2-(2-aminoethoxy)ethanol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2001 - 28 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Due to mortality in the high dose group (250 mg/kg) assigned to both the 48-hour harvest and the replacement animal groups, only four animals were available for harvest. This deviation had no impact on the integrity of this study.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-aminoethoxy)ethanol

EC Number:

213-195-4

EC Name:

2-(2-aminoethoxy)ethanol

Cas Number:

929-06-6

Molecular formula:

C4H11NO2

IUPAC Name:

2-(2-aminoethoxy)ethan-1-ol

Specific details on test material used for the study:

- Name of test material (as cited in study report): Diglycolamine (DGA)
- Physical state: transparent colorless liquid
- Analytical purity: responsability of the Sponsor
- Lot/batch No.: Lot 8043-70, recieved on 2001-03-28
- Storage condition of test material: ambient

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: CD-1 (ICR) BR strain
- Source: Charles River Laboratories, Kingston, NY (dose range finding study) + Charles River Laboratories, Raleigh, NC (definitive study)
- Age at study initiation: young adult mice, 7 weeks
- Weight at study initiation: 20-40g (weight variation did not exceed 20% of the mean weight of each sex). 28.4 -33.5 g in the final study.
- Assigned to test groups randomly, by a computer program
- Fasting period before study: no
- Housing: sanitary, polycarbonate cages containing Sani-Chips hardwood Chip Laboratory bedding.
- Housed separated by gender, up to 5 animals per cage during acclimation, and full dose group after randomization.
- Diet (e.g. ad libitum): commercial diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79 °F
- Humidity (%): 30-70%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark, 12 hrs light


IN-LIFE DATES: From: 17 April 2001 To: 08 May 2001

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % CMC (carboxymethyl cellulose)

Details on exposure:

one single intraperitoneal dose per mouse

Duration of treatment / exposure:

The animals were treated once and samples of bone marrow were taken 24 h and 48 h after the treatment.

Frequency of treatment:

The animals were treated once.

Post exposure period:

1st dose ranging study: 24h
2nd dose ranging study: 48h
final study: 24h and 48h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw (total dose)

No. of animals per sex per dose:

6 male animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

- Cyclophosphamide, dissolved in sterile deionized water
- Route of administration: oral, gavage
- Doses / concentrations: 80 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow of femur.
At least 2000 PCEs per animal were analyzed for the frequency of mironuclei. Cytotoxicity was assessed by scoring the numer of PCEs and normochromic erythrocytes (NCEs) in at least 500 erythrocytes for each animals.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
based on first dose range finding study with following doses: 500, 1000, 2000 mg/kg bw: all animals were found dead after 24h.
a second dose range finding study was performed with following doses: 62.5, 125, 250 mg/kg bw (3 animals per dose): animals were observed immediately after dosing, one hour after dosing and daily (for 2 days). 2 animals of the highest dose group died. Based on these results, the maximum tolerated dose was estimated to be 250 mg/kg bw.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Only males were used because there were no substantial differences in between the sexes in the dose range finding study.
Animals were dosed on April 30, 2001. Per dosing group (62.5, 125, 250 mg/kg, vehicle control and positive control), cells were harvested at 24 h after dosing. For both the 250 mg/kg and the vehicle control group, cells were harvested at 48 h after dosing in an extra group of 6 mice.
Animals were euthanized by CO2 inhalation followed by incision of the diaphragm.

DETAILS OF SLIDE PREPARATION:
Hind limb bones (tibias) were removed for marrow extraction. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).
Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in May-Grünwald solution followed by Giemsa, and protected by permanently mounted coverslips.

METHOD OF ANALYSIS:
Slides were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent icronucleated cells) was determined by analysing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE: NCE ratio was determined by scoring the number of PCEs and NCEs observed scoring at least the first 500 erythrocytes per animal.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although amond- and ring-shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and generally between 1/20th and 1/5th the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus.
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test article that did not induce both of these responses as considered negative. Statistical significance was not the only determinant of a positive response. Biological relevance of the results were also considered in the final evaluation.

Statistics:

Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogenous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was statistically significant (p <= 0.05), a Dunnet's t-test was used to determine which groups, if any, were statistically significantly different from the vehical control. Analyses were performed separately for each sampling time.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

signs of toxicity and mortality

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF first RANGE-FINDING STUDY
- Dose range: 500, 1000, 2000 mg/kg
- Clinical signs of toxicity in test animals: animals were found dead within 24h

RESULTS OF second RANGE-FINDING STUDY
- Dose range: 62.5, 125, 250 mg/kg
- Clinical signs of toxicity in test animals: no clinical signs in the lowest dosing groups (62.5 and 125 mg/kg). In the highest dosing group, slightly hypoactive 1h after dosing, rough haircoat 24h after dosing (+ 1/6 animals was found dead), clear discharge from eyes, hunched, labored respiration, hypoactive 48h after dosing.
- the maximum tolerated dose was estimated to be 250 mg/kg.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction
- % micronucleated PCEs (mean of 2000 per animal +/- S.E.):
62.5 mg/kg (24h harvest): 0.06 +- 0.02
125 mg/kg (24h harvest): 0.06 +- 0.02
250 mg/kg (24h harvest): 0.07 +- 0.03
250 mg/kg (48h harvest): 0.21 +- 0.08
vehicle control (24h harvest): 0.09 +- 0.04
vehicle control (48h harvest): 0.12 +- 0.03
positive control (24h harvest) 1.17 +- 0.12

- Appropriateness of dose levels and route: appropriate

- Statistical evaluation: the test article showed a statistically significant decrease in the PCE:NCE ratio at the 250 mg/kg dose level for the 24h harvest timepoint (cytotoxic to bone marrow). A statistically significant increase in micronucleated PCEs was not observed at any dose level or harvest timepoint. The positive control induced statistically significant increases in micronucleated PCEs as compared to that of the vehicle controls with a mean standard error of 1.17 +- 0.12 %.

Any other information on results incl. tables

Micronucleus Data Summary Table

 

Treatment	Dose	Harvest Time	% Micronucleated PCEs mean of 2000^a Per animals ± S.E. males	Ratio PCE:NCE Mean ± S.E. Males
Controls
Vehicle	0.5 % CMC	24 hr	0.09 ± 0.04	0.67 ± 0.09
		48 hr	0.12 ± 0.03	0.51 ± 0.02
Positive	CP 80 mg/kg	24 hr	1.17 ± 0.12*	0.55 ± 0.06
Test article	62.5 mg/kg	24 hr	0.06 ± 0.02	0.61 ± 0.05
	125 mg/kg	24 hr	0.06 ± 0.02	0.48 ± 0.05
	250 mg/kg	24 hr	0.07 ± 0.03	0.41 ± 0.04**
		48 hr	0.21 ± 0.08	0.41 ± 0.05

 

 

*Significantly greater than the corresponding vehicle control, ps0.01.

** Significantly less than the corresponding vehicle control, ps0.05.

^aOne animal from the 24-hour vehicle control group and two animais from the 62.5 mg/kg dose goup were scored out of >2000 PCE/animal. See individual animal data, Table 2.

CMC = Carboxymethy1 cellulose

CP = Cyclophosphamide

PCE = Polychromatic erythrocyte

NCE = Normochromatic erythrocyt

Applicant's summary and conclusion

Conclusions:

The test article, Diglycolamine (DGA), was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.