Reaction mass of N-(hydroxymethyl)hexadecan-1-amide and N-(hydroxymethyl)stearamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05. Sep. 2017 - 19. Feb. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N-(hydroxymethyl)hexadecan-1-amide and N-(hydroxymethyl)stearamide

EC Number:

911-553-0

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of N-(hydroxymethyl)hexadecan-1-amide and N-(hydroxymethyl)stearamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 103/16
- Expiration date of the lot/batch: June 2018
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: assumed stable , no indication of the contrary reported
- Solubility and stability of the test substance in the solvent/vehicle: assumed stable, no indication of the contrary reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumed non-reactiv , no indication of the contrary reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Method

Target gene:

his-, trp-

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the first experiment:
5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
The following nominal concentrations were prepared for the second experiment:
5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate.

Top dose according to Guideline

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

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:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 20 min
- Incubation time: 48 h

SELECTION AGENT (mutation assays): none

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: not applicable

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: number of colonies and bacterial background
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
none

- OTHER:

Rationale for test conditions:

according to Guideline

Evaluation criteria:

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.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant
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:

none applied

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted when the pre-incubation method was applied. Strain TA 100, 5000 µg/plate, no metabolic activation. Other concentrations or Strains were not affected.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that N-(hydroxymethyl) stearamide
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.

The test item N-(hydroxymethyl) stearamide 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.

In the first experiment, the test item (dissolved in ethanol) was tested up to concentrations

of 5000 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

the absence of metabolic activation.

Based on the first experiment, the test item was tested up to concentrations of 5000

μg/plate in the absence and presence of S9-mix in all bacteria strains using the preincubation

method.

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

The bacterial background lawn was not visible at the highest concentration (5000 μg/plate)

in the treatment without metabolic activation towards the bacteria strain TA100, and no

growth of this bacteria strain was observed, either.

Towards the other bacteria strains (TA97a, TA98, TA100 with metabolic activation, TA102

and TA1535) no cytotoxicity was observed.

The results of this experiment 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.