statistically significant only at high dose).

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Females exposed to 30 mg/kg bw/day showed statistically significant reduction in total movements (-73.5%) and ambulations (-83%), no such effect was seen in males. For males, grip strenth was decreased for all exposed rats compared to control group (appr. -23% and -21%, -27% and -17%, -29% and -20% for respectively fore and hind grip at 3, 10 and 30 mg/kg bw/day), however these effects were not statistically significant. Grip strenth was not affected in females.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females dosed at 30 mg/kg bw/day were found to have increased liver weights (appr. +23% and +25% for absolute and relative values respectively, both statistically significant). In parallel, relative liver weight was also slightly increased in males in the highest dose group (+7%, statistically significant). Lower thymus weights were noted in females (18% for absolute and 17% for relative weight, both statistically significant) at 30 mg/kg bw/day.
Other statistically significant changes in organ weights at the end of the treatment were considered to be of no toxicological significance as they remained within the range considered normal for rats of this age and strain or were in males considered to be the result of a test item-related effect on final body weight. These observations included effects on relative testes weights which were affected in a dose related manner compared to control males with +3%, +11% and +13% respectively for rats exposed to 3, 10 and 30 mg/kg bw/day (statistically significant only for two highest dose groups). Further observations included decreased weight of the brain in males only at 30 mg/kg bw/day (appr. -4% for absolute and +7.5% relative to body weight, both statistically significant). Relative kidneys weights were slightly increased for males in the highest dose group (+11%, statistically significant). At 10 mg/kg bw/ day, absolute and relative ovary weights were increased, but in absence of dose-relationship this was found to be not substance-related. No other findings were noted in males and females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Emaciation was noted in one female at 30 mg/kg bw/day at macroscopic examination. Swelling of the abdomen was noted for several rats. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of the two animals deceased before scheduled sacrifice was only possible to a limited agree due to the status. Findings consisted of many dark red foci of the thymus (correlated with congestion/hemorrhage) and many dark red foci of the thymus in one of the two rats. Therefore conclusion on cause of pre-scheduled deaths was not possible.
In all exposed females microscopic findings were noted in the liver, consisting of hepatocellular hypertrophy at minimal degree. The effects were found in 4/10 females at 3 and 10 mg/kg bw/day and in 8/10 females exposed at 30 mg/kg bw/day (all grade 1, minimal effects). Due to the fact that this effect was present at low severity (minimal) and in absence of effects in other parameters this observation was considered to be non-adverse at 3 and 10 mg/kg bw/day. In the thymus of 2/10 females at 30 mg/kg bw/day atrophy at minimal degree was observed. Based on the low severity, this effect was found to be non-adverse. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

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

Executive summary:

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

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

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

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 or test system 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

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 or test system 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.

A 90-days oral repeated dose toxicity study was conducted with the analogue substance TMAC, according to OECD guideline 408 and in accordance with GLP principles. Male and female rats were exposed to 0, 3, 10 and 30 mg TMAC/kg bw/day via oral gavage. Two males exposed to 30 mg/kg bw/day died during the study, on exposure days 57 and 80, respectively. In absence of clinical signs, effects on body weight and/or food consumption and observations during necropsy, no cause of death could be established. In the surviving exposed rats clinical signs consisted of lethargy, hunched and/or flat posture, piloerection and ptosis, especially at the highest dose. Severity and incidence decreased with dose. Males treated at 30 mg/kg bw/day showed statistically significant lower body weight (gain). Rats exposed to 30 mg/kg bw/day showed increase in alanine aminotransferase (males and females), aspartate aminotransferase (males only) levels, increased alkaline phosphatase levels (females only), bilirubin and bile acid levels. Cholesterol levels decreased for females treated with 30 mg/kg bw/day only. Females exposed to 30 mg/kg bw/day showed statistically significant reduction in total movements and ambulations, no other functional effects were observed. Females dosed at 30 mg/kg bw/day were found to have statistically significantly increased relative and absolute liver weights and lower thymus weights. Histopathology revealed hepatocellular hypertrophy in 4/10 females at 3 and 10 mg/kg bw/day and in 8/10 females exposed at 30 mg/kg bw/day (all grade 1, minimal effects). Due to the fact that this effect was present at low severity at 3 and 10 mg/kg/day and in absence of effects in other parameters this observation was considered to be non-adverse at these dose levels. In the thymus of females at 30 mg/kg bw/day atrophy at minimal degree was observed in 2/10 females with low severity. The findings in this study confirm the effects that were observed in the 28-days repeated dose oral toxicity study with TMAH. Taken all data together, the NOAEL of TMAC was determined to be 10 mg/kg bw/day based on adverse effects at 30 mg/kg bw/day.

Overall, the NOAEL for repeated dose oral toxicity for TMAH was considered to be 5 mg/kg bw/day for males and 10 mg/kg bw/day for females. The NOAEL was based on the 28-days oral repeated dose toxicity study, which is considered to be worst case.

 

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 bw/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 bw/day (males) and 30 mg/kg bw/day (males and females) and significant decreases in urine creatinine values at 10 and 30 mg/kg bw/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 bw/day TMAH. Inflammation, necrosis and squamous epithelial hyperplasia of the skin was found at 10 mg/kg bw/day in males and 5.5 mg/kg bw/day in females. Due to the significant effects on the skin a NOAEL for local effects was determined to be 5.5 mg/kg bw/day for males and 2.5 mg/kg bw/day for females. The NOAEL for systemic toxicity was established to be 10 mg/kg bw/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/day and female rats with 0, 2.5, 5.5, 10 and 50 mg/kg bw/day TMAH (5d/w; 6h/d) during 28 days. All rats exposed to 50 mg/kg bw/day 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/day 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/day: females; 2.5, 5.5 or 10 mg/kg bw/day). 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/day (10/10), 4/10 at the 5.5 mg/kg bw/day and 2/10 at 50 mg/kg bw/day dose levels. Based on these data, a systemic NOAEL of 2.5 mg/kg bw/day for females and 5.5 mg/kg bw/day 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 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/day, 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/day 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.