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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Both available in vitro studies indicate a negative result for the substance. The endpoint "In vitro gene mutation study in mammalian cells" is covered by Information from Read Across. Information is generated with the well established program "OECD QSAR Toolbox" version 4.2 for the two main components. The components are predicted to be negative for this endpoint.

Link to relevant study records

Reference

Reference 1

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

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:

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:

Target gene:

his-, trp-

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.09.2017 - 22.11.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”, adopted 14. Feb. 2017

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Primary cultures of human peripheral lymphocytes are used

Cytokinesis block (if used):

In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture) was determined in order to assess the cytotoxicity of the test item. rom these determinations, the test item concentrations which were evaluated for scoring of micronuclei were defined.

Metabolic activation:

with and without

Metabolic activation system:

S9 was obtained from Trinova Biochem GmbH, Gießen, and stored at – 80 ± 5 °C.
Batch nos.: 3755, 3691
Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.

Test concentrations with justification for top dose:

If neither cytotoxicity nor solubility are limiting factors, the maximum concentration should be 2 μL/mL, 2 mg/mL or 10 mM, whichever is the lowest. Usually, concentration intervals of approximately 2- to 3-fold will be appropriate. Therefore, testing was started with 2 mg/mL as highest concentration in the pre-experiment.
In the pre-experiment, 5 concentrations (2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.13 mg/mL) of the test item were used and tested with and without metabolic activation.
On the basis of the data found in the pre-experiment, 5 concentrations (125 µg/mL, 62.5 µg/mL, 31.3 µg/mL, 15.6 µg/mL, 7.8 µg/mL) of the test item were used in experiment I and tested without and with metabolic activation. The following concentrations were selected for scoring of micronuclei: 125 µg/mL, 62.5 µg/mL, 31.3 µg/mL.

The same concentrations as in experiment I were used and tested in experiment II (only without metabolic activation, extended exposure time).

Vehicle / solvent:

Ethanol absolute was chosen as solvent, because this solvent has no effect on the viability of cells, does not show genotoxicity and it was the only solvent in which the test item was not completely insoluble but formed a stable suspension.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

According to OECD 487.

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the five per cent level (p < 0.05).
For positive controls with high values of binucleated cells with micronuclei, the chi-squaretest was used.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item N-(hydroxymethyl) stearamide did not show genotoxic activity in this in vitro test for the induction of micronuclei.

Executive summary:

This study was performed to assess the genotoxic potential of N-(hydroxymethyl) stearamide to induce formation of micronuclei in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The study is considered as valid because the micronucleus induction of the solvent controls was in the range of the historical control data/literature data and the positive control compounds Mitomycin C (0.3 μg/mL), CPA (30 μg/mL) and Colchicine (0.035 μg/mL) showed distinct increases in the number of binucleated cells with micronuclei.

In experiment I in the approach without metabolic activation, only the highest test item concentration induced a statistically significant increase in the number of micronuclei in comparison to the solvent control. But since this value is still within the range of the historical control data of the solvent control, this effect is declared as not biologically relevant. A statistically significant dose dependant effect was not observed.

In the approach with metabolic activation, none of the tested concentrations induced a statistically significant increase in the number of micronuclei. All values remained well within the range of the historical control data of the solvent control.

In experiment II no increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations. In addition no statistically significant dose dependant effect was observed.

Finally, no relevant increase of the number of binucleated cells with micronuclei was detected at the evaluated concentrations in both experiments.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

Dec. 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: results derived from OECD QSAR Toolbox, with adequate documentation

Justification for type of information:

In General: The OECD QSAR Toolbox is a software providing databases and tools to characterise a given substance by structure and/or activity ("profiling").
In consequence the databases are screened for analogous with same profilers and categories are formed having same structural or activity profilers.
Therefore the applied approach can be interpreted as Structure-Activity-Relationship.

1. SOFTWARE
OECD QSAR Toolbox 4.2

2. MODEL (incl. version number)

no specific model used.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

CCCCCCCCCCCCCCCC(=O)NCO

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The software is released by OECD and used by ECHA.

5. APPLICABILITY DOMAIN
During category formation and sub-categorisation, a "domain" of analogous is build having same profiles as the target.

6. ADEQUACY OF THE RESULT
The predicted endpoint "in vitro Mammalian Cell Gene Mutation" is adequate to fulfil the information request "Mammalian Cell Gene Mutation"

For further information, please refer to the attached documentation.

