Registration Dossier

Diss Factsheets

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:
1980/09/03-1980/09/23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This GLP study was conducted similar to OECD guideline, however has significant deviations to warrant restriction.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Bone marrow was extracted 6 h post second dose. Sampling is recommended 18-24 h following final exposure. No mention of sampling in report other than final extraction. Additional deviations include 10 h light cycle and 4 mice/sex instead of 5
Principles of method if other than guideline:
Reported as SOP PH-309 and reference Schmid, W. (1975)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc O,O,O',O'-tetrakis(1,3-dimethylbutyl) bis(phosphorodithioate)
EC Number:
218-679-9
EC Name:
Zinc O,O,O',O'-tetrakis(1,3-dimethylbutyl) bis(phosphorodithioate)
Cas Number:
2215-35-2
Molecular formula:
Too complex
IUPAC Name:
zinc O,O,O',O'-tetrakis(1,3-dimethylbutyl) bis(phosphorodithioate)
Details on test material:
Test material is described as pale, yellow liquid with poor solubility in methyl cellulose. Two drops of 5% solution of Tween 80 was added to test sample to aid in suspension with the vehicle.

Test animals

Species:
mouse
Strain:
other: BS-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals from Blue Spruce Farms, 7-12 weeks old, were acclimated to the laboratory for 4 days. Animals were identified by cage number and uniquely numbered by ear tag. Animals were housed 4/box, according to sex, in plastic tote boxes deep fitted with galvanized mesh lids in accordance with the Institute of Laboratory Resources standards. Light cycle was 10h light, 14 h dark, temperature maintained between 67-77 °F, and humidity described as ambient. Wayne Lab Blox food and water were provided ad libitum.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
0.25% methylcellulose from Fisher Scientific Company, lot no. 784262.
Details on exposure:
20 ml/kg BW was administered by i.p. injection. Intraperitoneal was selected because it increases the likelihood of bioavailability.

Duration of treatment / exposure:
Duration of study was 4 weeks.
Frequency of treatment:
Two dose levels following a split dose regimen, separated by 24 h.

Post exposure period:
6 h after the second dose, the animals were sacrificed and femurs removed.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
4 mice/sex/group
Control animals:
yes, concurrent vehicle
Positive control(s):
Concurrently, triethylenemelamine was given in a split dose regimen at a dose of 0.5 mg/kg. TEM was obtained from Polysciences, Inc., lot no. 6-246. Used as comparison because of its known effects.

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
The femur was flushed with fetal calf serum until the bone appeared almost translucent and centrifuged at 1000 rpm for 5 min in an HS-4 Sorvall Centrifuge Head. The supernatant was removed and the sediment vortexed 5-10 sec to assure a homogenous mixture. A drop of the mixture was placed on a glass slide, smeared, and left to dry overnight. The smears were fixed in methanol, stained with May-Gruenwald solution undiluted 3 min, May-Gruenwald solution diluted 1:2 in distilled water 2 min, rinsed, Giemsa stain diluted 1:6 10 min, rinsed, blotted, cleared with xylene and mounted in permount with a cover glass.
Evaluation criteria:
The slides were screened for good preparation and 1000 polychromatic erythrocytes were counted for the presence of micronuclei.
Statistics:
Student "t" test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The 100 mg/kg dose group exhibited writhing, hypersensitivity and piloerection after the first dose. Following the second dose, the mice exhibited elevated gait, decreased body tone, piloerection, decreased activity, severe writhing, body drop, ptosis, tremors and cyanosis (1/8). The 50 mg/kg dose group exhibited piloerection, writhing and abnormal gait after the first dose and decreased activity, body tone, and cyanosis after the second dose. Only the positive control produced statistically significant increase in the number of micronuclei per 1000 polychromatic erythrocytes in the treated v control group. The mean value of micronucleated immature erythrocytes were 19.3 in the vehicle control, 64 in the positive control, 27.9 in the 100 mg/kg dose group and 16.9 in the 50 mg/kg dose group.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, the test article did not produce an increase in the number of micronuclei per 1000 polychromatic erythrocytes when administered i.p. to BS-1 mice.
Executive summary:

In an in vivo micronucleus test, male and female BS-1 mice were dosed with test substance at concentrations of 50 mg/kg/day and 100 mg/kg/day for two days. The negative and positive controls fulfilled the requirements for determination of a valid test. The test article did not produce an increase in the number of micronuclei per 1000 polychromatic erythrocytes. Based on the results of this study, the test substance would not be classified based on weight of evidence for the chemical class in accordance with the classification system of GHS. This toxicity study is classified as acceptable and satisfies the guideline requirement for in vivo mutagenicity test.

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