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

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1992-12-07 to 1993-04-16

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study was conducted according to specific laboratory protocol which was well documented and any protocol changes were amended and approved by the study director.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1994

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

This study consisted of 3 exposure groups and a control group, each having 5 female rabbits. Animals were exposed for 2 hours to either the test substance vapor or filtered air. Observations for clinical signs of toxicity, body weight, and gross pathology will be evaluated. Corneal thickness was measured in both eyes at several time periods during the study.

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

other: Corneal Thickness

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: Light yellow liquid
- Analytical purity: 98.5%
- Stability under test conditions: Stable at room temperature for the duration of the study.
- Storage condition of test material: The test substance was stored under well ventilated conditions at ambient temperature in an appropriate storage area.
- Other: One pint of test substance was used.
- Lot/batch No.: Lot No. 287389; BRRC Number 55-336
- Impurities (identity and concentrations): 0.06% dimethyl formamide, 1.06% 133 mw DMEE, 0.44% DMEE, 0.18 146mw amine, 0.03% 130mw amine, 0.55% pentamethyldiethylenetriamine, 0.14% 174mw amine, 0.03% 160mw amine, 0.03% 160mw amine, 0.44% 174mw amine, 0.4% all other impurities.
- Composition of test material, percentage of components: 96.6% Test substance, 0.06% dimethyl formamide, 1.06% 133 mw DMEE, 0.44% DMEE, 0.18 146mw amine, 0.03% 130mw amine, 0.55% pentamethyldiethylenetriamine, 0.14% 174mw amine, 0.03% 160mw amine, 0.03% 160mw amine, 0.44% 174mw amine, 0.4% all other impurities.

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products, Inc., Denver, PA
- Age at study initiation: The animals were approximately 5.5 to 6 months of age on the scheduled animal receipt date.
- Weight at study initiation: Range 3815.7 - 5526.1 g
- Fasting period before study: No
- Housing: All animals were individually housed prior to the start of exposure and during the study in stainless steel cages with wire mesh floors will be used throughout all phases of the study.
- Diet (e.g. ad libitum): AGWAY PROLAB Animal Diet Rabbit (Agway Inc.) was available as libitum except during exposure.
- Water (e.g. ad libitum): Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) will be available ad libitum, except during exposure, by an automatic watering system with demand control valves mounted on each rack.
- Acclimation period: Two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.11 - 21.11 deg C
- Humidity (%): 40-70%
- Air changes (per hr): At least 10 air changes each hour
- Photoperiod (hrs dark / hrs light): 12-hour photoperiod (approximately 0500 to 1700 hours for the light phase).

IN-LIFE DATES: From: 1992-11-03 To: 1993-02-16 (last group sacrificed on 1993-02-19)

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Two stainless steel chambers with glass doors and windows for animal observation. The total "volume" of the animals shall not exceed 5% of the volume of the test chamber.
- Method of holding animals in test chamber: Whole body exposure
- Source and rate of air: Laboratory pump
- Method of conditioning air: N/A
- System of generating particulates/aerosols: The test substance was metered with an appropriate laboratory pump to a heated glass evaporator to produce the vapor. The vapor was diluted to the desired concentration in the evaporator with filtered chamber supply air.
- Temperature, humidity, pressure in air chamber: Temperature was maintained at 22 + 2 deg C, and relative humidity maintained between 40 to 60%.
- Air flow rate: 1 L/min
- Air change rate: Approximately 13-14 air changes each hour, adequate oxygen content of at least 19%.
- Method of particle size determination: N/A
- Treatment of exhaust air: N/A

TEST ATMOSPHERE
- Brief description of analytical method used: Compositional analysis of the test substance was performed by the Sponsor. The rate of airflow was monitored continuously and recorded approximately every 30 minutes. All chambers were maintained at a slight negative pressure to prevent any test substance from entering the room containing the chambers and analytical verification by gas chromatographic technique.
- Samples taken from breathing zone: yes, chamber probes for sampling will be placed in the breathing zone of the animals. The nominal (estimated) chamber concentration will also be determined

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of the test substance was determined approximately twice during the exposure (contingent on analytical limitations) by a gas chromatographic technique. The control chamber atmosphere was also analyzed.

Duration of treatment / exposure:

The animals were exposed continuously for 2 hours to either the test substance vapor or filtered air.