Qualifier:

no guideline followed

Principles of method if other than guideline:

OECD QSAR Toolbox is used as a standard program for Read Across

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

not applicable for Read Across with OECD QSAR Toolbox

Target gene:

not applicable for Read Across with OECD QSAR Toolbox

Cytokinesis block (if used):

not applicable for Read Across with OECD QSAR Toolbox

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9

Test concentrations with justification for top dose:

not applicable for Read Across with OECD QSAR Toolbox

Vehicle / solvent:

not applicable for Read Across with OECD QSAR Toolbox

Details on test system and experimental conditions:

not applicable for Read Across with OECD QSAR Toolbox

Rationale for test conditions:

not applicable for Read Across with OECD QSAR Toolbox

Evaluation criteria:

not applicable for Read Across with OECD QSAR Toolbox

Statistics:

not applicable for Read Across with OECD QSAR Toolbox

Key result

Species / strain:

not specified

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

other: not applicable

Untreated negative controls validity:

other: not applicable

True negative controls validity:

other: not applicable

Positive controls validity:

other: not applicable

Key result

Species / strain:

not specified

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

other: not applicable

Untreated negative controls validity:

other: not applicable

True negative controls validity:

other: not applicable

Positive controls validity:

other: not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The component is predicted to be negative for in vitro Mammalian Cell Gene Mutation.

Executive summary:

The genotoxic potential of the component N-(hydroxymethyl)hexadecanamide, CAS 24537-30-2, has been assessed by using Read Across to analogeous substances.

The Read Across was performed with the well established programm OECD QSAR Toolbox.

A prediction for the Endpoints "Genotoxicity, in vitro, Mammalian Cell Gene Mutation, with S9" and "Genotoxicity, in vitro, Mammalian Cell Gene Mutation, without S9" was made.

The component is predicted to be negative for in vitro Mammalian Cell Gene Mutation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

Dec. 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: results derived from OECD QSAR Toolbox, with adequate documentation

Justification for type of information:

In General: The OECD QSAR Toolbox is a software providing databases and tools to characterise a given substance by structure and/or activity ("profiling").
In consequence the databases are screened for analogous with same profilers and categories are formed having same structural or activity profilers.
Therefore the applied approach can be interpreted as Structure-Activity-Relationship.

1. SOFTWARE
OECD QSAR Toolbox 4.2

2. MODEL (incl. version number)

no specific model used.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

CCCCCCCCCCCCCCCCCC(=O)NCO

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The software is released by OECD and used by ECHA.

5. APPLICABILITY DOMAIN
during category formation and subcategorisation, a "domain" of analogous is build having same profils as the target.

6. ADEQUACY OF THE RESULT
The predicted endpoint "in vitro Mammalian Cell Gene Mutation" is adequate to fulfill the information request "Mammalian Cell Gene Mutation"

For further information, please refer to the attached documentation.

Qualifier:

no guideline followed

Principles of method if other than guideline:

OECD QSAR Toolbox is used as a standard program for Read Across

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

not applicable for Read Across with OECD QSAR Toolbox

Target gene:

not applicable for Read Across with OECD QSAR Toolbox

Cytokinesis block (if used):

not applicable for Read Across with OECD QSAR Toolbox

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9

Test concentrations with justification for top dose:

not applicable for Read Across with OECD QSAR Toolbox

Vehicle / solvent:

not applicable for Read Across with OECD QSAR Toolbox

Details on test system and experimental conditions:

not applicable for Read Across with OECD QSAR Toolbox

Rationale for test conditions:

not applicable for Read Across with OECD QSAR Toolbox

Evaluation criteria:

not applicable for Read Across with OECD QSAR Toolbox

Statistics:

not applicable for Read Across with OECD QSAR Toolbox

Key result

Species / strain:

not specified

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

other: not applicable

Untreated negative controls validity:

other: not applicable

True negative controls validity:

other: not applicable

Positive controls validity:

other: not applicable

Key result

Species / strain:

not specified

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

other: not applicable

Untreated negative controls validity:

other: not applicable

True negative controls validity:

other: not applicable

Positive controls validity:

other: not applicable

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

The component is predicted to be negative for in vitro Mammalian Cell Gene Mutation.

Executive summary:

The genotoxic potential of the component N-(hydroxymethyl)octadecanamide, CAS 3370-35-2, has been assessed by using Read Across to analogeous substances.

The Read Across was performed with the well established programm OECD QSAR Toolbox.

A prediction for the Endpoints "Genotoxicity, in vitro, Mammalian Cell Gene Mutation, with S9" and "Genotoxicity, in vitro, Mammalian Cell Gene Mutation, without S9" was made.

The component is predicted to be negative for in vitro Mammalian Cell Gene Mutation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

As no effects are observed in the in vitro studies and predictions in the QSAR Toolbox indicates a negative result, the substance is considered to be not genotoxic and no classification is necessary.