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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Only minor deviations: weight variation in females at the commencement of the study > 20%; room temperature outside 22 ± 3°C
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dioxolane
EC Number:
211-463-5
EC Name:
1,3-dioxolane
Cas Number:
646-06-0
Molecular formula:
C3H6O2
IUPAC Name:
1,3-dioxolane
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Hoechst Celanese - batch number not provided
- Purity: Not provided

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc.
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: males: 24-36 grams at dose administration; females: 21-33 grams at dose administration.
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: up to five per cage
- Diet: ad libitum
- Water : ad libitum
- Acclimation period: at least 5 days (quarantined)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 74±6°F (20-27°C)
- Humidity (%): 50±20
- Air changes (per hr): not provided
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: not specified but the initiation date is October 2, 1989 and the review completed date is December 29, 1989

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 210, 105 and 52.5 mg/ml
- Lot/batch no.: JAN 3090J AND JUN1990J
Details on exposure:
The test article-vehicle mixture or the vehicle alone were administered by IP injection at a rate of 10 ml/kg body weight. The high dose for the micronucleus study was set at 80% of the LD50 which was 2100 mg/kg. The low dose was set at 1/4 the high dose in order to test within a narrower range.
In order to define the IP LD50, groups of 5 ICR mice of each sex were administered the test substance in corn oil by IP injection at 0, 1576, 2048, 2663, 3462 or 4500 mg/kg. Mortality at the end of a seven day observation period was used to calculate an IP LD50 of 2603 mg/kg.
Duration of treatment / exposure:
Single intraperitoneal injection
Frequency of treatment:
Once
Post exposure period:
24, 24 and 72 hours
Doses / concentrationsopen allclose all
Dose / conc.:
2 100 mg/kg bw (total dose)
Dose / conc.:
1 050 mg/kg bw (total dose)
Dose / conc.:
525 mg/kg bw (total dose)
Dose / conc.:
0 mg/kg bw (total dose)
No. of animals per sex per dose:
5 males and 5 females per concentration were sacrificed after 24, 48 and 72 hours (thirteen experimental groups - 65 males and 65 females - positive control animals sacrificed after 24 hours after dose administration - additional group of 5 males and 5 females as replacement animals)
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: intraperitoneal injection - dissolved in sterile distilled water
- Doses / concentrations: 0.25 mg/kg

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: the high dose for the micronucleus study was set at 80% of the LD50 which was 2100 mg/kg. The low dose was set at 1/4 the high dose in order to test within a narrower range.

DETAILS OF SLIDE PREPARATION: Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing a small volume of FBS. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a pasteur pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained.

METHOD OF ANALYSIS: Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei. The proportion of polychromatic erythrocytes to total erythrocytes and the number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes were also enumerated.
Evaluation criteria:
The test article is considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes is observed relative to the vehicle control. The positive response must be dose-dependent or must be observed at a single dose level at adjacent sacrifice time. If a single treatment group is significantly elevated at one sacrifice time, the assay is considered a suspected or unconfirmed positive and a repeat assay will be recommended.
Statistics:
Statistical significance will be determined using the Kastenbaum-Bowman tables which are based on the binomial distribution.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Bone marrow toxicity was observed in males and females at 24, 48 and 72 hours after administration of 2100 mg/kg.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
TEM induced a significant increase in micronucleated polychromatic erythrocytes in male and female mice relative to the vehicle control.
Remarks on result:
other:
Remarks:
12 male and 10 female mice receiving 2100 mg/kg were found dead prior to their scheduled sacrifice. Additional animals were dosed in a supplementary study for sacrifice at 72 hours after administration. In the supplementary study, 11/15 male and 11/15 female mice died prior to the scheduled sacrifice.
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): information summarized and presented for each treatment group by sacrifice time in tables 1 and 2.
- Clinical signs of toxicity in the high dose group induced prostration, lethargy, irregular breathing or ruffled fur.
- The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was not statistically increased in males and females, regardless of dose level or bone marrow collection time.

