Butanedioic acid, 2-hydroxy-, 1,4-bis[5,7,7-trimethyl-2-(1,3,3-trimethylbutyl)octyl] ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro studies on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells are available.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 7-10-C
- Expiration date of the lot/batch: not stated
- Purity test date: not stated


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Acetone, soluble at 200 mg/mL and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not expected

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none specific

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: National Institute of Health Sciences (NIHS)
- Suitability of cells: according to guideline
- Cell cycle length, doubling time or proliferation index: 9-12 h
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: Cells were cultured so as not exceed concentrations of 1.2 x l 06cells I mL.
- Methods for maintenance in cell culture if applicable: Cells that had been checked for mycoplasma contamination and other properties were used.


MEDIA USED
Three types of culture fluid, RPMI-0, RPMI-10 and RPMI-20, with different amounts of
serum added, were used. The compositions of each culture fluid are shown in the following
table. RPMI-0 for dilution or washing of the cells, RPMI-10 for normal culture, and RPMI-20
for colonization in 96-well plates were employed.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

In the short-term treatment with and without metabolic activation, the gene mutation test was
performed at 6 doses with 2 fold intervals (0.0625, 0.125, 0.250, 0.500, 1.00, 2.00 mg/mL).

Top dose was chosen based on Dose-Ranging study.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Culture conditions
1) Container : 75 cm2 or 150 cm 2 cultivation flask
2) Temperature and C02 concentration : 37°C and 5%
3) C02 incubator : SANYO Electric Co., Ltd. (Panasonic Corporation), type MC0-19AIC

In the short-term treatment and the continuous treatment method, the treatment solution was prepared
for a cultivation container as presented in the following two tables. The test was performed in the
manner of the Dose-ranging study. The negative control was performed using two cultures, and the test
substance treated group and the positive control were performed using one culture.

After test substance treatment end, the treatment solutions were centrifuged for 5 minutes at
approximately 200xg and the supernatant was removed by the aspirator. Ten mL of RPMI-0 culture
medium was added in the treatment container, and the cells were suspended by pipetting. The cell
suspensions were centrifuged under the same conditions. The supernatant was discarded and the cells
were suspended in 10 mL of RPMI-10 culture medium.

After having removed the test substance, the cell suspensions were measured cell concentration.
Then, the cell suspensions were prepared in RPMI-10 medium at 2 x 10 5 cells/mL (referred to as A in
the following table). The cell suspensions were further diluted to 8 cells/mL with 2 step dilutions as
presented in the following table, and the cell suspensions were dispensed 200 μL per well in 96-well
plates [Measurement of plating efficiency (PEO) and the relative survival (RSO)]. The plates were
incubated in the C02 incubator for 10-14 days then scored. The remaining cell
suspensions A were transferred to the 75 cm2 flasks, and cultured in the C02 incubator. As described
above, the cell suspensions were measured, diluted and cultured daily for 2 days (expression time).
From these result, the RSG was calculated.

After an expression time of 2 days following test substance treatment, 50 mL of a cell
suspension were prepared at 1 x 104 cells/mL. The cell suspensions were further diluted to
8 cells/mL with 2 step dilutions as presented in the following table, and the cell suspensions were
dispensed 200 μL per well in 96-well plates [Measurement of plating efficiency (PE2) and the
relative survival (RS2)]. For the remaining cell suspensions, trifluorothymidine (TFT) was added
to a final concentration of 3 μg/mL. These cell suspensions were dispensed 200 μL per well in
four 96-well plates per solvent control group and in two 96-well plates per each treatment group
[Detection of mutation frequency (MF)].
The PE2 plates were incubated in the C02 incubator for 10-14 days then scored. The MF plates were incubated in the C02 incubator for 12 days then scored.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

The judgment of the results confirmed a meaningful MF increase and whether or not it was
dose-dependent. The determination of increase in mutation frequency was evaluated by the Global
Evaluation Factor (GEF) note) judgment method. The test substance was considered to be clearly
positive if, in any of the conditions tested, the increase in MF above the concurrent background
exceeded the GEF and the increase was dose-dependent. The test substance was considered to be
clearly negative if, in all conditions tested, there was no dose-dependent response or, ifthere was
an increase in MF, it did not exceed the GEF. In addition, the result would not be considered
positive ifthe increase in MF occurred only at or below 10% RTG.
When there was no culture showing an RTG value between 10-20% RTG, the test substance was
considered to be negative if the following two circumstances were observed.
I. There was no dose-dependent response and no MFs above those seen in the concurrent
negative control or historical background ranges in a series of data points within 20% to
100% RTG. In addition, there was at least one data point between 20 and 25% RTG.
II. There was no dose-dependent response and no MFs above those seen in the concurrent
negative control or historical background ranges in a series of data points within 25% to
100% RTG. In addition, there was also a negative data point slightly below 10% RTG.
If the result obtained was difficult to make such clear judgment as described above, the
confirmatory test was performed in principle and comprehensively judgment was made,
accordingly. If there was no positive or negative conclusion in the results obtained in the
confirmatory test, the test substance response was concluded to be equivocal.
Note) GEF judgment method
The result was judged to be negative when the values of the test groups were below the value
which added 126 (xl0-6) to the MF.value of the negative control.

