Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984-1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study, GLP-compliant, available as unpublished report, no restrictions, adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Nichols W W, Moorehead P, Brewen G, 1972. Chromosome methodologies in mutation testing; ad hoc committee report. Toxicol. Appl. Pharmacol. 22, 269-275.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Preston R J, Au W, Bender M A, Brewen J G, Carano A V, Heddle J A, McFee A F, Wolff S, Wassom J S, 1981. Mammalian in vivo and in vitro cytogenetic assays: A report of the U.S. EPA’s Gene-Tox Program. Mutat. Res. 87, 143-188.
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Reference substance name:
Terphenyl
EC Number:
247-477-3
EC Name:
Terphenyl
Cas Number:
26140-60-3
Molecular formula:
C18H14
IUPAC Name:
1,1':4',1''-terphenyl
Constituent 2
Reference substance name:
Therminol 75
IUPAC Name:
Therminol 75
Details on test material:
- Name of test material (as cited in study report): Therminol 75 Heat Transfer Medium
- Supplier: Monsanto Company
- Substance type: mixed terphenyls and quaterphenyls
- Physical state: Light-brown soft solid
- Purity: N/A, complex mixture
- Lot/batch No.: Lot AE-01-S
- Storage condition of test material: Stored in original bottle placed in a secondary light-proof and unbreakable container at room temperature.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Inc., 1180C Day Road, Gilroy, CA 95020
- Age at study initiation: young adult
- Weight at study initiation: 175-275 g
- Assigned to test groups randomly: yes
- Housing: two or three per cage
- Diet (e.g. ad libitum): ad libitum, Certified Purina Lab Chow (#5002C)
- Water (e.g. ad libitum): ad libitum, fresh and purified water
- Acclimation period: minimum of 5 days after their arrival

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Lot/batch no. (if required): 790670
- Supplier: Fisher Scientific
- Description: Viscous, light-yellow liquid
- Storage conditions: Stored in brown glass bottle at 4°C
Details on exposure:
Preparation of the test article:
Therminol 75 and corn oil were first heated to 80°C in a water bath and mixed in a ratio of 1 part Therminol 75 to 2 parts corn oil. This mixture was allowed to cool to room temperature prior to further dilution with corn oil and administration to the animals. The initial heating was necessary to liquify the test article so that workable dilutions could be prepared. Fresh dilutions of Therminol 75 in corn oil were prepared for each pilot and definitive study.



Duration of treatment / exposure:
Single injection
Post exposure period:
All rats were observed for signs of toxicity immediately after administration of the test article, near the end of the working day on which animals were treated, and - when appropriate - each day prior to sacrifice.
Animals in the 24-hr pilot study were weighed on the day of dosing and at sacrifice. Animals in the initial and additional 14-day pilot study were weighed on the day of dosing, 7 days after dosing, and at sacrifice 14 days after dosing. In the definitive study, all animals were weighed on the afternoon prior to dosing, and animals sacrificed 24 hr after dosing were also weighed on the day of sacrifice.
In the definitive study, the groups of surviving rats were sacrificed 6, 12, and 24 hours after treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 2500, 5000 mg/kg bw
Basis:

No. of animals per sex per dose:
6 animals per sex per dose per time of sacrifice
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: intraperitoneal injection
- Doses / concentrations: 0.2 mg/kg body weight
- dissolved in Hanks' balanced salt solution (HBSS)
- Lot: 9558
- Supplier: Polysciences, Inc.; Catalog No. 1250
- Description: Light-colored powder
- Storage conditions: Stored at 4°C in original and secondary containers

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on the results obsreved from the dose range-finding study and the two pilot studies.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately 2 to 3 hr prior to sacrifice, each rat was injected ip with colchicine dissolved in HBSS (4 mg/kg body weight). Skin and muscle tissue were carefully removed from the femur immediately after sacrifice.

DETAILS OF SLIDE PREPARATION:
Slides were prepared by dropping cell suspensions onto water-dipped slides, passing the slides through the flame of an alcohol lamp several times, and checking the cell density using a phase-contrast microscope. If cell density was too thick, more fixative (methanol: glacial acetic acid, 3:1) was added to dilute the suspension. Based on the amount of cell suspension, six or fewer slides were prepared from each tube.
The slides were placed in 7% Giemsa (Gurr's R66 in m/15 Sorensen's buffer, pH 6.8) for 5 to 20 min, rinsed in distilled water, soaked in xylene, and mounted with Permount to make permanent slides for scoring under bright-field or phase-contrast optics (100x oil objective).

