N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-08-25 - 1984-11-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP study with methods similar to OECD 411 conducted according to internal laboratory methods.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

EC Number:

221-220-5

EC Name:

N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)

Cas Number:

3033-62-3

Molecular formula:

C8H20N2O

IUPAC Name:

{2-[2-(dimethylamino)ethoxy]ethyl}dimethylamine

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)
- Substance type: Pure active substance
- Physical state: liquid, yellow/transparent
- Analytical purity: 99.6%
- Lot/batch No.: Reference no. 51DKE115 / Sample no. 45-190

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rabbits (70 of each sex) received from Langshaw Farms, Augusta, MI.
- Age at study initiation: 12-13 wks
- Weight at study initiation: 2-3 kg
- Fasting period before study:
- Housing: Individually housed in stainless steel cages with wire floors
- Diet (e.g. ad libitum): Oregon State University diet provided by Langshaw Farms. For the first 5 days, each rabbit received 6 oz of diet; the next 5 days 7 oz received; the next 5 days 8 oz received. After study initiation food was available ad libitum.
- Water (e.g. ad libitum): Water (Municipal Authority of Westmoreland County, Greensburg, PA), ad libitum to all animals.
- Acclimation period: Approximately two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8-21.1 deg C
- Humidity (%): 40-60%
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A

IN-LIFE DATES: From: 1982-08-25 To: 1982-09-24

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: Deionized water

Details on exposure:

TEST SITE
- Area of exposure: Dorsal area of trunk
- % coverage: 25-30% of body area
- Type of wrap if used: 4-inch square, 8-ply gauze patch held together on the back by using strips of two sided tape.
- Time intervals for shavings or clipplings: Fur was clipped shortly before study initiation and trimmed as necessary during the study.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dose was wipped away witha gauze patch dampened with warm water and then blotted with a disposable wiper.
- Time after start of exposure: Six hours after dosing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.0 mL
- Concentration (if solution): 2.0%, 0.7%, 0.2%
- Constant volume or concentration used: yes, constant volume for several dose concentration groups.
- For solids, paste formed: no

VEHICLE
- Justification for use and choice of vehicle (if other than water): dieoniced water
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, certain rabbits because of their tendency to remove the sheeting and gauze patches, had extra tape placed over the polyethylene sheeting or wore Elizabethan collar during exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of all concentrations and deionized water were analyzed on a monthly basis.

Duration of treatment / exposure:

13 weeks (90 days)

Frequency of treatment:

6 hrs/day, 5 days/wk

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 other: % (nominal per unit area)

Remarks:

1.98 % analytical; male/female

Doses / concentrations 2

Dose / conc.:

0.7 other: % (nominal per unit area)

Remarks:

0.75% analytical; male/female

Doses / concentrations 3

Dose / conc.:

0.2 other: % (nominal per unit area)

Remarks:

0.25% analytical; male/female

Doses / concentrations 4

Dose / conc.:

0 other: %

No. of animals per sex per dose:

10/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure concentration levels for this 90-day dermal study were based on the results of a 9-day study where the test substance was given as aqueous dilutions in deionized water using constant volumes, and a water control group was used. Each rabbit received 1 mL/day of the dilutions. The concentrations of the test substance were 2.0% (8 mg/kg/day nominal), 0.7% (2.8 mg/kg/day nominal), and 0.2% (0.74 mg/kg/day nominal). All percentages were calculated on a weight/ weight basis. Water was chosen as the diluent and negative control substance because the test substance is soluble in water at the concentrations used. Since the lowest concentration in the 9-day study, 2.5% produced local and systemic toxcity, the upper concentration chosen for the subchronic study was below that concentration.
- Rationale for animal assignment (if not random): By a formal randomization system
- Rationale for selecting satellite groups: High dose and control groups for male and female rabbits were used to assess the reversibility of any induced toxic effects.
- Post-exposure recovery period in satellite groups: one month
- Section schedule rationale (if not random): At random

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (on weekdays)
- Cage side observations checked: Toxicologic and pharmaccologic signs and dermal irritation; mortality and morbidity only (weekends)

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: N/A

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Immediately prior to dosing

BODY WEIGHT: Yes
- Time schedule for examinations: First day of exposure, weekly thereafter and preceding sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes; measured weekly for males and twice weekly for females, after week 1. food consumption was not measured during the recovery period.

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
- Time schedule for examinations: N/A

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to study initiation and again before the 90-day sacrifice. Blood was also taken from the male rabbits prior the the recovery sacrificed, but not remale rabbits.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters checked: Hematocrit, hemoglobin, erythrocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total leukocyte count, differential leukocyte count, platelet count and reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to study initiation and again before the 90-day sacrifice. Blood was also taken from the male rabbits prior the the recovery sacrificed, but not remale rabbits.
- Animals fasted: No data
- How many animals: All animals
- Parameters checked: Glucose, creatinine, bilirubin (total), serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, albumin, total protein, globulin, gamma-glutamyl transpeptidase, creatine phosphokinase, calcium, sodium, potassium, chloride, total CO2 and phosphorus.

