Fatty acids, C18-unsatd., reaction products with ammonia-ethanolamine reaction by-products

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Berol amine 715; Limited reporting; not all strains used; 2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have.

GLP compliance:

yes

Remarks:

Following GLP principles (Statement SD, QA inspection)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products

EC Number:

629-757-0

Cas Number:

1224966-15-7

Molecular formula:

UVCB, no structural formula can be set

IUPAC Name:

Fatty acids, C18 unsaturated, reaction product with ammonia-ethanolamine reaction by-products

Details on test material:

The test material was in the form of a dark brown viscous liquid and was stored in the dark under ambient conditions. The pH value for the test substance was 11.2.

Method

Target gene:

All these strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium.

S. typhimurium TA 1535 his G46 rfa -Δ uvr B -is derived from the his G46 strain but is a deep rough * excision repair deficient ** strain which is highly sensitive to base-pair substitution mutagens.
S. typhimurium TA 1537 his C3076 rfa -Δ uvr B -is a strain derived from a his C3076 mutant and carries deep rough and excision repair deficiency mutations. This strain is highly sensitive to frameshift mutagens.
S. typhimurium strain TA 1538 his D30S2 rfa -Δ uvr B-is a strain derived from a his D3052 mutant, and also carries deep rough and excision repair deficiency mutations. This strain is highly sensitive to frameshift mutagens.
S. typhimurium TA 98 his D3052 rfa - Δ uvr B - R + is also derived from a his 03052 mutant, but in addition to the deep rough and excision repair deficiency mutations it also carries a resistance transfer factor. This additional factor makes TA 98 very sensitive to the weaker frame-shift mutagens.
S. typhimurium TA 100 his G46 rfa -Δ uvr B -R + derived from the his G46 strain and carries the deep rough and excision repair deficiency mutations and also a resistance transfer factor. This additional factor makes TA 100 particularly sensitive to basepair substitution mutagens.
*All the five strains described above carry a mutation known as deep rough which affects the cell membrane so that the normal lipopolysaccharide cell coat is defective. These strains therefore allow a greater permeability of test agents across this membrane and into the cell.
** In bacteria almost all the primary dramage caused to DNA bya mutagen is repaired by excision and recombinatim repair systems so that normalIy only a small percentage of the potential mutagenic damage is expressed. The five tester strains used here lack this excision repair thus making them more sensitive to mutagens.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

preliminary toxixity test: 33, 100, 333, 1000, 3333 and 10000 µg/plate
mutation tests: 1, 10, 33, 100 and 333 µg/plate

Vehicle / solvent:

Acetone (Koch-Light Laboratories, Colnbrook, Berkshire)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

2-Aminoanthracene (Lot NO. 122837) from the Aldrich Chemical Company, Wembley, Middlesex, England. Sodium azide (Lot No. 042676) 9-aminoacridine (Lot No. W333JE) and 2-nitrofluorene (Lot No. A3A) frin IIT Research Institute, Chicago USA.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: none
- Exposure duration: 48h

NUMBER OF REPLICATIONS: the mutation assay was performed in triplicate and an independent repeat was also done.


NUMBER OF CELLS EVALUATED: colonies of 0.1 mm or more in diameter were counted with a Biotran III automated counter (New Brunswick Inc., N.J., U.S.A.) at maximum sensitivity.

DETERMINATION OF CYTOTOXICITY
- Method: number of colonies and the background lawn was detemined

Evaluation criteria:

A positive mutagenic response is recorded if there is:

i) for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98, at least a doubling of the mean concurrent control values at some
concentration of the test substances and for S. typhimurium strain TA 100, a 1.5-fold increase over the control value.

ii) a dose related response, although at high dose levels this relationship may be inverted because of, for example, (1) toxicity
to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where
mutagens require metabolic activation by the liver.

iii) a reproducible effect in independent tests.

Statistics:

not applicable

Results and discussion

Test results

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:

toxicty was observed from 100 µg/plate in TA 1535 and TA 98 and from 333 µg/plate in TA 1537 nad TA 100

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Inadequate responses were obtained with 2-aminoanthracene with strains TA 1537 and TA 100 in the first test, so it was decided to retest
Berolamine 715 with these strains in the presence of S-9 mix (Test 3). The results of this test confirmed that the test substance was not
mutagenic in these strains in the presence of S-9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have. Although structure does not give rise for this specific concern.

2-Aminoanthracene was used as the sole indicator of the efficacy of the S9-mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In the Ames test the test substance was not mutagenic in any of the 5 strains used in this study either in the presence or absence of S-9 mix.

Executive summary:

In the Ames test the test substance was not mutagenic in any of the 5 strains used in this study either in the presence or absence of S-9

mix. Toxicity to the background lawn of bacteria was recorded at concentrations above 100 µg/plate.With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs in the strains tested in this study.