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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Three in vitro genetic toxicity studies were run on DMEE and all reported negative findings, with and without metabolic activation (i.e., a Salmonella typhimurium/Escherichia coli Reverse Mutation Assay, a Chromosome Aberration Test in human lymphocytes, and an L5178Y Mouse Lymphoma Assay). In addition, an in vivo mouse micronucleus assay conducted according to EPA OPP 84-2 is available on a structurally similar substance, 2-(2-aminoethoxy)ethanol (CAS# 929-06-6). The in vivo study reported negative results as well.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
EPA OPP 84-2
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River laboratories, Kingston,NY
- Diet (e.g. ad libitum): ad libitum (PMI Feeds,Inc. Certified Rodent Diet #5002)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature : 64-79 °F ( 18-26 °C)
- Humidity (%): 30 - 70
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
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.
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 male animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
Tissues and cell types examined:
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: Pretest were performed

DETAILS OF SLIDE PREPARATION:
Preparation of Slides. Following centrifugation to pellet the tissue, the supematant 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. For control of bias, all slides were coded prior to analysis.
Slides prepared from the bone marrow collected from five animals per group, if available, at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing 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. The historical background frequency of micronucleated cells were expressed as percent micronucleated cells based on the number of PCEs analyzed. The historical background frequency of micronuclei in the Cr1:CD-lB(ICR) BR strain at this laboratory is about 0.0 to 0.4%, which is within the range reported in the published data (Salamone and Mavoumin, 1994). The critena for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond- and ring-shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and generally between one-twentieth and one-fifth the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure pennatted the differentiation by color of PCEs and NCEs (bluish-gray and red, respectively).


Evaluation criteria:
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 homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was statistically significant (p <0.05), a Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time. The crteria 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 was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevante of the results in the final evaluation.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

The test article, Diglycolamine (DGA), induced signs of clinical toxicity and mortality in the treated animals and was cytotoxic to the bone marrow (i.e., a statistically significant decrease in the PCE:NCE ratio) at the 250 mglkg dose level for the 24-hour harvest timepoint. A statistically significant increase in micronucleated PCEs was not observed at any dose level or harvest timepoint. The positive control, cyclophosphamide, induced statistically significant increases in micronucleated PCEs as compared to that of the vehicle controls with a mean and Standard error of 1.17 I 0.12%.

Micronucleus Data Summary Table

 

Treatment

Dose

Harvest Time

% Micronucleated PCEs mean of 2000a 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 animals 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

 

Conclusions:
Interpretation of results (migrated information): negative
The test article, Diglycolamine (DGA), was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Negative findings for DMEE were reported in three reliable and well-conducted in vitro studies (i.e., a Salmonella typhimurium/Escherichia coli Reverse Mutation Assay, a Chromosome Aberration Test in human lymphocytes, and an L5178Y Mouse Lymphoma Assay):

 

1. In a Klimisch 1 study conducted under GLP and according to OECD Guideline 471 (Bacterial Reverse Mutation Assay), DMEE did not produce a dose-dependent mutagenic response in any of the S. typhimurium orE. colistrains tested. Under the conditions of this assay, DMEE was not mutagenic at 20, 100, 500, 2500 or 5000 µL/plate dose levels.

 

2. In a Klimisch 1 study conducted under GLP and according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test), doses of 41.6, 83.3, 166.5, 333, 666, or 1332 ug/mL DMEE did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation. DMEE was reported to be non-clastogenic under the conditions of this study.

 

3. In a Klimisch 1 rated Mouse Lymphoma Assay conducted under GLP and according to OECD 476 (In vitro Mammalian Cell Gene Mutation Test), L5178Y mouse lymphoma cells were exposed to doses of DMEE ranging from 20 to 1332 ug/mL. DMEE did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation.

In conjunction with the negative in vivo findings for 2-(2-aminoethoxy)ethanol (CAS# 929-06-6), which differs from DMEE only by the absence of two methyl groups on the terminal nitrogen, sufficient evidence is available to support a lack of mutagenic effects following exposure to DMEE.


Justification for selection of genetic toxicity endpoint
A reliable GLP-compliant in vivo mouse micronucleus assay conducted according to EPA OPP 84-2 is available on a structurally similar substance, 2-(2-aminoethoxy)ethanol (CAS# 929-06-6) as a read-across study. DMEE, 2-(2-(dimethylamino)ethoxy)ethanol, and 2-(2-aminoethoxy)ethanol are close structural analogues which differ only by the presence of two methyl groups on the terminal nitrogen. Male CD-1 mice were given a single intraperitoneal dose of 1, 62.5, 125, or 250 mg/kg test substance. The test substance induced signs of clinical toxicity and mortality in the treated animals and was cytotoxic to the bone marrow at the 250 mg/kg dose level for the 24-hour harvest time point. A statistically significant increase in micronucleated PCEs was not observed at any dose level or harvest time point. The use of this study for read-across purposes is further supported by negative findings for DMEE in three reliable and well-conducted in vitro studies (i.e., a Salmonella typhimurium/Escherichia coli Reverse Mutation Assay, a Chromosome Aberration Test in human lymphocytes, and an L5178Y Mouse Lymphoma Assay – see discussion below).

Justification for classification or non-classification

Classification for genetic toxicity is not warranted according to the criteria of EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.