URINALYSIS: Yes
- Time schedule for collection of urine: For non-recovery male rabbits urine was collected during weeks 6, 10, and 13. Urine was collected from recovery male and female rabbits during week 12. Urine was collected from non-recovery female rabbits during weeks 5, 9, and 13. Urine was not collected from males or females during the recovery period or prior to the recovery sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Volume, specific gravity, total protein, glucose, ketones, bilirubin, blood, urobilinogen, and amount and nature of sediment.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, upon necropsy the following tissues were examined grossly and fixed in commercially prepared 10% neutral buffered formalin from all dosage groups and control groups: All lesions, Brain - 3 coronal sections (cerebrum, cerebellum, brain stem), Spinal cord* - 3 levels, Sciatic nerve, Eyes*, Pituitary, Salivary glands (submaxillary), Heart and aorta, Thymus, Thyroid (with parathyroids), Trachea, Lungs with mainstem bronchi, Esophagus
Stomach, Small and large intestine, Adrenals, Pancreas, Liver and gall bladder, Kidneys, Testes and epididymis (males), Prostate, Accessory sex glands, Uterus and ovaries (females), Mammary gland*, Urinary bladder, Spleen, Mediastinal or mesenteric lymph node, Sternum* with marrow, Gastrocnemius muscle, Any other target tissue, and Treated and untreated skin.
* Tissues with asterisk were examined microscopically only if indicated by other findings.

HISTOPATHOLOGY: Yes, Histopathological examination was limited to all tissues from the high dose and control groups and target tissues from the other groups (treated skin only).

Other examinations:

ORGAN WEIGHTS: At the 90-day sacrifice, weights were recorded for the liver, kidneys, brain, testes, and adrenals of all animals. Based on the evaluation of these weights, no organ weights were recorded at the recovery sacrifice.

Statistics:

For body weight changes, food consumption values, and organ weights, both absolute and relative to final body weight, group means were compared by use of Bartlett’s test for homogeneity of variances, and when Bartlett’s test showed homogeneity, a standard analysis of variance. If F for analysis of variance was significant, Duncan’s multiple range test was used to delineate which groups differed from the controls. If Bartlett’s test showed heterogeneity, Levene’s test for equal variances was used, and if variances were equal, a standard analysis of variance was performed. Then the analysis of variance test showed a signification F value, polled variance t-tests were used. When levene’s test showed unequal variances, means were compared by Welch and Brown-Forsythe analysis of variance test for groups with unequal variances. When either of these tests showed significance, separate variance t-tests were used to differentiate between groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

-The incidence of erythema, edema, desquamation and fissuring were directly related to the concentration of the test substance, which is reflected in the similar pattern of Draize scores. Open sores at the application site occurred primarily at the highest concentration.
- The signs of skin irritation subsided gradually after termination of treatment and in most cases had disappeared by the time of sacrifice of the recovery animals.

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Description (incidence):

- There were no deaths or signs of systemic toxicity among the male rabbits of any dose group or controls. Skin irritation in males was observed including: erythema, desquamation, edema and fissuring or cracking of the skin occurring in a dose-related pattern.
- One female rabbit was sacrificed moribund after 12 days on study and a second female was found dead after 20 days. All other female rabbits survived until schedule sacrifice. Clinical signs included erythema, edema, desquamation, and some fissuring.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- There were no treatment-related effects on body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- There were no treatment-related effects on food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- There was a statistically significant increase in absolute but not relative testicular weight at the 0.7% concentration. Since there was no significant effect at the highest concentration, the observed difference was not considered biologically significant. No other significant differences were observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- There were no biologically significant effects with respect to clinical pathology. Occasional statistically significant differences were considered not biologically important because they were not dose-related or because the differences occurred with respect to only one of the two control groups.
- No biologically significant gross lesions were observed. The only treatment-related lesions were found on the treated skin. Epidermal vacuolization occurred in 10 males and 10 females at the highest concentration (2%) of the test substance, and in 2 males and 7 females at 0.7% of the test substance. No lesions were observed in the control groups. Acanthosis occurred at approximately the same incidence in all the male dosage groups, but was more pronounced at the highest concentration of the test substance. Among the females the condition was twice as prevalent at the two highest concentrations of the test substance as in the controls. In addition, the lesion was generally more severe in the high dose animals than in the controls or lower dosage groups. The incidence of dermatitis was increased in females at all concentrations of the test substance, but the incidence among the treated males was not increased.
- No other treatment-related lesions were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 8 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: There was an absence of any indication of systemic toxicity at all dose levels. All dose levels tested resulted in varying levels of irritation at the exposure site.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Daily recurrent application of the test substance to intact skin of rabbits, over a period of 90 days at concentrations of 2.0, 0.7, and 0.2% produced local dermal inflammation (including: erythema, edema, desquamation, fissuring, and by microscopic examination epidermal vacuolization and acanthosis were observed) but no signs of toxicity.