Statistics:

Any statistical method was not used for the data analysis.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Gene Mutation test by short-term treatment process
In the short-term treatment with and without metabolic activation, the gene mutation test was
performed at 6 doses with 2 fold intervals (0.0625, 0.125, 0.250, 0.500, 1.00, 2.00 mg/mL).
In the short-term treatment with and without metabolic activation, Oil membrane-like
precipitates derived from the test substance were observed on the surface of treatment solution at
the dose of 1.00 and 2.00 mg/mL at the end of treatment.
Regarding cytotoxicity, the test substance was nontoxic in the short-term treatment with and
without metabolic activation. In the short-term treatment without metabolic activation, the RTG at
the highest dose (2.00 mg/mL) was 112%. In the short-term treatment with metabolic activation,
the RTG at the highest dose (2.00 mg/mL) was 109%.
Precipitates were observed at doses of 1.00 and 2.00 mg/mL in the short-term treatment with and
without metabolic activation, but these suspended substances were properly removed by the
supernatant removal operation, and there was no effect on the subsequent operations. In addition,
the test substance was nontoxic at these doses. Based on the above results, in the short-term
treatment with and without metabolic activation, all the dose levels tested were included in the
analysis of the gene mutation frequencies and evaluated.
In the result of these evaluations, in the short-term treatment with and without metabolic
activation, no dose-dependent response and no increase in the total gene mutation frequency
(T-MF) of the test substance treated groups were observed as the following table. Therefore, the
test substance was judged to be "negative" both in the short-term treatment with and without
metabolic activation.

Gene Mutation test by continuous treatment process
In the 24 hours treatment of continuous treatment process, the gene mutation test was performed
at 6 doses with 2 fold intervals (0.0625, 0.125, 0.250, 0.500, 1.00, 2.00 mg/mL).
In the results of the test, slight turbidity caused by suspended substances derived from the test
substance was observed on the treatment solution at the dose of 0.250 and 0.500 mg/mL at the end
of treatment. Then, Oil membrane-like precipitates derived from the test substance were observed
on the surface of treatment solution at the dose of 1.00 and 2.00 mg/mL at the end of treatment.
Regarding cytotoxicity, the test substance was nontoxic at any dose tested. The RTG at the
highest dose (2.00 mg/mL) was 98%.
Precipitates were observed at doses of 0.250 to 2.00 mg/mL, but these suspended substances
were properly removed by the supernatant removal operation, and there was no effect on the
subsequent operations. In addition, the test substance was nontoxic at these doses. Based on the
above results, all the dose levels tested were included in the analysis of the gene mutation
frequencies and evaluated.
In the result of these evaluations, no dose-dependent response and no increase in the T-MF of
the test substance treated groups were observed as the following table. Therefore, the test
substance was judged to be "negative" in the continuous treatment.

Conclusions:

From the results obtained, the test substance was comprehensively concluded
as "negative".

Executive summary:

Diisostearyl malate was tested for its potentials to induce mutation at the thymidine kinase

locus of mouse lymphoma L5178Y cells in vitro.

As the result of gene mutation tests, in the short-term treatment with and without metabolic

activation, no dose-dependent response and no increase in the T-MF were observed. Also in

the continuous treatment without a metabolic activation, no dose-dependent response and no

increase in the T-MF were observed.

From the results obtained in these tests, the test substance was comprehensively concluded

as "negative". Therefore, under these test conditions, the test substance have no potential for

mutagenicity in the mouse lymphoma thymidine kinase locus gene mutation test. Results from

both of the positive and negative controls under all treatment conditions met acceptance

criteria, indicating that these tests were carried out appropriately.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identity: SALACOS 222
Batch No.: 7-2-D
Aggregate State at
Room Temperature: Liquid
Colour: Colourless to pale yellow
Molecular Weight: 639
Purity: > 98 %
Solubility in Water: Insoluble
Stability in Solvent: Not indicated by the sponsor
Storage: At room temperature
Expiration Date: April 30, 2010

Target gene:

no gene targeted in this assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP,
Technical University Darmstadt, 64287 Darmstadt, Germany) were stored in liquid nitrogen
in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the
same cell culture batch in experiments. Before freezing each batch was screened for
mycoplasm contamination and checked for karyotype stability. Consequently, the
parameters of the experiments remain similar because of standardized characteristics of the
cells.
Thawed stock cultures were propagated at 37° C in 80 cm² plastic flasks (GREINER,
72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded into 15 mL of
MEM (Minimal Essential Medium; SEROMED; 12247 Berlin, Germany) supplemented with
10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells
were sub-cultured twice weekly. The cell cultures were incubated at 37° C in a humidified
atmosphere with 1.5 % carbon dioxide (98.5 % air).

