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Repeated dose toxicity: dermal

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Administrative data

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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetramethylammonium hydroxide (TMAH)
- Substance type: aqueous solution (25%)
- Physical state: clear liquid
- Analytical purity: not reported
- Lot/batch No.: Run No. 10806113
- Specific density: 1.014 g/ml at 20°C
- Storage: in septum-capped containers at room temperature (appr. 22°C) under a hood

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.