Reaction mass of N,N’-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide), Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]- and Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 29 August 2012 to 17 September 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, 12-hydroxy-,reaction products with ethylenediamine

IUPAC Name:

Octadecanoic acid, 12-hydroxy-,reaction products with ethylenediamine

Constituent 2

Reference substance name:

100545-48-0

Cas Number:

100545-48-0

IUPAC Name:

100545-48-0

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Octadecanoic acid, 12-hydroxy-,reaction products with ethylenediamine

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

These strains were used to detect base changes and frameshift mutations as follows:
- Base change mutagens: S. typhimurium TA1535 and TA100, and E. coli WP2 uvrA (pKM101).
- Frameshift mutagens: S. typhimurium TA1537 and TA98.

Additional strain / cell type characteristics:

other: The genotype of each bacterial strain were: S. typhimuriumTA1535: hisG46, rfa ∆ uvrB; TA1537: hisC3076, rfa ∆ uvrB; TA98: hisD3052, rfa ∆ uvrB pKM101; TA100: hisG46, rfa ∆ uvrB pKM101; E.Coli WP2 uvrA: trpE ochre, uvrA pKM101.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, from male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Plate incorporation assay: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (with and without metabolic activation)
Pre-incubation assay: 50, 150, 500, 1500 and 5000 μg/plate (with and without metabolic activation)

Vehicle / solvent:

Water containing 0.15% bacteriological agar

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Sodium azide: (2 μg/plate for strains TA100 and TA1535), 9-aminoacridine: (50 μg/plate for strain TA1537), 2-nitrofluorene: (2 μg/plate for strain TA98) and 4-nitroquinoline-1-oxide: (2 μg/plate for strain WP2 uvrA (pKM101)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

2-Aminoanthracene: 5 μg/plate for strains TA100 and TA1535 and 10 μg/plate for strain WP2 uvrA (pKM101); Benzo[a]pyrene: 5 μg/plate for strains TA98 and TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate incorporation method: (Test 1)
- Aliquots of 0.1 mL of the test substance solutions (at 5, 15, 50, 150, 500, 1500 and 5000 μg/plate), positive control or vehicle control (water + 0.15% agar) were placed in glass tubes. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10 h bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labeled with a unique code, identifying the contents of the dish. Triplicates were used for each treatment.
- Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 h. After the incubation period, the appearance of the background bacterial lawn was examined and revertant colonies were counted using an automated colony counter. Any toxic effects of the test substance were detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration was selected for use in the second test. If toxic effects were observed, a lower concentration would be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least one precipitating concentration should be included in the second test.

Preincubation method: (Test 2)
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Evaluation criteria:

- Test substance is considered to have mutagenic activity: If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

- Test substance is not considered to have mutagenic activity: If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.

- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

- Test is considered valid: If the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls. Mean viable cell counts in the 10-h bacterial cultures must be at least 10E09/mL.

Statistics:

The statistical procedures used were usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance was considered along with statistical significance.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Test 1:
- No evidence of toxicity was obtained following exposure to the test substance. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Test 2:
- No evidence of toxicity was obtained following exposure to the test substance.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test substance at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

- The results in detail obtained with the test substance and positive control compounds are presented in appendix 2 of the attached background material.

 - The absence of colonies on sterility check plates confirmed the absence of microbial  contamination of the S9 mix, buffer and test substance formulation.

- The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10⁹/mL in all cases, and therefore met the acceptance criteria.

- The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

-  Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance showed no evidence of mutagenic activity in the bacterial system, with and without emetabolic activation.

Executive summary:

An in vitro Ames assay was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471, EU Method B.13/14 and EPA OPPTS 870.5100 in compliance with GLP.

Tester strains TA98, TA100, TA1535 and TA1537 ofSalmonella typhimuriumand strain WP2uvrA ofEscherichia coliwere evaluated in the presence and absence of metabolic activation. The test was performed in two independent assays using the plate incorporation and the pre-incubation methods. Water containing 0.15% bacteriological agar was selected as the vehicle of choice. In the first test, the dose levels were: 5, 15, 50, 150, 500, 1,500 and 5,000 μg/plate (with and without metabolic activation). In the second test, the dose levels were 50, 150, 500, 1,500 and 5,000 μg/plate (with and without metabolic activation). No positive mutagenic responses were observed with any of the strains in the absence and presence of S9 mix. Neither any precipitate nor appreciable toxicity was seen.

Under the study conditions, the test substance therefore showed no evidence of mutagenic activity in the bacterial system.