METHOD OF ANALYSIS:
Slides were evaluated for mitotic index (based on at least 1000 cells/animal), and 60 cells per animal, when possible, were evaluated for chromosomal aberrations.
For analysis of the slides, score sheets were used to record the cytogeneticist's name, date, microscope used, coded slide number, project number, quality of the slide, vernier settings, MI, chromosome number, and numbers of various categories of chromatid and chromosomal aberrations for each cell scored. Chromatid and isochromatid gaps were recorded for each cell but were not considered chromosomal aberrations in the analysis of the data. After analysis was completed, the slides were decoded and the score sheets were summarized.
Evaluation criteria:
Positive. A test article was considered to have elicited a positive response in the in vivo bone marrow cytogenetic assay if either the mean aberrant cell frequency or the mean chromosomal aberration frequency per cell, or both, was significantly greater (P < 0.05) in the test article-treated animals than in the negative control animals.
Negative. A test article was considered to have elicited a negative response if the results obtained by each cytogeneticist were in general agreement and the criteria for a positive response were not met.
Inconclusive. The results of this assay were considered inconclusive if there was reason to believe that the concentrations of the test article selected for evaluation were inappropriate (i.e., excessive cytotoxicity or lack of any toxicity).
Statistics:
The following statistics were calculated for each animal: MI, the total number of chromosomal aberrations and the frequency of chromosomal aberrations per cell, the number and frequency of aberrations in each category, the number and frequency of cells with structurally aberrant chromosomes, the number and frequency of aneuplold (hyperploid) cells, the number and frequency of polyploid cells, and the number and frequency of severely damaged cells (i.e., cells with > or = 5 chromosomal aberrations).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 4, 20, 100, 500, or 2500 mg/kg
- Clinical signs of toxicity in test animals: There was no significant effect of treatment with Therminol 75 on body weight during the 14-day period of the study. No significant clinical signs of toxicity were noted during the study, and no animals died as a result of treatment with Therminol 75.

PILOT STUDIES
- Dose range: 0, 500, 240, 3350, 5000 mg/kg
- Clinical signs of toxicity in test animals: In the 24-hour pilot study, there was no consistent, significant effect of treatment with Therminol 75 on animal body weights or mitotic indices. No significant clinical signs of toxicity were observed.
In the 14-day pilot study, the mean body weight gain was depressed for males treated with 3350 and 5000 mg/kg Therminol 75. None of the animals died. The only signs of toxicity observed were rough fur for several animals and swelling at the site of injection of Therminol 75 in one male and two females treated with 5000 mg/kg. An LD50 value for Therminol 75 could not be calculated because there were no deaths.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Moderate exudates from one eye were observed in one female (5000 mg/kg dose group) and one male (500 mg/kg dose group) on the afternoon after dosing. The eyes of these animals were clear the next day. No other signs of toxicity were observed.
- Cytological observations: At 6, 12, and 24 hour posttreatment, no significant differences in any of the three measures of cytological damage evaluated were observed between the negative control and Therminol 75-treatment groups in male or female rats .
- Appropriateness of dose levels and route: Based on results from the pilot studies.
- Statistical evaluation: The three measures of cytological damage that were statistically evaluated were the MI, the percentage of chromosomally aberrant cells, and the frequency of chromosomal aberrations per cell.

Any other information on results incl. tables

Cytogenetic evaluation of bone marrow cells treated with Therminol 75:

 

Female rats

6-hour sacrifice

12-hour sacrifice

24-hour sacrifice

Negative control

5000 mg/kg Therm 75

Negative control

5000 mg/kg Therm 75

Negative control

5000 mg/kg Therm 75

0.2 mg/kgTEM

Number of animals

5

5

5

5

5

5

5

Mitotic Index * (Mean % ±SEM)

2.44 ± 0.60

2.61 ± 0.60

5.14 ± 0.98

3.52 ± 1.18

2.20 ± 0.51

3.77 ± 0.61

1.49 ± 0.42

Number of cells analyzed

300

300

300

300

298

300

300

Number of aberrant cells° (Mean % ±SEM)

1 (0.33 ± 0.33)

0

0

0

1 (0.34 ± 0.34)

0

134 (44.67 ± 9.09)

Number of cells with structurally abnormal chromosomes (Mean % ±SEM)

0

0

0

0

1 (0.34 ± 0.34)

