Reaction products of behenic acid, Eicosadioic acid and Polyglyceryl-10

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

One Guideline Study "bacterial reverse mutation assay" available

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 July 2004 - 22 September 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

Identity: Nomcort HK-P
Batch No.: 2-10-A
·Aggregate States
at Room Temperature: solid
·colour: whitish
Purity: 100 %
Stability in Solvent: not indicated by the sponsor
Storage: room temperature
Expiration Date: April 29, 2007

Target gene:

his-, trp-

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix: Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33; 100; 333; 1000; 2500; and 5000 μg/plate
Concentration range in the main test (without metabolic activation): 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

On the day of the experiment, the test item Nomcort HK-P was dissolved in dry THF
(MERCK, D-64293 Darmstadt; purity > 99.5 % ). The solvent was chosen because of its
solubility properties and its relative non-toxicity to the bacteria (5).
The test item precipitated in the overlay agar at 1000 μg/plate and above with and without
metabolic activation in both experiments. The undissolved particles had no influence on
the data recording.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar
plates:
25 μL Test solution at each dose level, solvent (negative control) or
100 μL reference mutagen solution (positive control),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test
without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL Overlay agar
In the pre-incubation assay 25 μL test solution or 100 μL reference mutagen solution, 500
μL S9 mix I S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a
test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 ml overlay agar
( 45° C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in
the dark (2).

Rationale for test conditions:

according to Guideline

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of
revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or
thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded
at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically
relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is
regarded as an indication of a mutagenic potential if reproduced in an independent second
experiment. However, whenever the colony counts remain within the historical range of
negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required (2).

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the
experimental conditions reported, the test item did not induce gene mutations by base pair
changes or frameshifts in the genome of the strains used.
Therefore, Nomcort HK-P is considered to be non-mutagenic in this Salmonella typhimurium
and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Nomcort HK-P to induce gene

mutations in the plate incorporation test (experiment I) and the pre-incubation test

(experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and

TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver

microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations:

33; 100; 333; 1000; 2500; and 5000 μg/plate

The plates incubated with the test item showed normal background growth up to

5000 μg/plate with and without metabolic activation in both independent experiments.

Minor toxic effects, evident as a reduction in the number of revertants, were observed at

5000 μg/plate with metabolic activation in strains TA 1535 and TA 98 in experiment I. In

experiment II, a slight toxic effect was observed without metabolic activation in strain TA

1535 at 5000 μg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was

observed following treatment with Nomcort HK-Pat any dose level, neither in the presence

nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation

rates with increasing concentrations in the range below the generally acknowledged

border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase

of induced revertant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)