Registration Dossier

Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: older study; not conducted in accordance with GLP guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1973
Report Date:
1973

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
One hundre:l male rats (Sprague-Dawley), 10-12 weeks old, were purchased from Carwort:1 Farms, Inc., and randomly assigned to five groups of 20 males each. Following a period of acclimation each group was randomly assigned one of the fo:~lowing treatments: 20, 200 or 2000 mgjkg sucrose acetate isobutyrate (experimental groups), 25 mg/kg Apholate (positive control) or corn oil only (nege.tive control). All treatments were administered by gavage as single dose~ in corn oil. The low dose of SAIB used is equivalent to the amount of the compound per day that would be ingested if a 50 kg person drank two quarts of soft drink containing the proposed use level of SAIB.
Duration of treatment / exposure:
single oral dose
Frequency of treatment:
single oral dose
Post exposure period:
Within two hours after treatment, the males were mated with two untreated, virgin females for the following week. Subsequent matings using naive females were conducted during the third, fifth and seventh weeks after treatment. Mating during these weeks represent samples of post meiotic, meiotic and pre-meiotic stages of spermatogenesis, respectively.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
20 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Positive control(s):
25 mg/kg Apholate

Examinations

Tissues and cell types examined:
Seventy-two hours after treatment, blood was collected from five males of each group to be cultured for cytogenetic studies. The leukocytes were cultured using a modification of the micro-blood culture method described by Noguchi. Ten metaphase plates per rat (50 per group) were examined under the light microscope and scored for chromosome number, achromatic gaps, chromatid deletions or exchanges, chromosome deletions or exchanges and miscellaneous aberrations.

Twelve to fourteen days from the mid-week of their caging and presumptive mating all females were sacrificed with an overdose of ether. The uteri were exposed and the total implantation sites were counted and categorized as comprising viable fetuses, early deaths or late deaths. Early deaths are brown or black necrotic spots on the implants containing no embryo. Late fetal deaths are implantation sites containing macerated or partially macerated fetuses and placenta which may be white or pale. The ovaries were removed and the corpora lutea. counted under a dissecting microscope.
Statistics:
The statistics analyzed using the analyses of variance test were:
Insemination index (number of males that inseminated a. female/number of males per group x 100), the pregnancy index (number of pregnant
females/number of females mated x 100), the implantation index (number of implantation sites/total number of corpora lutea x 100), the mean total implantation sites and the early deaths per pregnancy. The data on the early deaths per pregnanc-y were transformed by the Freeman-Tukey Poisson transformation before being subjected to the analysis of variance test.

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
not specified
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Sex:
female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Early deaths per pregnancy for all mating periods after treatment were evaluated. These data were transformed using the Freeman-Tukey Poisson transfonnation before analysis using the analysis of variance test. The only statistical differences seen were an increase in early deaths in the positive control group (Apholate, 25 mg/kg p.o.) during the 3rd and 5th weeks of mating. Reduction of total implantations, an indication of pre-implantation
loss, was examined from two aspects; both the mean implantations per pregnancy and an implantation index (total implantations/total corpora lutea x 100) were calculated for each group at each mating period. These statistics were compared with their concurrent control groups using the analyses of variance test.

The mean implants per pregnancy of the high dose group of SAIB (2000 mg/kg) and the positive control group (Apholate) were statistically lower than the control groups during the 3rd and 7th weeks of mating. In the case of the positive control group this decrease in implants correlated with a significant increase in early fetal deaths during the 3rd mating week, but not during the 7th week. None of the SAIB groups had significantly increased early deaths during any of the mating periods. Published iata from control rats in respect to mean total implants appear to be scanty mainly because most dominant lethal assays are done in mice; therefore a normal range or limit has not been as well established in rats as it has in mice.

In this study, though some of the groups had statistically lower mean implants than the respective controls, none of these were fewer than the lower limit of eight per pregnancy in mice established by Epstein, et al. Also, all were within the ranges seen in rats from teratology studies conducted in this laboratory.10 The implantation indices reflected the same situation as described for the mean implantations per pregnancy. Pre-implantation losses, whether measured as mean implants/pregnancy or as the percent of corpora lutea implanted are only an indirect measure of dominant lethal mutations since these losses can be due not only to genetic causes but to non-genetic causes as well. The number of males in both the middle and high dose groups of SAIB that successfully inseminated a female during the first mating period was somewhat lower than in the control group. A possible explanation for this could have been the proximity of mating and the mechanical effects of administering large doses of a compound. When tested statistically, neither dose level was significantly different from the control group. The pregnancy rates during all mating weeks were not significantly different when compared vii th the concurrent control groups. However, the rates of all dose groups during the first week of mating, including the negative controls and excluding the positive controls, were somewhat lower than during the succeeding periods of mating. This may have been due to a combinat:i.on of the slightly lower inseminations in the case of the two SAIB groups and to the naivity of both the males and females of all dose groups.