Frequency of treatment:

Single exposure

Post exposure period:

N/A

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

0.1 ppm (nominal)

Remarks:

group I

Doses / concentrations 3

Dose / conc.:

1 ppm (nominal)

Remarks:

group II

Doses / concentrations 4

Dose / conc.:

5 ppm (nominal)

Remarks:

group III

Doses / concentrations 5

Dose / conc.:

10 ppm (nominal)

Remarks:

group IV

Doses / concentrations 6

Dose / conc.:

12 ppm (nominal)

Remarks:

group V

Doses / concentrations 7

Dose / conc.:

15 ppm (nominal)

Remarks:

group VI

Doses / concentrations 8

Dose / conc.:

25 ppm (nominal)

Remarks:

group VII

Doses / concentrations 9

Dose / conc.:

30 ppm (nominal)

Remarks:

group VIII

No. of animals per sex per dose:

5 female animals/group for a total of 8 groups

Control animals:

yes

Details on study design:

Shortly after their arrival at the laboratory, the animals were transported to the room selected for the study. Once in the room, the animals were removed from the shipping cartons and examined. All animals with evidence of disease or physical abnormalities wer e discarded and the reason for rejection was recorded. If an unusually large number of animals shows evidence of disease or physical abnormalities, the entire shipment of animals would be rejected for use in the study. A total of 3 animals will be randomly selected for a pretest health screen.

During the acclimation period, animals were fed the same diet used during the study. Animals were observed twice daily for any clinical signs of disease or abnormality. Individual detailed physical examinations were conducted weekly.

Examinations

Examinations:

- Mortality and clinical observation: The animals were observed twice daily for any clinical signs of disease or abnormality; Animals will be observed on a group basis during the exposure period. Observed mortality and/or signs of toxicity will be recorded on the day observed.

- Body weight: Individual body weights were measured on the morning prior to the exposure and at the end of the study (just preceding sacrifice).

- Corneal thickness measurements: Corneal thickness was measured using a DGH-2000 ultrasonic pachymeter (DGH Technology). The corneal thickness was measured for both eyes of rabbits while restraining animals temporarily in a cat bag. The measurements were be made prior to the exposure and immediately following the exposure. In addition, the measurements will be made approximately at 1 hour or less (if feasible) and between 3-4 hours, 5-6 hours, and approximately at 24, 48, and 72 hours following exposure and continued until the measurement values are within the range of the pretest value.

- Eye irritation observations: Eye irritation readings were made at approximately 1, 3, 24, and 48 hours after exposure.

- Anatomic pathology examinations: After the last corneal thickness measurement (approximately 3 days following the exposure), all surviving animals were sacrificed by caudal ear vein injection of an overdose of sodium pentobarbital. All animals, including those that died or were sacrificed during the study, were given a complete necropsy. Eyes will be removed and fixed for possible future microscopic evaluations.

Positive control:

N/A

Results and discussion

Details on results:

Mean analytical concentrations of 4.6, 8.8, 12.2, 14.9, 21.1, and 24.8 ppm were measured for the 5, 10, 12, 15, 25, and 30 ppm target concentrations, respectively. The analytical values for the 0.1 and 1.0 ppm target concentration groups were determined to be 0.1 and 0.9 ppm, respectively. Because of sample line blockage, these latter two values were calculated from the nominal concentrations and analytical/nominal concentration ratios obtained for a similar trial exposure.

No mortality occurred during the study. No treatment-related effects on body weight were observed. The following clinical observations were noted post-exposure: perinasal wetness (215 animals in the 12 ppm group and all 5 animals in the 25 ppm group), and periocular encrustation (115 rabbits from the 15 ppm group).

No increases in corneal thickness were noted for the 0.1 and 1.0 ppm groups. However, corneal thickness was slightly decreased, especially at the 3- and 5-hr post-exposure periods for these groups; the significance of these decreases in unknown. A decrease in corneal thickness was observed in rabbits in the 5 ppm group at the 3- and 5-hr post-exposure period, followed by a slight increase at the 48-hr post-exposure period. In general, concentration-related increases in corneal thickness were observed immediately following and within one hour of exposure to 10 ppm and higher test substance vapor concentrations. The thickness generally reached a maximum level at the 3-hr post-exposure measurement period in these groups and generally decreased at the 24-hr post exposure period. Slight (marginal) increases in corneal thickness continued throughout the post exposure period.

No signs of eye irritation or corneal opacities were noted in the 0.1 or 1.0 ppm exposure groups. In rabbits exposed to 5 ppm test substance vapor, minor corneal opacity was noted. Rabbits exposed to between 10 and 30 ppm test substance vapor exhibited corneal opacity (generally dullness or no defined edge of cornea of the eye), redness, and discharge. These observations generally corresponded with the changes in thickness of the cornea of rabbits. However, there was no clear concentration-dependent response relationship observed between the eye irritation data and the changes in corneal thickness in these groups. No exposure-related effects were observed at necropsy in any group.

Applicant's summary and conclusion

Conclusions:

A 2-hr exposure of rabbits to the test substance vapor at concentrations between 8.8 and 24.8 ppm produced increased corneal thickness and eye irritation. Rabbits exposed to 5 ppm A-99 vapor demonstrated a slight change in corneal thickness. No increased corneal thickness was noted for rabbits exposed to 0.1 or 0.9 ppm of the test substance vapor.