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EC number: 211-776-7 | CAS number: 694-83-7
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- 5 October 1984 - 22 February 1985
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: GLP study, the study is comparable to OECD 476. The purity is not indicated and analytical certificate is not available.
Cross-referenceopen allclose all
- Reason / purpose:
- reference to same study
- Reason / purpose:
- reference to other study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 985
- Report Date:
- 1985
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- no
- Type of assay:
- mammalian cell gene mutation assay
Test material
Reference
- Name:
- Unnamed
- Type:
- Constituent
- Details on test material:
- - Name of test material (as cited in study report): Hexamethylene Diamine
- Physical state: clear liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Lot/batch No.: no data
- Stability under test conditions: no data
- Storage condition of test material: in the plastic container received from the sponsor, maintained in frozen aliquots in a Revco Ultra-low Freezer.
Method
- Target gene:
- Hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
serum free medium F12 (for wash)
F12FCM5 (for plate) - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- At dose levels of 25, 50, 100, 175 and 250 µg/mL of treatment volume without metabolic activation preparation.
At doses levels of 50, 100, 300, 450 and 600 µg/mL of treatment volume with a 5% concentration of metabolic activation preparation.
See below Table 7.6.1/1 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water supplied by Pharmakon Research International.
- Justification for choice of solvent/vehicle: no data
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Ethylmethanesulfonate (EMS) and Dimethylnitrosamine (DMN)
- Remarks:
- Positive controls: EMS was used at 200 µg/mL without S9 activation and DMN was used at 100 µg/mL with S9 activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- in suspension for preliminary cytotoxicity and mutagenicity screen
- in agar (plate incorporation) for the main test
DURATION
- Preincubation period: No
- Exposure duration: 5 h
- Selection time (if incubation with a selection agent): 7 days
SELECTION AGENT (mutation assays): hypoxanthine
NUMBER OF REPLICATIONS:
Triplicate
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- - The test substance was tested to the level of at least 10% survival or to a level deemed acceptable by the sponsor.
- At least three of the five test substance concentrations should produce toxicity ranging from 10 to 100% survival in the cytotoxicity assay.
- ideally, at least one concentration should induce toxicity of less than 50% survival in the assay.
- The plating efficiency of the solvent control was at least 50%.
- The spontaneous mutation frequency of the solvent control was within 2 standard deviations of the historical mean frequency for Pharmakon Research International, Inc. - Statistics:
- Dose-response analyses were performed on transformed mutation frequency data by the one-way analysis of variance method outlined by Snee and Irr (1981). Dose-response was considered significant if p < 0.01.
