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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-05-28 to 1993-01-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The GLP study was conducted according to internal study methods (BRRC Project No. 91-15-18271) and in accordance with Organization for Economic Cooperation and Development Guidelines for Testing Chemicals Health Effects Test Guidelines, Office of Toxic Substances, U. S. Environmental Protection Agency (TSCA) 40 CFR Part 798.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The study was conducted according to internal study methods (BRRC Project No. 91-15-18271) and in accordance with Organization for Economic Cooperation and Development Guidelines for Testing Chemicals Health Effects Test Guidelines, Office of Toxic Substances, U. S. Environmental Protection Agency (TSCA) 40 CFR Part 798.

GLP compliance:

yes

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

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 actuve substance
- Physical state: Clear liquid
- Analytical purity: 96.2%
- Storage condition of test material: Stored at room temperature
- Lot/batch No.: Lot No. 84505, Sample No. 54-109
- Impurities (identity and concentrations): 0.5% DMEA, 0.08% 117 MW Amine, 1.7% 133 MW Dimethylamine, 0.06% 146 MW Amine, 0.03% 160 MW Amine, 0.9% DMAE- Dimethylaminoethyl ethanediamine (EDA), 0.15% 174 MW Amine (combined), 0.3% Other purities.
- Composition of test material, percentage of components: 96.2 Test substance, 0.5% DMEA, 0.08% 117 MW Amine, 1.7% 133 MW Dimethylamine, 0.06% 146 MW Amine, 0.03% 160 MW Amine, 0.9% DMAE- Dimethylaminoethyl ethanediamine (EDA), 0.15% 174 MW Amine (combined), 0.3% Other purities.

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, (Portage, MI)
- Age at study initiation: Approximately 5 weeks old
- Weight at study initiation: 23.5 to 28.8 g for males and 19.7 to 22.6 g for females
- Assigned to test groups randomly: [Yes, under following basis: Following the pretest body weight, the animals were assigned to 3 treatment groups, a vehicle control group, and a positive control group using a nonstratified randomization procedure based on body weight. At the time of group assignment, only animals with body weights within two standard deviations of the population mean for each sex were included.]
- Fasting period before study: N/A
- Housing: Group housed in shoe-box type plastic cages (30.0 X 20.0 X 12.5 cm)
- Diet (e.g. ad libitum): Pelleted, certified AGWAY PROLAB Animal Diet Rat, Mouse, Hamster 3000 (Agway Inc.) was available d libitum
- Water (e.g. ad libitum): Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) was available ad libitum
- Acclimation period: Approximately 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 4 deg C
- Humidity (%): 40-70%
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): 12-hour photoperiod (approximately 0500 to 1700 hours for the light phase)

IN-LIFE DATES: From: 1991-05-27 To: 1991-06-07

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Sterile, distilled, deionized water
- Justification for choice of solvent/vehicle: N/A
- Concentration of test material in vehicle: N/A
- Amount of vehicle (if gavage or dermal): 10 ml/kg
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required): BRRC Sample Nos. 53-101A through U
- Purity: N/A

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Each test substance dosing solution was prepared by diluting the test substance in water. Concentrations were not adjusted for percent active ingredient of test substance. They were prepared on the day of treatment and were stored at room temperature prior to use. The TEM positive control dosing solution was prepared by dissolving it in ethanol then diluting it with sterile, distilled, deionized water. It was
prepared on the day of treatment and was stored at room temperature prior to use.

DIET PREPARATION
- Rate of preparation of diet (frequency): On the day of treatment
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A

- Justification for dose concentration: Based upon mortality data obtained in a range-finding study, the acute intraperitoneal LD50 for the combined sexes was calculated to be 181 mg/kg (no 95% confidence interval was calculated since none of the dose levels had partial survival). The doses for the definitive micronucleus assay were selected by the study director as approximately 25%, 50%, and 80% of the LD50 or 45, 90, and 145 mg/kg of the test substance.

