Dodecamethylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

One Guideline study on reverse bacterial mutagenicity available

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. August 2018 - 08. January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name 1,12-Diaminododecane
Batch no. B1-5-10
Appearance White flakes with different size
Composition 1,12-Diaminododecane, mono-constituent
Purity 99.15%
Homogeneity homogeneous
Expiry date 29.01.2019
Storage Room Temperature (20 ± 5°C)
The following additional information was relevant to the conduct of the study, according to
OECD 471:
CAS No. 2783-17-7
EINECS-No. 220-489-6
Stability H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: 0.1 - 1g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Target gene:

his-, trp-

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Metabolic activation system:

produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraper-itoneally

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the first experiment:
1250 μg/plate, 375 μg/plate, 125 μg/plate, 37.5 μg/plate and 12.5 μg/plate
The following nominal concentrations were prepared for the second experiment:
1250 μg/plate, 625 μg/plate, 313 μg/plate, 156 μg/plate, 78 μg/plate, 39 μg/plate and 20 μg/plate

In a non-GLP pre-test, the solubility of the test item was tested in demineralized water, di-methyl sulfoxide (DMSO), acetone, ethanol and tetra hydrofurane.
The solid test item is not sufficiently soluble in all solvents.
In consultation with the sponsor and based on the non-GLP pre-test, demin. water was cho-sen as vehicle, because this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the first and the second experiment, a stock suspension containing 12.5 g/L of the test item in demin. water was prepared and was warmed up to about 70°C. The test item suspension was not sterile filtrated before use.
The stock suspension was used to prepare the geometric series of the concentrations to be tested.

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was tested in demineralized water, di-methyl sulfoxide (DMSO), acetone, ethanol and tetra hydrofurane.
The solid test item is not sufficiently soluble in all solvents.
In consultation with the sponsor and based on the non-GLP pre-test, demin. water was cho-sen as vehicle, because this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the first and the second experiment, a stock suspension containing 12.5 g/L of the test item in demin. water was prepared and was warmed up to about 70°C. The test item suspension was not sterile filtrated before use.
The stock suspension was used to prepare the geometric series of the concentrations to be tested.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene

Details on test system and experimental conditions:

Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
• 100 μL test suspension at each dose level, solvent (negative control) or reference mutagen solution (positive control)
• 500 μL S9 mix (see chapter 6.5.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension (see chapter 6.4.2, test system, culture of the strains)
• 2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
• 100 μL test suspension at each dose level, solvent (negative control) or reference mutagen solution (positive control)
• 500 μL S9 mix (see chapter 6.5.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 μL bacteria suspension (see chapter 6.4.2, test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous re-vertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

no statistical analysis performed.

Key result

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the results of this study it is concluded that 1,12-Diaminododecane is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Executive summary:

Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines (see chapter 5, page 9).

The test item 1,12-Diaminododecane was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

Experiment 1:

In the first experiment, the test item (suspended in demin. water) was tested up to concen-trations of 1250 μg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and absence of metabolic activation.

Experiment 2:

Based on the first experiment, the test item was tested up to concentrations of 1250 μg/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation.

The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

no studies available

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

as the substance is evaluated as not mutagenic, no mode of action identified

Justification for classification or non-classification

The available Information is conclusive but not sufficient for classification.