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:
11th September to 11th October 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed per recognized guideline following GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
122384-85-4/68855-45-8
Phenol, dodecyl-, sulfurized, calcium salts
Testing was performed on a commercial sample of this material. Typical purity of this material as distributed in commerce is 60% alkyl phenol sulfide and 40% highly refined lubricant base oil.

Test animals

Species:
mouse
Strain:
other: Crl:CD-1®(ICR) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI.
- Age at study initiation: All animals were eight weeks and three days old at the time of dosing.
- Weight at study initiation: The weight range of the animals used in the micronucleus assay was 23.7-35.1 and 21.2-28.2 grams for the males and females, respectively.
- Assigned to test groups randomly: Yes
- Housing: Animals were housed five per cage during quarantine, and housed at least five per cage at randomization. Sanitized caging was used for housing the animals.
- Diet/water (e.g. ad libitum): A commercial diet (Purina® Certified Laboratory Pellets ® # 5002) and water were available ad libitum for the duration of the study. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed on a retrospective basis for specified microorganisms, pesticides, alkalinity, heavy metals, and halogens.
- Acclimation period: Animals were quarantined for seven days before being placed on study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 6 °F
- Humidity (%): 55 ± 15%
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
The vehicle control, peanut oil (Sigma, Lot # 83H0848), was administered concurrently with the test article at a volume of 10 mL/kg.
Details on exposure:
Dosing suspensions were prepared just prior to dosing and were prepared by making a 500 mg/mL stock for the high dose (5000 mg/kg ). This was prepared by adding peanut oil (Sigma, Lot # 83H0848) to the test material resulting in a dark brown viscous solution. Dilutions of this stock were prepared for the 2500 and 1250 mg/kg and dose levels. Volumes dosed were 10 mL/kg and were based upon individual animal weights. All dosing stocks were continuously mixed during the dosing procedure.
Duration of treatment / exposure:
The animals dosed with the test article and the vehicle control were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow.
Frequency of treatment:
Once intraperitoneally
Post exposure period:
24, 48 and 72 hours post-dose
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1250, 2500 and 5000 mg/kg b.wt.
Basis:
nominal conc.
The dose volume was 10 mL/kg b.wt. for all groups.
No. of animals per sex per dose:
Five mice/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide at a dose of 60 mg/kg b.wt.

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose levels in the main study were selected based on results from the dose selection study. the dose levels selected were1250, 2500 and 5000 mg/kg.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanized by CO2/O2 inhalation followed by penetration of the thorax. A limb bone was removed from each hind leg and the adhering soft tissue and epiphyses were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 - 5 mL bovine serum (one tube for each animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, and stained in May-Grunwald solution followed by Giemsa (Schmid, 1975). The air-dried slides were coverslipped.

METHOD OF ANALYSIS:
The slides were coded for analysis, and scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ratio. Standard forms were used to record these data. One thousand PCEs per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this Crl:CD-1®(ICR) BR strain is about 0.0 - 0.4%.

The frequency of PCEs versus NCEs was determined by scoring the number of PCEs and NCEs observed in the optic fields while scoring at least the first 1000 erythrocytes.

Evaluation criteria:
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ringshaped micronuclei occasionally occurred. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).
Statistics:
Results for each sex / harvest time were analyzed by ANOVA on either untransformed (when variance homogeneous) or rank transformed (when
variance heterogeneous) micronucleus cell count data. If significance was observed with ANOVA, a Dunnett’s t-test was used to determine which
dose groups were different from the negative control. A Cochran-Armitage test for linear trend was used to evaluate dose-response.

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:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Dose levels of 1625, 2750, 3875 and 5000 mg/kg were administered by intraperitoneal injection for the dose selection study.
- Clinical signs of toxicity in test animals:
All animals were examined after dosing and daily throughout the duration of the study (three days) for toxic effects and/or mortalities. All animals appeared normal immediately after dosing and remained healthy until the end of the observation period. No mortality occured.
- Rationale for exposure: Based on these results, the maximum tolerated dose was estimated to be >5000 mg/kg.


RESULTS OF DEFINITIVE STUDY
All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest times. All test article dosed groups appeared normal immediately after dosing and remained healthy until the appropriate harvest times.
The test article induced no statistically significant increases in micro-nucleated polychromatic erythrocytes over the levels observed in the vehicle controls in either sex or at any of the harvest times. The test article did not induce a statistically significant change in the PCE:NCE ratio. The positive control, Cyclophosphamide, induced statistically significant increases in micronucleated PCEs in both sexes as compared to the vehicle controls, with means and standard errors of 2.38% ± 0.54% and 4.28% ± 0.79% for the males and females, respectively.

Any other information on results incl. tables

There was no mortality and all animals appeared normal without sign of adverse effect until sacrifice. Mean percentage of micronucleated PCEs was within the range of laboratory historical controls for all treatment and vehicle control groups.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test article did not induce a statistically significant increase in micro-nuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.

Executive summary:

In a study conducted in accordance with GLP to OECD guideline 474, based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg. In the micronucleus assay, the test article was suspended in peanut oil and dosed by intraperitoneal injection at 1250, 2500 and 5000 mg/kg. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals dosed with the test article and the vehicle control were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. The animals dosed with the positive control were euthanized approximately 24 hours after dosing for extraction of the bone marrow.

One thousand PCEs per animal were scored. The test article did not induce a statistically significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test. The test article did not induce a statistically significant change in the PCE:NCE ratio.

The test article did not induce a statistically significant increase in micro-nuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse micronucleus assay.