Duration of treatment / exposure:

72 hours

Frequency of treatment:

Single dose

Post exposure period:

3 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

45 mg/kg diet

Remarks:

nominal concentration

Doses / concentrations 2

Dose / conc.:

90 mg/kg diet

Remarks:

nominal concentration

Doses / concentrations 3

Dose / conc.:

145 mg/kg diet

Remarks:

nominal concentration

No. of animals per sex per dose:

5/sex/ treated with 45 or 90 mg/kg
8/sex/treated with 145 mg/kg
5/sex/treated with either the vehicle control (water, 10.0 ml/kg) or positive control (triethylenemelamine, 0.30 mg/kg)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: triethylenemelamine (Lot No. 84505, BRRC Sample No. 52-793)
- Justification for choice of positive control(s): Consistently produced positive responses in prior tests; Peripheral blood was collected 30 hours
after treatment.
- Route of administration: Single intraperitoneal injection
- Doses / concentrations: 0.30 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow, polychromatophilic erythrocytes (PCE) and normochromic erythrocytes (NCE).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The test substance doses of approximately 25%, 50%, and 80% of the LD50, or 45, 90, and 145 mg/kg were selected by the Study Director and were based upon the results of the range-finding study that was a part of this experimental protocol.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Blood samples were collected approximately 30, 48 and 72 hours after dosing. Blood samples were collected from the positive control animals approximately 30 hr after dosing.

DETAILS OF SLIDE PREPARATION: Slides were coded with the randomly-assigned animal numbers and read without knowledge of the treatment group to prevent bias.

METHOD OF ANALYSIS: Blood samples of one to a few drops were collected by nicking the tail with a scalpel. One or two blood smears were made per animal per sampling time. Micronuclei in peripheral polychromatic erythrocytes were stained with Curres 8-66 Ciemsa diluted in phosphate buffer. Slides were coded with the randomly-assigned animal numbers and read without knowledge of the treatment group to prevent bias. The PCE:NCE ratio for approximately 1000 total cells will be calculated, recorded, and shown in the final report to provide an estimate of the cytotoxicity of the test agent. Excessive cytotoxicity in this test system will be defined by a PCE:NCE ratio of 0.01 or lower. Mice with an excessive reduction in the PCE:NCE ratio will be identified in the raw data and final report.

OTHER: N/A

Evaluation criteria:

One to a few drops of blood were collected from the tail by nicking it with a scalpel. One or two blood smear slides were prepared/animal/sampling time. Micronuclei in peripheral blood polychromatophilic erythrocytes were stained with Gurr's R-66 Giemsa diluted in phosphate buffer. Slides were coded with the randomly-assigned animal number and read without knowledge of treatment group to prevent bias. Slides were identified by BRRC project number, animal number and sampling interval.

The PCE:NCE ratio for a total of 1000 cells for each animal was calculated to provide an estimate of test substance cytotoxicity. A minimum of 1000 PCE for each animal were scored for the presence of micronuclei unless the cytotoxicity of the test substance prevented this.

Statistics:

Data were statistically evaluated using the Mann-Whitney U test. All statistical analyses were performed using BMDP Statistical Software. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance. Various models of calculators, computers, and computer programs may have been used to analyze data for this study. Since various models round or truncate numbers differently, values in some tables may differ slightly from those in other tables or from independently calculated data. The integrity of the study and interpretation of the data were unaffected by these differences.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 32, 64, 128,256 and 512 mg/kg
- Solubility: N/A
- Clinical signs of toxicity in test animals: The aniamls were observed for signs of toxic and/or pharmacological effects for 72 hr after dosing. Blood was taken from the tail vein at 48 hr and 72 hr after dosing from the 128 mg/kg and water control animals, and was used for determining the ratio of polychromatophilic erythrocytes (PCE) to normochromic erythrocytes (NCE). Body weights were measured before dosing and before sacrifice on the fourth day after dosing (approximately 72 hr postdosing), 5 of 5 males and females were found dead in the 256 and 512 mg/kg dose groups after 72 hours. By the end of the 72 hr observation period, the body weights of the 128 mg/kg males were statistically significantly reduced; whilst those of the females of this dosage group were also reduced, this was not statistically significant. By 72 hr, body weight gains for both males and females were reduced.
- Evidence of cytotoxicity in tissue analyzed: 48 hr sample for 0 mg/kg= 27.6 +/- 2.1 PCE/NCE (male) and 38.0 +/- 8.0 PCE/NCE (female); 72 hr sample for 0 mg/kg= 27.0 +/- 2.6 PCE/NC (male) and 33.0 +/- 6.2 PCE/NCE; 48 hr for 128 mg/kg= 28.6 +/- 5.0 (male) and 35.8 +/- 9.6 (female); 72 hr for 128 mg/kg= 26.4 +/- 5.6 (male) and 29.4 +/- 8.8 (female). The findings showed no evidence for bone marrow toxicity from the test substance given ip at a dosage of 128 mg/kg.