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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No additional data
- Remarks on result:
- other: strain/cell type: CHO-K1-BH4
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table 7.6.1/2: Preliminary cytotoxicity Screen
| Relative Cell Survival (%) | |||||
S.9 concentrations | ||||||
Test article | µg/mL | 0% | 1% | 2% | 5% | 10% |
Untreated | - | 100a | 77.6 | 103.6 | 99.5 | 102.0 |
HMD | 3 | 105.0 | 144.1 | 130.2 | 96.1 | 100.3 |
HMD | 9 | 109.9 | 116.2 | 128.8 | 123.6 | 107.9 |
HMD | 30 | 117.6 | 112.1 | 99.7 | 101.6 | 96.1 |
HMD | 91 | 98.0 | 108.3 | 117.6 | 109.1 | 103.5 |
HMD | 304 | 53.2 | 115.3 | 109.8 | 97.6 | 89.0 |
HMD | 912 | 0 | 0 | 0 | 0 | 0 |
HMD | 3039 | 0 | 0 | 0 | 0 | 0 |
HMD | 9116 | 0 | 0 | 0 | 0 | 0 |
aAbsolute cloning efficiency = 63.5 %
Table 7.6.1/3: Preliminary mutagenicity Screen
Compound | µg/mL | S.9 (±) | Relativea Initial Survival (%) | Total No. of Mutants (5 plates) | Cloning Efficiency (%) | Mutant Frequency (Mutants/106clonables cells) | Mean Mutation Frequency |
| 1% S.9 Concentration | ||||||
Untreated | - - | - - | 99.6 100.4 | 13 8 | 108.3 97.5 | 12.0 8.2 |
10.1 |
- - | + 1% + 1% | 102.9 98.9 | 4 9 | 96.0 86.8 | 4.2 10.4 |
7.3 | |
HMD | 100 100 | + 1% + 1% | 85.2 86.8 | 6 2 | 63.8 96.0 | 7.2 2.1 |
4.6 |
400 400 | + 1% + 1% | 2.4 3.1 | 4 11 | 91.5 83.0 | 4.4 13.3 |
8.9 | |
600 600 | + 1% + 1% | - - | - - | - - | - - |
- | |
| 2% S.9 Concentration | ||||||
Untreated | - - | - - | 99.6 100.4 | 13 8 | 108.3 9705 | 12.0 8.2 |
10.1 |
- - | + 2% + 2% | 88.5 102.9 | 12 5 | 92.8 95.2 | 12.9 5.3 |
9.1 | |
HMD | 100 100 | + 2% + 2% | 91.2 94.9 | 6 10 | 87.7 96.8 | 6.8 10.3 |
8.6 |
400 400 | + 2% + 2% | 55.4 53.2 | 4 3 | 96.3 85.8 | 4.2 3.5 |
3.9 | |
600 600 | + 2% + 2% | 35.7 25.8 | 5 2 | 111.5 87.7 | 4.5 2.3 |
3.4 | |
| 5% S.9 Concentration | ||||||
Untreated | - - | - - | 99.6 100.4 | 13 8 | 108.3 97.5 | 12.0 8.2 |
10.1 |
- - | + 5% + 5% | 97.7 91.0 | 9 5 | 99.5 92.2 | 9.0 5.4 |
7.2 | |
HMD | 100 100 | + 5% + 5% | 85.5 86.0 | 5 10 | 86.7 105.7 | 5.8 9.5 |
7.7 |
400 400 | + 5% + 5% | 66.1 52.6 | 13 4 | 99.5 98.0 | 13.1 4.1 |
8.6 | |
600 600 | + 5% + 5% | 23.8 27.6 | 9 3 | 98.5 80.5 | 9.1 3.7 |
6.5 | |
| 10% S.9 Concentration | ||||||
Untreated | - - |
| 99.6 100.4 | 13 8 | 108.3 97.5 | 12.0 8.2 |
10.1 |
- - | + 10% + 10% | 95.4 83.9 | 12 11 | 105.5 100.7 | 11.4 10.9 |
11.1 | |
HMD | 100 100 | + 10% + 10% | 86.2 77.9 | 10 10 | 90.2 85.7 | 11.1 11.7 |
11.4 |
400 400 | + 10% + 10% | 68.9 62.6 | 7 8 | 81.8 92.5 | 8.6 8.6 |
8.6 | |
600 600 | + 10% + 10% | 37.4 38.3 | 7 16 | 89.3 87.2 | 7.8 18.3 |
13.0 | |
DMN
| 100 100 | + 10% + 10% | 28.9 28.1 | 146 126 | 61.5 64.2 | 237.4 196.3 |
216.9 |
200 200 | - - | 72.4 53.2 | 161 158 | 83.2 71.2 | 193.5 221.9 |
207.6 | |
| 0% S.9 Concentration | ||||||
Untreated | - - | - - | 98.4 101.6 | 5 8 | 92.5 97.0 | 5.4 8.2 |
6.8 |
HMD | 50 50 | - - | 103.8 96.2 | 2 3 | 76.2 85.3 | 2.6 3.5 |
3.1 |
100 100 | - - | 60.7 64.8 | 8 2 | 75.3 89.5 | 10.6 2.2 |
6.4 | |
200 200 | - - | 51.5 39.5 | 4 3 | 76.8 95.3 | 5.2 3.1 |
4.1 | |
250 250 | - - | 28.4 22.7 | 11 22 | 87.0 78.5 | 12.6 28.0 |
20.3 | |
EMS | 200 200 | - - | 54.1 52.3 | 156 172 | 70.2 69.8 | 222.2 246.4 |
