1,2-Ethanediamine, N1-(2-aminoethyl)-, 2-hydroxy-2-phenylethyl and stearyl derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in Ames assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 1993 to 29 September 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Use of Sodium Azide (NaA) instead of Methyl Methane Sulfonate (MMS) as positive control on TA100 without metabolc activation. The test article appearance was not lquid to viscous as mentioned in the signed protocol.

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:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
3.86
- Expiration date of the lot/batch:
August 1994
- Identification: Steaiyl-Diphenoxyethyl-Dimethylentriamin (FAT 92267/A)
- Dispensary code no: 515-93

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature

Target gene:

Salmonella typhimurium histidine (his) reversion system.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 from liver of rats treated with AROCLOR 1254.

Test concentrations with justification for top dose:

- Preliminary study only on strain TA 100: 6173/1235/247/49/10 µg/plate corresponding to active ingredient concentrations of 5000/1000/200/40 and 8 µg/plate.
- Main study number 1 (in all strain): 6173/1235/247/49/10 µg/plate corresponding to active ingredient concentrations of 5000/1000/200/40 and 8 µg/plate.
- Main study number 2 (in all strain): 6173/3087/1543/772/386 µg/plate corresponding to a active ingredient concentrations of 5000/2500/1250/625/313 µg/plate.

Vehicle / solvent:

- Identification: Absolute ethanol
- Supplier: Prolabo
- Batch number: 92113 and 92350.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Absolute ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation: TA98, TA1538, TA1537, TA1535, TA100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation: TA98 TA1538

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation: TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation: TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation: TA 100

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

NUMBER OF REPLICATIONS: Triplicate

All the preparations were used on the day of formulation.

Treatment design:
After treatment each tube of molten agar contains:
- 2 ml of molten agar
- 100 µl of bacterial culture
- 50 or 100 µl of the test article formulated depending on the vehicle used
- 500 µl of S9 Mix (if appropriate)

The mixture will be poured onto VB medium constituting an agar upper layer. The petri dishes will be incubated at 37 °C ± 1 °C for 48 hours minimum.

Evaluation criteria:

The assay will usually be considered valid if the following criteria are met:
- The mean negative control counts fall within the normal range.
- The positive control chemicals induce clear increases in revertant numbers.
confirming discrimination between different strains, and an active Sc Mix preparation
- No more than 5 % of the plates in the assay are lost through contamination or some other unforeseen event.

The test article will be considered to be clearly mutagenic if:
- The number of revertants in presence of the test article is greater than or equal to twice the number of revertants in the negative control at one dose level (the upper dose before toxicity) or more with a dose correlation
- The positive trends/effects are reproducible.

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at 1235 and 6173 microgr./plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at 1235 and 6173 microgr./plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

- PRELIMINARY STUDY:

A precipitate appeared during incorporation of the test article solutions at concentrations of 6173 and 1235 µg/plate.

 

- MAIN STUDY No1:

A precipitate was noted during incorporation at 1235 and 6173 µg/plate. On a few occasion the colonies were counted manually instead of automatically.

*Without metabolic activation:

Signs of toxicity were observed at 6173 microgr./plate in all the tester strains and at 1235 µg/plate in TA1535, TA 1537 and TA 1538 only. For all tested concentrations, the mean number of colonies was less than twice the mean number of colonies in the negative control except with TA1537 at 6173 µg/plate where severe toxicity was noted. In this case, the increase of the mean number of colonies cannot be interpreted as the result of a mutagenic activity. All data were acceptable and no positive increase in the number of revertants per plate containing the test article was observed with any of the tester strains. All criteria for a valid study were met.

*With metabolic activation:

Signs of toxicity were noted in all the tester strains at 6173 µg/plate. For all tested concentrations, the mean number of colonies was less than twice the mean number of colonies in the negative control with each of the tester strains. All data were acceptable and no positive increase in the number of revertants per plate containing the test article was observed with any of the tester strains. All criteria for a valid study were met. 

