Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 84-2
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.