234.3 |
aAbsolute cloning efficiency: - Mean for 1-2-5-10% = 79.9
- Mean for 0% = 68.4
Table 7.6.1/4: Mutagenicity Data
Compound | µg/mL | S.9 (±) | Relativea Initial Survival (%) | Total No. of Mutants (5 plates) | Cloning Efficiency (%) | Mutant Frequency (Mutants/106clonables cells) | Mean Mutation Frequency |
| 1% S.9 Concentration | ||||||
HMD
| 25 25 25 | - - - | 109.4 105.6 99.5 | 4 5 7 | 90.2 94.3 86.0 | 4.4 5.3 8.1 |
6.0 |
50 50 50 | - - - | 83.4 98.6 74.3 | 1 0 16 | 86.3 93.7 84.7 | 1.2 0b 18.9 |
6.7 | |
100 100 100 | - - - | 66.0 62.4 47.4 | 4 3 2 | 84.7 86.0 79.2 | 4.7 3.5 2.5 |
3.6 | |
175 175 175 | - - - | 58.3 54.9 38.7 | 1 4 0 | 88.7 93.3 86.7 | 1.1 4.3 0b |
1.8 | |
250 250 250 | - - - | 35.5 15.5 39.6 | 6 4 3 | 93.5 83.8 74.8 | 6.4 4.8 4.0 |
5.0 | |
|
| ||||||
HMD | 50 50 50 | + 5% + 5% + 5% | 81.1 82.1 98.9 | 9 3 14 | 84.8 87.7 105.8 | 10.6 3.4 13.2 |
9.0 |
100 100 100 | + 5% + 5% + 5% | 85.3 83.1 78.4 | 8 6 2 | 91.5 92.7 87.0 | 8.7 6.5 2.3 |
5.9 | |
300 300 300 | + 5% + 5% + 5% | 76.7 69.3 73.5 | 3 5 7 | 100.5 108.3 94.5 | 3.0 4.6 7.4 |
5.0 | |
450 450 450 | + 5% + 5% + 5% | 55.4 47.1 40.3 | 2 3 5 | 91.8 76.7 88.7 | 2.0 3.8 6.0 |
4.0 | |
600 600 600 | + 5% + 5% + 5% | 36.1 40.6 30.5 | 1 15 12 | 91.8 76.7 86.7 | 1.1 19.6 13.5 |
11.4 | |
aAbsolute cloning efficiency: Mean = 93.5
bNot scorable mutants.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay
Under the test conditions, Hexamethylene diamine was found not to induce mutations in this in vitro CHO/HGPRT mammalian cell forward gene mutation assay. - Executive summary:
The ability of Hexamethylene Diamine to induce mutations at the hypoxanthine-guanine phosphoribosyl transférase (hgprt) locus in Chinese hamster Owary (CHO) cells (clone K1 designated CHO-K1-BH4) was tested following directives of the OECD guideline 476 and in compliance with GLP.
A Preliminary Cytotoxicity Screen was performed to evaluate HMD for cytotoxicity in CHO at 8 dose levels with and without metabolic activation. Hexamethylene Diamine was then evaluated in a Preliminary Mutagenicity Screen at several dose levels, with and without metabolic activation. Based on the results of the Preliminary Mutagenicity, the test article mutagenicity was tested with and without a 5% concentration of metabolic activation preparation.
Hexamethylene Diamine was evaluated in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at dose levels of 25, 50, 100, 175 and 250 µg/mL of treatment volume without metabolic activation preparation and at dose levels of 50, 100, 300, 450 and 600 µg/mL of treatment volume with a 5% concentration of metabolic activation preparation.
The positive control articles, EMS and DMN, indicate that the integrity of the test system was valid.
No statistically significant increases of mutants in HMD treated cultures were observed when compared to the negative controls. Results for Hexametylene Diamine were negative in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at the dose levels and S9 concentrations tested.
Under the test conditions, HMD didn't induce mutagenicity in mammalian cells in the absence and the presence of activation system.
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