There did not appear to be any significant increase in chromosome numbers or aberrations in any of the groups. However, it should be noted that this was a preliminary study designed as an adjunct to the dominant lethal assay reported here and not a separate, well-timed cytogenetic test. There does appear to be an unusually high incidence of tetraploidly which however oecurred in all groups. We have not seen this reported in the literature and since our experience with cytogenetic tests in this strain of rat is limited, we offer no explanation.

Any other information on results incl. tables

Early fetal deaths per week of mating
pregnancy at different group 1 3 5 7
periods of mating positive control 1.59 3.28* 3.28* 0.88
2000 mg/kg SAIB 1.06 1.47 0.87 0.73
200 mg/kg SAIB 1.05 0.63 1.21 0.85
20 mg/kg SAIB 0.32 1.59 1.1 0.93
negative control 0.96 0.92 0.94 0.62
*Statistically different from negative controls (p≤ 0.05)
week of mating
mean implantations per group 1 3 5 7
pregnancy at different positive control 12 9.2* 13.3 10.5*
periods of mating 2000 mg/kg SAIB 14 10.6* 13.8 11.5*
200 mg/kg SAIB 10.9 12.1 13.5 12.9
20 mg/kg SAIB 11.9 11.7 12.8 12.1
negative control 11.3 13.2 13.3 14.2
*Statistically different from negative controls (p≤ 0.05)
Percent of total corpora week of mating
lutea implanted at  group 1 3 5 7
different periods of positive control 80.8 68.1 87.3 72.7
mating 2000 mg/kg SAIB 92.9 70.8 91.1 79.6
200 mg/kg SAIB 70.6 81.2 90.4 91.8
20 mg/kg SAIB 82 80.6 88.4 82
negative control 72 83.6 93.7 98.9
Percentage inseminations week of mating
at different periods of  group 1 3 5 7
mating positive control 95 90 85 75
2000 mg/kg SAIB 70 100 95 95
200 mg/kg SAIB 75 85 90 85
20 mg/kg SAIB 80 90 85 95
negative control 85 90 95 85
Percentage pregnancies week of mating
at different periods of group 1 3 5 7
mating positive control 72.5 62.5 62.5 65
2000 mg/kg SAIB 42.5 85 77.5 82.5
200 mg/kg SAIB 55 75 85 65
20 mg/kg SAIB 55 72.5 72.5 75
negative control 57.5 62.5 77.5 72.5
examination of metaphase metaphase tetraploidy chromosome gap fragment increased decreased condensed unequal
plates in leukocyte plates break number number arm length
cultures counted
positive control 20 3 1 0 1 0 2 0 0
2000 mg/kg SAIB 50 3 0 0 0 0 5 0 1
200 mg/kg SAIB 50 5 2 0 2 0 3 0 0
20 mg/kg SAIB 50 8 0 0 0 0 2 0 1
negative control 50 5 0 0 1 0 4 2 0

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
An increase in early fetal deaths per pregnancy is a direct and unequivocal measure of dominant lethal mutations. In this study, the only increases in early fetal deaths per pregnancy were observed, as expected, in the positive control group. Therefore, it is concluded that quantities of sucrose acetate isobutyrate ranging from the proposed use level to 100 times this level; administered per os as single doses did not cause dominant lethal
mutations in rats.
Executive summary:

One hundred male rats (Sprague-Dawley), 10-12 weeks old, were randomly assigned to five groups of 20 males each. Following a period of acclimation each group was randomly assigned one of the following treatments: 20, 200 or 2000 mg/kg sucrose acetate isobutyrate (experimental groups), 25 mg/kg Apholate (positive control) or corn oil only (negative control). All treatments were administered by gavage as a single dose in corn oil. Within two hours after treatment, the males were mated with two untreated, virgin females for the following week. Subsequent matings using naive females were conducted during the third, fifth and seventh weeks after treatment. Mating during these weeks represent samples of post meiotic, meiotic and pre-meiotic stages of spermatogenesis, respectively. Twelve to fourteen days from the mid-week of their caging and presumptive mating all females were sacrificed with an overdose of ether. The uteri were exposed and the total implantation sites were counted and categorized as comprising viable fetuses, early deaths or late deaths. The major statistics of interest in the dominant lethal assay are pregnancy rate, early deaths per pregnancy and mean total implantations, and an increase in early fetal deaths per pregnancy is a direct and unequivocal measure of dominant lethal mutations. In this study, the only increases in early fetal deaths per pregnancy were observed, as expected, in the positive control group. Therefore, it is concluded that quantities of sucrose acetate isobutyrate ranging from the proposed use level to 100 times this level; administered orally in single doses did not cause dominant lethal mutations in rats.