- Rationale for exposure: N/A
- Harvest times: 48 and 72 hours
- High dose with and without activation: N/A
- Other: The control group received ip distilled water (10 ml/kg).

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): N/A
- Induction of micronuclei (for Micronucleus assay): Blood samples of one to a few drops were collected by nicking the tail with a scalpel. One or two blood smears were made per animal per sampling time. Micronuclei in peripheral polychromatic erythrocytes were stained with Curres 8-66 Ciemsa diluted in phosphate buffer. Slides were coded with the randomly-assigned animal numbers and read without knowledge of the treatment group to prevent bias. The PCE:NCE ratio for approximately 1000 total cells will be calculated, recorded, and shown in the final report to provide an estimate of the cytotoxicity of the test agent. Excessive cytotoxicity in this test system will be defined by a PCE:NCE ratio of 0.01 or lower. Mice with an excessive reduction in the PCE:NCE ratio will be identified in the raw data and final report.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of PCE/NCE was determined to assess any bone marrow cytotoxicity, and the number of micronucleated erythrocytes was counted and expressed as the proportion of polychromatophilic erythrocytes with micronuclei. There were no significant decreases in PCE/NCE ratios at any dosage, indicating an absence of bone marrow cytotoxicity. There were no significant changes in the incidence of micronucleated polychromatophils with either sex at any dosage, when compared with the water-alone controls. The positive control, TEM, produced highly statistically significant increases in micronucleated polychromatophils with evidence of cytotoxicity.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: See Statistics section above

Any other information on results incl. tables

The above dosage-mortality data allowed the calculation of a 72 hr-ip LD50 of 181 mg kg- for both male and female Swiss-Webster mice. By the end of the 72 hr observation period, the body weights of the 128 mg kg males were statistically significantly reduced; whilst those of the females of this dosage group were also reduced, this was not statistically significant. By 72 hr, body weight gains for both males and females were reduced.

Results of the mouse peripheral blood micronucleus test with the test substance given by intraperitoneal injection

Group	Dosage	Sex	PCE/NCE (% Controls)^a			Micronuclei (% PCE with MN)
			30 hr	48 hr	72 hr	30 hr	48 hr	72 hr
Water^b	10 ml/kg	Male				0.40	0.30	0.44
		Female	-	-		0.24	0.12	0.24
Test substance	45 ml/kg	Male	113.0	126.4	101.5	0.46	0.26	0.34
		Female	106.4	107.8	104.2	0.28	0.3	0.16
	90 ml/kg	Male	111.6	114.4	112.5	0.34	0.20	0.34
		Female	88.5	97.4	109.7	0.28	0.02	0.36
	145 ml/kg	Male	103.4	116.8	105.9	0.48	0.26	0.42
		Female	91.1	94.2	113.2	0.20	0.16	0.24
Positive control^c	0.3 ml/kg	Male	46.6		-	2.90
		Female	39.5			2.10

^ARatio of polychromatophilic erythrocytes to normochromic erythrocytes as % of control

(water) values.

^BControl group.

^CTEM = triethylenemelamine positive control (30 hr sample only).

Applicant's summary and conclusion

Conclusions:

The test substance did not produce significant, treatment-related increases in the incidence of micronucleated polychromatophilic erythrocytes among male or female Swiss-Webster mice assessed at concentrations of 45, 90, or 145 mg/kg did not produce significant, treatment-related increases in the incidence of micronucleated polychromatophilic erythrocytes among male or female Swiss-Webster mice assessed at 30, 48, or 72 hours after treatment with a single dose by intraperitoneal injection. Therefore, the test substance was not considered to be an inducer of micronuclei in male or female Swiss-Webster mice under the conditions of this in vivo assay.