Any other information on results incl. tables

Table 1: Micronucleated polychromatic erythrocytes in bone marrow: summary




























































































































































































































 



 



 



 



 



Micronucleated polychromatic erythrocytes



Treatment



Sex



Time (hr)



Number of mice



PCE/Total Erythrocytes



Number per 1000 PCE (Mean ± SD)



Number per PCE scored



Corn oil 10 ml/kg



M



24



5



0.59



0.2 ± 0.42



1/5000



48



5



0.62



0.2 ± 0.42



1/5000



72



5



0.62



0.2 ± 0.42



1/5000



F



24



5



0.59



0.2 ± 0.42



1/5000



48



5



0.62



0.0 ± 0.00



0/5000



72



5



0.66



0.0 ± 0.00



0/5000



C-121


525 mg/kg



M



24



5



0.53



0.2 ± 0.45



1/5000



48



5



0.62



0.2 ± 0.45



1/5000



72



5



0.54



0.4 ± 0.55



2/5000



F



24



5



0.57



0.4 ± 0.55



2/5000



48



5



0.64



0.0 ± 0.00



0/5000



72



5



0.65



0.6 ± 0.89



3/5000



C-121


1050 mg/kg



M



24



5



0.53



0.2 ± 0.45



1/5000



48



5



0.58



0.0 ± 0.00



0/5000



72



5



0.43



0.0 ± 0.00



0/5000



F



24



5



0.60



0.2 ± 0.45



1/5000



48



5



0.67



0.4 ± 0.89



2/5000



72



5



0.54



0.0 ± 0.00



0/5000



C-121


2100 mg/kg



M



24



5



0.31



0.6 ± 0.89



3/5000



48



3



0.26



0.3 ± 0.58



1/5000



72



0 (a)



 



 



 



F



24



5



0.44



0.0 ± 0.00



0/5000



48



5



0.41



0.4 ± 0.55



2/5000



72



0 (a)



 



 



 



TEM


0.25 mg/kg



M



24



5



0.57



18.4 ± 5.90



92/5000*



F



24



5



0.57



20.8 ± 7.63



104/5000*



(a): no surviving animals at this time point


Table 2: Micronucleated polychromatic erythrocytes in bone marrow: summary – supplemental study

























































 



 



 



 



 



Micronucleated polychromatic erythrocytes



Treatment



Sex



Time (hr)



Number of mice



PCE/Total Erythrocytes



Number per 1000 PCE (Mean ± SD)



Number per PCE scored



Corn oil


10 ml/kg



M



72



5



0.59



0.4 ± 0.55



2/5000



F



72



5



0.60



0.2 ± 0.45



1/5000



C-121


2100 mg/kg



M



72



4



0.37



0.0 ± 0.00



0/4000



F



72



4



0.35



0.0 ± 0.00



0/4000


Applicant's summary and conclusion

Conclusions:
The negative and positive controls fulfilled the requirements for determination of a valid test.
Under the conditions of the assay described in this report, C-121 did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

Male and female ICR mice were exposed to 525, 1050 or 2100 mg/kg of C-121 which was administered as a single IP injection. The high dose level was calculated to be 80% of the LD50. Bone marrow cells, collected 24, 48 and 72 hours after treatment were examined microscopically for micronucleated polychromatic erythrocytes. Marked reduction in the ratio of polychromatic erythrocytes to total erythrocytes was observed in male and female mice in the high dose groups indicating test article-induced bone marrow toxicity. A total of 23 of 35 male mice and 21 of 35 female mice in the high dose group died prior to the scheduled sacrifice. All mice in the 1050 and 525 mg/kg dose groups survived to treatment. No significant increases in micronucleated polychromatic erythrocytes were observed at 24, 48 and 72 hours after dose administration in males or females. The results of the assay indicate that under the conditions described in this report, C-121 did not induce a significant increase in micronculeated polychromatic erythrocytes in male or female ICR mice. C-121 was concluded to be negative in the mouse micronucleus assay.

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