Registration Dossier

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:
Not stated in report
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to guideline and/or standard method but was non-GLP.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Benzyltrimethylammonium chloride was obtained from Fluka Chemical Corporation (Ronkonkoma, NY) in one lot (306793/1). Information on the identity, purity, and stability of the bulk chemical was provided by the manufacturer; identity was confirmed by the study laboratory. Reports on analyses performed in support of the benzyltrimethylammonium chloride studies are on file at the National Institute of Environmental Health Sciences.

The manufacturer identified the chemical, an off-white to yellow crystalline powder, as benzyltrimethylammonium chloride by nuclear magnetic resonance spectroscopy. The purity of lot 306793/1, determined by argentometric titration, was 100.4% or greater. The study laboratory confirmed the identity of the chemical with infrared spectroscopy. The spectrum was consistent with a literature reference for benzyltrimethylammonium bromide (Aldrich, 1990).

Based on the manufacturer's stability information, the bulk chemical was stored at room temperature in sealed containers flushed with nitrogen to expel moisture.

Reference:
Aldrich Catalog/Handbook of Fine Chemicals 1990-1991 (1990), p. 146. Aldrich Chemical Company, Inc., Milwaukee, WI.

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Male and female B6C3F1 mice were obtained from Taconic Farms (Germantown, NY). Upon receipt, the mice were 4 weeks old. Animals were quarantined for 12 to 15 days and were 6 weeks old on the first day of the studies. Before the studies began, five male and five female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood was collected from five male and five female untreated mice at the end of the 13-week studies. The sera were analyzed for antibody titers to rodent viruses (Boorman et al., 1986; Rao et al., 1989b). All results were negative.

Feed and water were available ad libitum. Female mice were housed five per cage; male mice were housed individually.

References:
Boorman, G.A., Hickman, R.L., Davis, G.W., Rhodes, L.S., White, N.W., Griffen, T.A., Mayo, J., and Hamm, T.E., Jr. (1986). Serological titers to murine viruses in 90-day and 2-year studies. In Complications of Viral and Mycoplasmal Infections in Rodents to Toxicology Research and Testing (T.E. Hamm, Jr., Ed.), pp. 11-23. Hemisphere Publishing Corporation, Washington, DC.

Rao, G.N., Piegorsch, W.W., Crawford, D.D., Edmondson, J., and Haseman, J.K. (1989b). Influence of viral infections on body weight, survival, and tumor prevalence of B6C3F1 (C57BL/6N × C3H/HeN) mice in carcinogenicity studies. Fundam. Appl. Toxicol. 13, 156-164.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Core study groups of 10 male and 10 female mice received benzyltrimethylammonium chloride in deionized water by gavage at doses of 0, 12.5, 25, 50, or 100 mg/kg, 5 days per week for 13 weeks. Feed and water were available ad libitum. Female mice were housed five per cage; male mice were housed individually.
Duration of treatment / exposure:
5 days per week for 13 weeks
Frequency of treatment:
5 days per week for 13 weeks
Post exposure period:
Not specified in report.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 12.5, 25, 50, or 100 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females/dose level
Control animals:
yes, concurrent vehicle
Positive control(s):
No data.

Examinations

Tissues and cell types examined:
normochromatic erythrocytes (NCEs)
Details of tissue and slide preparation:
At the end of the 13-week toxicity study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared
and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 1,000 normochromatic erythrocytes (NCEs) in up to 10 animals per dose group.
Evaluation criteria:
A final call of positive for micronucleus induction is preferably based on reproducibly positive trials. Results of the 13-week studies were accepted without repeat tests, because additional test data could not be obtained. Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dose 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. 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 dose group is less than or equal to 0.025 divided by the number of dose groups.

Reference
Integrated Laboratory Systems (ILS) (1990). Micronucleus Data Management and Statistical Analysis Software, Version 1.4. ILS, P.O. Box 13501, Research Triangle Park, NC 72207.

Results and discussion

Additional information on results:
In vivo, benzyltrimethylammonium chloride induced a significant dose-related increase in the frequency of micronucleated normochromatic erythrocytes in the peripheral blood of male and female mice administered 12.5 to 100 mg/kg by gavage for 13 weeks (Table 1). Micronucleus analyses yielded positive trends (P

Any other information on results incl. tables

Table 1 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with Benzyltrimethylammonium Chloride by Gavage for 13 Weeks

 Compound  Dose(mg/kg)  Number of Mice with ErythrocytesScored Micronucleated NCEs/1,000 NCEsb   Pairwise P Valuec
 Male        
 Waterd    10  3.7 + 0.6  
 BTMAC  12.5  10  2.5 + 0.5  0.937
   25  10  2.8 + 0.6  0.868
   50  10  5.2 + 0.9  0.056
   100  9  6.6 + 1.1  0.003
         
       P < 0.001e  
 Female        
 Water    10  2.0 + 0.3  
 BTMAC  12.5  10  2.5 + 0.6  0.228
   25  10  3.0 + 0.3  0.078
   50  10  3.9 + 0.3  0.007
   100  9  6.4 + 0.6  0.000
         
       P < 0.001  

b Mean ± standard error

c Pairwise comparison to solvent control; significant at P<0.006 (ILS, 1990)

d Solvent control

e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test, significant at P<0.025 (ILS, 1990)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
Benzyltrimethylammonium chloride induced significant increases in the frequency of micronucleated normochromatic erythrocytes in peripheral blood of male and female mice in the 13-week study.
Executive summary:

The potential for formation of micronucleated normochromatic erythrocytes was investigated in male and female B6C3F1 mice following 13 weeks of exposure. Significant increases in the frequency of micronucleated normochromatic erythrocytes were found in the peripheral blood of male and female mice administered benzyltrimethylammonium chloride by gavage for 13 weeks.