- MAIN STUDY N°2:

From the results obtained in the first study, a closer range of doses near to the top limit dose were used in this study: 386 / 772 / 1543 / 3087 and 6173 microgr./plate in both the presence and absence of metabolic activation system (corresponding to active ingredient concentrations of 313, 625, 1250, 2500 and 5000 µg/plate, respectively). The highest dose-level corresponded approximately to an active ingredient concentration of 5000 µg/plate which is the required maximum dose-level. A precipitate was constantly noted after the incorporation at 1543 / 3087 and 6173 µg/plate. Precipitation was also occasionally seen at lower dose levels. As a result of this precipitate, colonies were systematically counted manually at 6173 µg/plate and on a few occasions only at 3087 and 1543 µg/plate.

*Without metabolic activation:

Toxicity was observed in all the tester strains at the two highest dose-levels except with TA98 at 3087 µg/plate. For all concentrations, the mean number of colonies was less than twice the mean number of colonies in the negative control with each of the tester strains. All data were acceptable and no positive increase in the number of revendants per plate containing the test article was observed with any of the tester strains. All criteria for a valid study were met. 

*With metabolic activation:

Toxicity was observed in all the tester strains at the highest dose-level of 6173 µg/plate. For all concentrations, the mean number of colonies was less than twice the mean number of colonies in the negative control with each of the tester strains. All data were acceptable and no positive increase in the number of revertants per plate containing the test article was observed with any of the tester strains. All criteria for a valid study were met.

Conclusions:

FAT 92267/A is not mutagenic in Ames assay.

Executive summary:

The bacterial reverse mutation assay was performed following OECD Guideline 474. The test was performed under GLP. The mutagenic potential of FAT 92267/A was evaluated using Salmonella typhimurium in the absence and presence of a liver microsomal system. The test article (FAT 92267/A) was tested on 5 strains of Salmonella typhimurium (TA98, TA 100, TA 1535, TA 1537, TA 1538), with and without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strain TA 100 without metabolic activation. The 5 concentrations (6173, 1235, 247, 49 and 10 µg/plate corresponding to active ingredient concentrations of 5000, 1000, 200, 40 and 8 µg/plate, respectively) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near to the top limit dose (6173, 3087, 1543, 772 and 386 µg/plate corresponding to active ingredient concentrations of 5000, 2500, 1250, 625 and 313 µg/plate respectively). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study. Under the experimental conditions employed, it can be concluded that FAT 92267/A, did not cause an increase in the number of revertants per plate of any tester strains used TA 98, TA 100, TA 1535, TA 1537 and TA 1538 either in presence or absence of a metabolic activation system in both the two independent studies performed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The bacterial reverse mutation assay was performed following OECD Guideline 471. The test was conducted under GLP conditions.The mutagenic potential of FAT 92267/A was evaluated using Salmonella typhimurium in the absence and presence of a liver microsomal system. The test article (FAT 92267/A) was tested on 5 strains of Salmonella typhimurium (TA98, TA 100, TA 1535, TA 1537, TA 1538), with and without metabolic activation. A range of sub-toxic concentrations was determined in a preliminary study on the strain TA 100 without metabolic activation. The 5 concentrations (6173, 1235, 247, 49 and 10µg/plate corresponding to active ingredient concentrations of 5000, 1000, 200, 40 and 8 µg/plate, respectively) were tested in triplicate on the strains mentioned above with and without metabolic activation. The results were confirmed in a second independent study, using a closer range near to the top limit dose (6173, 3087, 1543, 772 and 386µg/plate corresponding to active ingredient concentrations of 5000, 2500, 1250, 625 and 313µg/plate respectively). A negative control (vehicle) and a positive control (specific standard mutagen) were included in each study. Under the experimental conditions employed, it can be concluded that FAT 92267/A, did not cause an increase in the number of revertants per plate of any tester strains used TA 98, TA 100, TA 1535, TA 1537 and TA 1538 either in presence or absence of a metabolic activation system in both the two independent studies performed.

Justification for classification or non-classification

Based on the findings of the genetic toxicity study, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.