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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is a GLP study and is recently (2010) performed according to standard methods, there it is considered reliable, adequate and relevant.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
secondary source
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Principles of method if other than guideline:
The micronucleus test was consistent with the US EPA guidelines for studies of this type (OPPTS 870.5395) and with OECD 474. The testing was in accordance with Good Laboratory Practice Guidelines of the OECD (OECD, 1997) and the U.S. EPA (CFR, 2007). The study was an extension of the combined repeated dose/reproductive and developmental toxicity screening in rats.

Bone marrow was collected from all animals at terminal sacrifice and flushed into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged, the majority of the HI FBS was decanted, and the pellet was re-suspended. Bone marrow smears were prepared by placing single drops of suspension on microscope slides (minimum of two per preparation). The slides were coded, air dried, fixed in methanol and allowed to air dry a second time.

Coded slides were stained with acridine orange (Hayashi et al., 1983). A total of 1000 erythrocytes/slide were evaluated (both polychromatic (PCE) and normochromatic erythrocytes (NCE)), and the PCE/NCE ratio was calculated. The number of micronucleated PCEs from a total of 2000 PCEs was then determined for each animal.

The percentages of PCEs , micronucleated cells in NCEs, and the ratios of PCEs to total erythrocytes in the test substance- and vehicle-treated groups were compared using ANOV A (Snedecor and Cochran, 1980). If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett Test (Dunnett, 1964) was used to compare each test substance-treated group to the vehicle control group. In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA (Snedecor and Cochran, 1980).
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Naphthenic acids
EC Number:
215-662-8
EC Name:
Naphthenic acids
Cas Number:
1338-24-5
Molecular formula:
For the acidic (naphthenic) fraction: CnH2n+zO2, where n = carbon number and z = homologous group series number: z = 0 when no ring structures are present, z = -2 when 1 ring is present, z = -4 when 2 rings are present etc. For the non-acidic fraction: not applicable
IUPAC Name:
Naphthenic acids
Test material form:
other: liquid
Details on test material:
The test sample used in the current program was a blend of naphthenic acids from three sources. The samples were dried under a stream of nitrogen and then re-dissolved in 0.5 mL dichloromethane. The samples were analyzed by GC-MS (Young et al., 2008) and the total ion current mass spectra were collected and tablulated (Holowenko et al., 2002).

Based on these data it was determined that there were no significant differences among these samples (Fedorak, 2009). The data indicated that the test material contained constituents with carbon numbers predominantly in the range of C6-C16 (corresponding to a molecular weight range of approximately 116-250) and with a ring distribution of approximately 0 rings (24%), 1 ring (39%), 2 rings (31%), 3 rings (5%) and 4 rings (1%).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: no data

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
Duration of treatment / exposure:

Approximately 30 days
Frequency of treatment:
Daily
Post exposure period:
None
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 900 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
6 males/6 females
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral gavage
- Doses / concentrations: 60 mg/kg/day

Examinations

Tissues and cell types examined:
bone marrow , polychromatic and normochromatic eryhtrocytes
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
Bone marrow was collected from all animals at terminal sacrifice

DETAILS OF SLIDE PREPARATION:
Bone marrow was collected from all animals at terminal sacrifice and flushed into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged, the majority of the HI FBS was decanted, and the pellet was re-suspended. Bone marrow smears were prepared by placing single drops of suspension on microscope slides (minimum of two per preparation). The slides were coded, air dried, fixed in methanol and allowed to air dry a second time. Coded slides were stained with acridine orange (Hayashi et al., 1983).

METHOD OF ANALYSIS:
A total of 1000 erythrocytes/slide were evaluated (both polychromatic (PCE) and normochromatic erythrocytes (NCE)), and the PCE/NCE ratio was calculated. The number of micronucleated PCEs from a total of 2000 PCEs was then determined for each animal.

The percentages of PCEs , micronucleated cells in NCEs, and the ratios of PCEs to total erythrocytes in the test substance- and vehicle-treated groups were compared using ANOV A (Snedecor and Cochran, 1980). If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett Test (Dunnett, 1964) was used to compare each test substance-treated group to the vehicle control group. In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA (Snedecor and Cochran, 1980).
Statistics:
The percentages of PCEs , micronucleated cells in NCEs, and the ratios of PCEs to total erythrocytes in the test substance- and vehicle-treated groups were compared using ANOV A (Snedecor and Cochran, 1980). If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett Test (Dunnett, 1964) was used to compare each test substance-treated group to the vehicle control group. In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA (Snedecor and Cochran, 1980).

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:
Systemic toxicity: No treatment-related bone marrow effects.
Genotoxic effects: Not mutagenic. The frequencies of micronuclei in in bone marrow from rats treated with refined naphthenic acids did not differ statistically from those in the sham and vehicle control groups. A significant increase in micronucleus frequency was found in material harvested from rats treated with the positive control, cyclophosphamide providing evidence that the test had worked as expected.

Any other information on results incl. tables

Table 1. Summary of results of micronucleus data from rats following repeated treatment with refined naphthenic acids. 

Treatment

Gender

Total MN PCEs/2000 PCEs (N=5)

% MN PCEs

Total MN NCEs/2000 NCEs (N=5)

Ratio of PCEs to Total Erythrocytes

Corn Oil

Males

8

0.08+0.08

3

0.54+0.07

 

Females

8

0.08+0.12

4

0.69+0.11

 

 

 

 

 

 

Sham Control

Males

6

0.06+0.04

3

0.52+0.11

 

Females

8

0.08+0.08

2

0.55+0.17

 

 

 

 

 

 

Naphthenic Acid

 

 

 

 

 

100 mg/kg/day

Males

7

0.07+0.07

1

0.53+0.09

 

Females

4

0.04+0.04

7

0.65+0.16

300 mg/kg/day

Males

4

0.04+0.04

3

0.49+0.67

 

Females

5

0.06+0.05

5

0.67+0.13

900 mg/kg/day

Males

8

0.08+0.08

5

0.61+0.11

 

Females

5

0.06+0.05

5

0.75+0.19

 

 

 

 

 

 

Positive Control (Cyclophosphamide)

60 mg/kg/day

Males

128

1.28+0.14a

13

0.40+0.21

 

Females

97

0.97+0.19a

16

0.51+0.12a

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Naphthenic acids did not induce chromosomal aberrations under the conditions of the test
Executive summary:

A micronucleus test was conducted in male and female Wistar rats with refined Naphtenic acids dosed at 100, 300 and 900 mg/kg bw by oral gavage.A total of 1000 erythrocytes/slide were evaluated (both polychromatic PCE and normochromatic erythrocytes NCE), and the PCE/NCE ratio was calculated. The number of micronucleated PCEs from a total of 2000 PCEs was then determined for each animal.The frequencies of micronuclei in in bone marrow did not differ statistically from those in the sham and vehicle control groups. A significant increase in micronucleus frequency was found in material harvested from rats treated with the positive control, cyclophosphamide providing evidence that the test had worked as expected.