0

131 (43.67 ± 9.01)

Number of cells (%) with:

 

 

 

 

 

 

 

Chromosome deletions

0

0

0

0

0

0

0

Chromosome exchanges

0

0

0

0

0

0

0

Chromatid deletions

0

0

0

0

1 (0.34)

0

36 (17.48)

Chromatid exchanges

0

0

0

0

0

0

6 (2.91)

Aneuploidy

1 (0.33)

0

0

0

0

0

1 (1.46)

Plyploidy

0

0

0

0

0

0

0

Severe damage

0

0

0

0

0

0

94 (31.33)

Types of aberrations per cell:

 

 

 

 

 

 

 

Overall frequency of aberrations** (Mean % ±SEM)

0.003 ± 0.003

0

0

0

0.003 ± 0.003

0

1.820 ± 0.508

Chromosome deletions

0

0

0

0

0

0

0

Chromosome exchanges

0

0

0

0

0

0

0

Chromatid deletions

0

0

0

0

1 (0.003)

0

65 (0.316)

Chromatid exchanges

0

0

0

0

0

0

8 (0.039)

Number of cells (%) with:

 

 

 

 

 

 

 

Chromatid gaps

1 (0.33)

1 (0.33)

2 (0.67)

2 (0.67)

0

0

7 (3.40)

Chromosome gaps

0

0

0

0

0

0

0

 

 

Male rats

6-hour sacrifice

12-hour sacrifice

24-hour sacrifice

Negative control

5000 mg/kg Therm 75

Negative control

5000 mg/kg Therm 75

Negative control

5000 mg/kg Therm 75

0.2 mg/kgTEM

Number of animals

5

5

5

5

5

5

5

Mitotic Index * (Mean % ±SEM)

4.57 ± 1.15

5.38 ± 1.45

6.06 ± 2.97

7.24 ± 1.88

6.10 ± 0.68

4.75 ± 0.50

2.53 ± 0.37

Number of cells analyzed

300

290

300

300

300

300

300

Number of aberrant cells° (Mean % ±SEM)

0

0

1 (0.33 ± 0.33)

1 (0.33 ± 0.33)

1 (0.33 ± 0.33)

0

117 (39.00 ± 6.47)

Number of cells with structurally abnormal chromosomes (Mean % ±SEM)

0

0

0

0

0

0

116 (38.67 ± 6.76)

Number of cells (%) with:

 

 

 

 

 

 

 

Chromosome deletions

0

0

0

0

0

0

0

Chromosome exchanges

0

0

0

0

0

0

0

Chromatid deletions

0

0

0

0

0

0

30 (13.45)

Chromatid exchanges

0

0

0

0

0

0

19 (8.52)

Aneuploidy

0

0

1 (0.33)

1 (0.33)

1 (0.33)

0

1 (0.45)

Plyploidy

0

0

0

0

0

0

0 (0.00)

Severe damage

0

0

0

0

0

0

77 (25.67)

Types of aberrations per cell:

 

 

 

 

 

 

 

Overall frequency of aberrations** (Mean % ±SEM)

0

0

0.003 ± 0.003

0.03 ± 0.003

0.003 ± 0.003

0

1.547 ± 0.309

Chromosome deletions

0

0

0

0

0

0

0

Chromosome exchanges

0

0

0

0

0

0

0

Chromatid deletions

0

0

0

0

0

0

49 (0.220)

Chromatid exchanges

0

0

0

0

0

0

29 (0.130)

Number of cells (%) with:

 

 

 

 

 

 

 

Chromatid gaps

0

1 (0.34)

0

2 (0.67)

0

1 (0.33)

6 (2.69)

Chromosome gaps

0

0

0

0

0

0

1 (10.45)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The substance does not induced chromosomal damage in the in vivo micronucleus test.
Executive summary:

This study assessed the ability of Therminol 75 administered by intra-peritoneal injection to induce chromosomal damage in bone marrow cells for Fischer 344 rats. In the definitive study, rates were given Therminol 75 at doses of 0, 500, 2500 and 5000 mg/kg body weight. Groups of animals were sacrificed 6, 12 and 24 hrs after treatment. Cells from animals exposed to 0 and 5000 mg/kg Therminol 75 and those from animals in the positive control groups were evaluated microscopically for mitotic index and chromosomal abnormalities. On the basis of those results, it could be concluded that Therminol 75 dose not induce chromosomal damage in male and female Fischer 344 rats under the conditions used in this study.