Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1999
Report Date:
1998
Reference Type:
publication
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- dicyclohexylcarbodiimide (CASRN 538-75-0; MW 206.32)
- Source and lot/batch No.of test material: Aldrich Chemical Co. (Milwaukee, WI)
- Purity: 99+%

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 weeks
- Assigned to test groups randomly: yes
- Housing: individually housed
- Diet: NIH-07 Open Formula Diet, pelleted form (Zeigler Bros., Inc., Gardener, PA), ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3ºC
- Humidity (%): 50 ± 15%
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle.

Administration / exposure

Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: the test item is moisture sensitive, so ethanol was the preferred solvent.
- Concentration of test material in vehicle: 1.5, 3.0, 6.0, or 12.0 mg/kg
- Dosing volume: 2.0 ml/kg bw.
Details on exposure:
TEST SITE: skin, not specified.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 ml/kg bw.
- Concentration (if solution): 1.5, 3.0, 6.0, or 12.0 mg/kg.
- Constant volume or concentration used: yes
- For solids, paste formed: no
Duration of treatment / exposure:
90 days (13 weeks).
Frequency of treatment:
5 days per week.
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
12 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex per dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Because the blood samples were taken from animals on test in a subchronic toxicity study, there was no positive control group.

Examinations

Tissues and cell types examined:
Normochromatic erythrocytes (PCE) from peripheral blood.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Doses were selected based on preliminary toxicity tests (no further data).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
- Treatment: 10 mice per dose group per sex, were administered the test item dissolved in ethanol via skin painting, five times per week for 13 weeks.
- At termination, the animals were examined for gross pathology and histopathology; blood chemistry and hematology parameters were also measured.

DETAILS OF SLIDE PREPARATION:
- At the end of the exposure period, blood samples were obtained from the retro-orbital sinus under anesthesia prior to euthanasia, and smears were immediately prepared and fixed in absolute methanol. Coded slides from each animal were stained with acridine orange [Tice et al. (1989)].

METHOD OF ANALYSIS:
- All slides were evaluated at 1,000x magnification using epi-illuminated fluorescence microscopy (450–490 nm excitation, 520 emission).
- the frequency of MN-NCE among 1,000 NCE per animal and the percentage of PCE among 1,000 erythrocytes was determined in each of 10 mice per sex per dose group.
Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups.
Statistics:
The frequency of micronucleated cells among PCEs was analyzed by a statistical software package (Micronucleus Assay Data Management and Statistical software package (ver. 1.4) [Margolin and Risko, 1988; ILS, 1990]) that tested for increasing trend over dose groups with a one-tailed CochranArmitage trend test, followed by pairwise comparisons between each dosed group and the control group (ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. The %PCE data were analyzed by an analysis of variance (ANOVA) test based on individual animal data; pairwise comparisons between the exposure and control groups were made using a two-tailed Student’s t-test.

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
positive
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Key result
Sex:
female
Genotoxicity:
positive
Remarks:
weak
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): positive results were seen in male mice based on a trend test analysis (P 5 0.003), although none of the treated groups showed MN-NCE frequencies that were significantly higher than the corresponding control group. In female mice, increased frequencies of MN-NCE were seen in all four treatment groups; however, only the mice receiving 3.0 mg DCC/kg/day showed significantly elevated MN-NCE frequencies, and the trend P value was not significant. The results in females were judged weakly positive.
- Ratio of PCE/NCE (for Micronucleus assay): Neither sex showed chemical-induced effects on the %PCE in blood.

Any other information on results incl. tables

Table 2. Frequency of MN-PCE and %PCE in Peripheral Blood of Male and Female B6C3F1 Mice.

Dose

(mg/kg)

N

MN-PCE/

1000 PCE

(mean ± SE)

P value*

% PCE

(mean ± SE)

P value*

Males

0

10

3.6 ± 0.43

 

4.8 ± 0.25

 

1.5

10

3.1 ± 0.48

0.720

4.3 ± 0.08

0.114

3.0

10

5.3 ± 0.50

0.036

4.4 ± 0.11

0.264

6.0

10

5.7 ± 0.70

0.015

4.1 ± 0.14

0.048

12.0

10

5.7 ± 0.68

0.015

4.3 ± 0.14

0.107

 

 

P=0.003**

 

P = 0.071***

 

Females

0

10

2.7 ± 0.63

 

4.3 ± 0.19

 

1.5

10

3.7 ± 0.58

0.105

4.9 ± 0.14

0.009

3.0

10

5.1 ± 0.67

0.003

4.6 ± 0.16

0.085

6.0

10

4.3 ± 0.68

0.027

4.9 ± 0.22

0.015

12.0

10

4.4 ± 0.50

0.022

4.5 ± 0.22

0.191

 

 

P = 0.078**

 

P = 0.096

 

*: Comparison of individual dose groups to the concurrent control; significant at P 0.006. **: Trend test, one-tailed. Significant at P0.025. ***: ANOVA, significant at P ≤ 0.025.

Applicant's summary and conclusion

Conclusions:
The test item gave a positive result in male and female B6C3F1 mice.
Executive summary:

A subchronic peripheral blood micronucleus study in male and female B6C3F1 mice was performed for the test item, by a method similar to OECD 407 (GLP study). Ten animals per sex per dose group were exposed to 0 (control), 1.5, 3, 6, 12 mg/kg bw of test item in ethanol by skin painting, five days a week for 13 weeks. At the end of the exposure period, blood samples were obtained from the retro-orbital sinus under anesthesia prior to euthanasia, and smears were immediately prepared, fixed in absolute methanol and stained with acridine orange. Peripheral blood analysis included assessment of the frequency of MN-PCE among 1000 PCE and the %PCE among 1000 erythrocytes. After 13 weeks of dermal administration, the test item induced significant increases in the frequency of micronucleated normochromatic erythrocytes in peripheral blood of mice, based on the results of a trend analysis, although none of the treated groups showed a significant increase in the frequency of micronucleated NCEs compared to the control. In females, the frequency of all treated groups was greater than the control, but only significant at 3 mg/kg bw, without a significant trend. No effect was observed on the percentage of polychromatic erythrocytes, suggesting no measurable induced toxicity. Based on the available information, the test item gave a positive result under test conditions.