3',5'-dichloro-4'-ethyl-2'-hydroxypalmitanilide

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

Study period:

from 1989, june 12th to 1989, july 25th

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to the Test Guidelines described in the EEC Directive 84/449/EEC.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

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

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3',5'-dichloro-4'-ethyl-2'-hydroxypalmitanilide

EC Number:

406-200-8

EC Name:

3',5'-dichloro-4'-ethyl-2'-hydroxypalmitanilide

Cas Number:

117827-06-2

Molecular formula:

C24H39Cl2NO2

IUPAC Name:

N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)hexadecanamide

Details on test material:

RCC NOTOX Substance : 1763
RCC NOTOX old project : 1343
Description : Solid
Purity : 98.5%
Instructions for test article storage : At room temperature in the dark in a tightly sealed container
Stability of test article : Stable for one year under storage conditions
Safety precautions : Gloves, goggles and face mask will be suf’ficient to ensure personnel health and safety
Disposal Category : III
Stability in vehicles : Unknown

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The concentration of cytochrome P—450 (expressed per g wet liver) in the S9—preparation is derived from the sodium dithionite difference spectrum of CO—saturated sampies, using an extinction coefficient of 91 mM-1.cm-1 between Amax and A490nm

Test concentrations with justification for top dose:

100 µg/l
333 µg/l
1000 µg/l
3330 µg/l
5000 µg/l

Vehicle / solvent:

The test substance has been dissolved in dimethylsulphoxide of spectroscopic
quality (Merck). Test substance concentrations were prepared directly prior to
use.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

The vehicle of the test article

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Saline = Physiological saline (Medital Pharma Ned. B.V.)

Positive controls:

yes

Remarks:

TA1535 at 1 ug/plate

Positive control substance:

sodium azide

Remarks:

Without metabolic activation (—S9—mix)

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

Saline = Physiological saline (Medital Pharma Ned. B.V.)

Positive controls:

yes

Remarks:

TA1537 at 60 ug/plate

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation (—S9—mix)

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

Saline = Physiological saline (Medital Pharma Ned. B.V.)

Positive controls:

yes

Remarks:

TA98 at 2 ug/plate

Positive control substance:

other: daunomycine

Remarks:

Without metabolic activation (—S9—mix)

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

TA100 at 650 ug/plate

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (—S9—mix)

Controls 6

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

TA1535 and TA1537 at 5 ug/plate

Positive control substance:

other: 2 aminoanthracene

Remarks:

With metabolic activation (+S9—mix)

Controls 7

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

TA98 and TA100 at 0,5 ug/plate

Positive control substance:

other: 2 aminoanthracene

Remarks:

With metabolic activation (+S9—mix)

Details on test system and experimental conditions:

TEST SYSTEM
Test System : Salmonella typhimurium bacteria
Rationale : Recognized by the international guidelines as the recommended test system (e.g. EPA, OECD, EEC).
Source : Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (1987)
The characteristics of the individual strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3O76 Frameshift
TA98 hisD3O52/R_factor* Frameshift
TA1535 hisG46 Base—pair substitutions
TA100 hisG46IR_f’actor* Base—pair substitutions
*: R—factor = plasmid pKM1O1 (increases error—prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide celicoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair systern (deletion of the ultraviolet— repair B gene)
Stock cultures of the four strains were stored in liquid nitrogen (—196°C).
Strains were regularly checked for their histidine—requirement,
crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV—
sensitivity and the number of spontaneous revertants.
CELL CULTURE
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth
(Oxoid no. 2) and incubated in a shaking bath (37°C, 150 spm), until the cultures reached an
0.0. of 0.4 at 700 nm (10e9 cells/ml). Freshly grown cultures of each strain were used for a test.
Agar plates
Agar plates (diameter 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per
liter: 18 g purified agar (Oxoid, code L28) in Vogel—Bonner Medium E (*), 10 g glucose, 0.5
mg biotin and 0.6 mg histidine. (*) Vogel, H.J. and Bonner, 0.M. Acetylornithinase of Escherichia coli: partial
purification and some properties. J. Biol. Chem. 218 (1956) 97—106.
Top agar
Vogel—Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar.
Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar
tubes were autoclaved for 20 min at 120°C. Prior to the viability determination the top
agars were supplemented with 1.55 mg histidine per top agar.
Environmental conditions
All incubations have been carried out in the dark at 37°C. The temperature was monitored
during the experiment.

Evaluation criteria:

Selection of dose levels
Selection of’ an adequate range of doses was based on a preliminary toxicity
test with strain TA100, both with and without S9—mix. Nine concentrations
have been tested in duplicate for toxicity. The highest concentration of
test article used in the subsequent mutagenesis assay was that which gave a
reduced survival on the non—selective plates. 1f no toxicity was observed,
the highest dose level used in the mutagenesis assay was 5 mg/plate unless
the test article exhibited limited solubility or was not uniformly
dispersible in the solvent of choice.
Direct plate incorporation assay
Five different doses (with approximately half—log steps) of the test
substance have been tested in triplicate in each strain. The test substance
has been tested both with and without S9—mix in each strain, in two
independent experiments. Top agar in top agar tubes was melted and heated
to 45°C. The following solutions were successively added to 3 ml top agar:
0.1 ml of a fresh bacterial culture (10e9 cells/ml) of one of the tester
strains, 0.1 ml of a dilution of’ the test substance in dimethylsulphoxide,
and either 0.5 ml S9—mix (in case of activation assays) or 0.5 ml 0.1 M
phosphate buffer (in case of non—activation assays). The ingredients were
mixed on a Vortex and the content of the top agar tube was poured onto a
selective agar plate. After solidification of the top agar, the plates were
turned and incubated in the dark at 37°C for 48 h .
Colony counting
The revertant colonies (histidine independent) have been counted
automatically with an Artek model 880 colony counter or nianually, if less
than 40 colonies per plate were present. Plates with sufficient test
article precipitate to interfere with automated colony counting have been
counted manually.

Statistics:

No formal hypothesis testing has been done.
A test substance was considered negative (not mutagenic) in the Ames test
if:
a) The total number of revertants in any tester strain at any concentration
was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one
independently repeated experiment.
A test substance was considered positive (mutagenic)in the Ames test if:
a) It induced at least a 2—fold, dose related increase in the number of
revertants with respect to the number induced by the solvent control in
any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 was considered to be not
significant. If the test substance showed in the first test only a
positive response at one or two concentrations, the assay was repeated
with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one
independently repeated experiment.
The preceding criteria were not absolute and other extenuating factors
might enter into the final evaluation decision.

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:

no cytotoxicity

Positive controls validity:

valid

Additional information on results:

TOXICITY OF THE TEST SUBSTANCE
The survival of the TA100 culture is determined by comparing the number of
colonies on the plates containing the test substance with those on the solvent
control plate.
Both in the absence and presence of’ S9—mix the survival of strain TA100 is not
reduced up to test substance concentrations of 5000 mg/plate. Based
on these data, the test substance was tested up to a concentration of 5000
mg/plate in the absence and presence of S9—mix.
THE AMES SALMONELLA/MICROS0ME PLATE TEST
All bacterial strains showed negative responses over the entire dose
range of the test substance, i.e. no dose—related, two—fold, increase in the
number of’ revertants in two independently repeated experiments. The negative
and strain—specific positive control values fell within our laboratory
background historical ranges indicating that the test conditions were optimal
and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the resuits of’ this study It is conciuded that the test substance can
be considered as not mutagenic in the Ames Salmonella/microsome assay.