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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study methodology was broadly consistent with modern OECD test guideline 476, and a claim of GLP compliance was made, however as the guideline itself was not directly followed, and as the study report is missing a few very important details (test substance purity, for example), the study cannot be considered reliable without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
: No lot number, purity or expiry date was indicated.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
C,C'-azodi(formamide)
EC Number:
204-650-8
EC Name:
C,C'-azodi(formamide)
Cas Number:
123-77-3
Molecular formula:
C2H4N4O2
IUPAC Name:
diazene-1,2-dicarboxamide
Details on test material:
- Name: Celogen AZ 130
- Description: yellow powder
no data on purity or batch number
- Source: Pharmakon Research
- Date of recepetion: February 23, 1984
- Storage: at room temperature in the container received from the sponsor.

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell culture: Chinese hamster ovary cells, clone K1, subclone BH4, designated CHO-K1-BH4, from Dr. AbrahamW. Hsie, Biology Division, Oak Ridge National Laboratories, PO Box Y, Oak Ridge, Tennessee 37380
- Type and identity of media: F12FCM5 (Ham's F12 supplemented with 5% dialyzed and heat-inactivated bovine serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors
Test concentrations with justification for top dose:
cytotoxicity: 0.01,0.04, 0.13, 0.4, 1.3, 4.0, 13.3, 40, 133 and 400 µg/ml
mutation assay: 5, 16.7, 50, 167 and 500 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: solubility

All required dilutions were made with DMSO, Lot #741643, supplied by Fisher Scientific.
Dilutions were prepared the day of the test. Dosing solutions were used within two hours of preparation.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: ethyl methanesulfonate [EMS, 200 ug/ml (Sigma M0880)] with S9: dimethylnitrosamine [DMN, 100 ug/ml (Aldrich N2,500-1)]
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period:none
- Exposure duration: 19h
- Expression time (cells in growth medium): 8days

NUMBER OF REPLICATIONS:5

DETERMINATION OF CYTOTOXICITY
- Method: relative cell survival
Evaluation criteria:
The mutant frequency, expressed as TGr mutants/10^6 clonable cells, was calculated by dividing the total number of mutant clones by the number of cells plated, corrected for the cloning efficiency (average of three plates) of the cells at the time of mutant selection.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Celogen AZ, was evaluated in a cytotoxicity prescreen with [2% (v/v)] and without S-9 at dose levels of 0.01, 0.04, 0.13, 0.4, 1.3, 4, 13.3, 40, 133 and 400 µg/ml. The 400 µg/ml dose approached the limit of solubility of the test article. The doses evaluated produced no detectable cytotoxicity to the CHO cell line without metabolic activation and produced 63.1% relative cell survival with metabolic activation at the 400 µg/ml level.
Based upon these findings, Celogen AZ, was evaluated in duplicate cultures in the mutation assay at dose levels of 5, 16.7, 50, 167 and 500 µg/ml with and without S-9.
There were no dose-dependent increases in the mutant frequencies of the cultures treated with the test article. The 500 µg/ml dose approximated the limit of solubility of the test article in DMSO. Therefore, the test article did not exhibit mutagenic activity under the conditions of the assay. This study fulfilled all the criteria of an acceptable assay.

Any other information on results incl. tables

 Mutagenicity Data 
 Control Cultures             
 compound  µg/ml    S-9  Relative Initial Survival (%)  Total No. of Mutants (5 plates)   Cloning Efficiency (%)  Mutant Frequency (Mutants/10^6 clonable cells) 
 Untreated    (-)  108.2  1  68.8   1.5 
 Untreated    (-)  91.8  0  60.3   0.0 
 Untreated    (+)  90.0  0  74.3  0.0 a
 Untreated    (+) 91 0  71. 7 0.0 a
 DMSO 10 (-) 118.2 1 82.2 1.2
 DMSO 10 (-) 86.2 1 72.3 1.4
 DMSO 10 (+) 82 2 65.2 3.1
 DMSO 10 (+) 98.9 8 50.3 15.9
 EMS 200 (-) 88.4 175 57.5 304.3
 EMS 200 (-) 82.7 149 43.3 344.1
 DMN 100 (+) 12.5 90 30.6 294.1
 DMN 100 (+) 10.9 74 41.2 179.6
 Celogen AZ  5 (-) 99 1 57.8 1.7
 Celogen AZ  5 (-) 105.6 1 75.3 1.3
 Celogen AZ  16.7 (-) 82.4 1 63.2 1.6
 Celogen AZ  16.7 (-) 64.9 2 57.5 3.5
 Celogen AZ  50 (-) 88.8 1 58.5 1.7
 Celogen AZ  50 (-) 83.2 0 46.3 0
 Celogen AZ  167 (-) 109.1 0 64.3 0.0 a
 Celogen AZ  167 (-) 76.1 0 58.7 0.0 a
 Celogen AZ  500 (-) 48.7 1 80.7 1.2
 Celogen AZ  500 (-) 54.5 10 65.2 15.3
 Celogen AZ  5 (+) 86.6 1 56.5 1.8
 Celogen AZ  5 (+) 74.2 1 60.2 1.7
 Celogen AZ  16.7 (+) 96.8 0 53 0
 Celogen AZ  16.7 (+) 116.4 1 54 1.8
 Celogen AZ  50 (+) 92 1 65.5 1.5
 Celogen AZ  50 (+) 90.4 2 34.8 5.7
 Celogen AZ  167 (+) 22.7 1 64.2 1.6
 Celogen AZ  167 (+) 26 1 64.2 1.6
 Celogen AZ  500 (+) 7.5 0 70.8 0.0 a
 Celogen AZ  500 (+) 5.7 0 65.2 0.0 a
a = No scorable mutants            

Applicant's summary and conclusion

Conclusions:
Celogen AZ did not exhibit mutagenic activity under the conditions of the assay.
Executive summary:

Celogen AZ, was evaluated in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in cultured Chinese hamster ovary (CHO) cells (Pharmakon Research International 1984, PH314 -UN-003 -84). Cytotoxicity of the test article was first estimated in a prescreen by exposing CHO cells to 10 levels of Celogen AZ in the presence and absence of metabolic activation. The metabolic activation mixture (S-9) contained 2% (v/v) of an Aroclor 1254-induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors. Doses of 0.01, 0.04, 0.13, 0.4, 1.3, 4, 13.3, 40, 133 and 400 µg/ml were evaluated. The 400 µg/ml dose approached the limit of solubility of the test article. The doses evaluated produced no detectable cytotoxicity to the cell line without metabolic activation and resulted in 63.1% relative cell survival with metabolic activation at the 400 µg/ml dose.

Based upon these findings, Celogen AZ was evaluated in duplicate cultures in the mutation assay at dose levels of 5, 16.7, 50, 167 and 500 µg/ml with and without S-9. The 500 µg/ml dose approximated the limit of solubility of the test article in DMSO.

There were no dose-dependent increases in the mutant frequencies of the cultures treated with the test article. Therefore, the test article did not exhibit mutagenic activity under the conditions of the assay. This study fulfilled all the criteria of an acceptable assay.