Cytokinesis block (if used):

Colcemid was added (0.2 Mg/mL culture medium) to the cultures

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

6.3, 12.5, 25, 50, 100, 200 µg/mL
The highest concentration used in the cytogenetic experiments was chosen with regard to
the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top
concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of
control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case
of non-toxicity the maximum concentration should be 5 mg/mL, 5 ML/mL or 10 mM,
whichever is the lowest, if formulation in an appropriate solvent is possible.
With respect to the solubility of SALACOS 222, in the pre-test, 3500 Mg/mL (approx. 5.5 mM)
of SALACOS 222 was applied as top concentration for treatment of the cultures. Test item
concentrations between 27.3 and 3500 Mg/mL (with and without S9 mix) were chosen for the
evaluation of cytotoxicity. Precipitation of the test item after 4 hrs treatment was observed at
109.4 Mg/mL and above.
Since no relevant toxicity was observed in the pre-test on toxicity , the
test item was tested up to a concentration exhibiting clear test item precipitation as
recommended in the OECD Guideline 473. Therefore, 200 Mg/mL was chosen as top
treatment concentration in the absence and the presence of S9 mix in Experiment I.
Dose selection of Experiment II was also influenced by the occurrence of precipitation. In the
range finding experiment, no reduced cell numbers were observed after 24 hrs exposure up
to the highest concentration. Therefore, 200 Mg/mL was chosen as top treatment
concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix,
200 Mg/mL was chosen as top treatment concentration with respect to the results obtained in
Experiment I.

Vehicle / solvent:

Acetone
On the day of the experiment (immediately before treatment), the test item was dissolved in
acetone (E. MERCK, 64293 Darmstadt, Germany; purity 99.5 %). The final concentration of
acetone in the culture medium was 0.5 % (v/v). The solvent was chosen to its solubility
properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours / 18 hours / 28 hours
- Expression time (cells in growth medium): 15.5 or 25.5 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 2.5 hours

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: two

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Colcemid was added (0.2 Mg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs,
respectively after the start of the treatment. The cells on the slides were treated 2.5 hrs later,
in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in
the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid
(3:1 parts, respectively). Per experiment two slides per group were prepared. After
preparation the cells were stained with Giemsa (E. Merck, 64293 Darmstadt, Germany).

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe
der Industrie, Cytogenetik" [5]) using NIKON microscopes with 100x oil immersion
objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were
recorded as structural chromosome aberrations. Gaps were recorded as well but not
included in the calculation of the aberration rates. 100 well spread metaphase plates per
culture were scored for cytogenetic damage on coded slides, except for the positive control
in Experiment II, in the absence of S9 mix, at the 28 hrs preparation interval without
metabolic activation, where only 50 metaphase plates were scored.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the
analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphase plates per culture was
determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means
a near tetraploid karyotype).

DETERMINATION OF CYTOTOXICITY
For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per
test item and solvent control group, not treated with colcemid, were set up in parallel. These
cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine
microscopically the cell number within 10 defined fields per coded slide. The cell number of
the treatment groups is given in percentage compared to the respective solvent control.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

A test item is classified as non-clastogenic if:
the number of induced structural chromosome aberrations in all scored dose groups is in
the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells,
excluding gaps).
and/or
no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
the number of induced structural chromosome aberrations is not in the range of the
laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
and
either a concentration-related or a significant increase of the number of structural
chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher’s exact test (9) (p < 0.05).
However, both biological and statistical significance should be considered together. If the
criteria mentioned above for the test item are not clearly met, the classification with regard to
the historical data and the biological relevance is discussed and/or a confirmatory
experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study,
it is important to include the polyploids and endoreduplications. The following criterion is
valid:
A test item can be classified as aneugenic if:
Y the number of induced numerical aberrations is not in the range of the laboratory’s
historical control data range (0.0 – 5.2 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (9) (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

did not induce structural chromosome aberrations as determined by the chromosome
aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, SALACOS 222 is considered to be non-clastogenic in this chromosome
aberration test with and without S9 mix, when tested up to precipitating concentrations.

Executive summary:

The test item SALACOS 222, dissolved in acetone, was assessed for its potential to induce

structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two

independent experiments. The following study design was performed:

Without S9 mix With S9 mix

Exp. I Exp. II Exp. I Exp. II

Exposure period 4 hrs 18 hrs 28 hrs 4 hrs 4 hrs

Recovery 14 hrs - - 14 hrs 24 hrs

Preparation interval 18 hrs 18 hrs 28 hrs 18 hrs 28 hrs

In each experimental group two parallel cultures were set up. Per culture 100 metaphase

plates were scored for structural chromosome aberrations, except for the positive control, in

Experiment II, at preparation interval 28 hrs, without metabolic activation, where only

50 metaphase plates were scored.

The highest applied concentration in the pre-test on toxicity (3500 Mg/mL; approx. 5.5 mM)

was chosen with regard to the solubility properties of the test item in an appropriate solvent

with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data

and the occurrence of precipitation. The chosen treatment concentrations are described in

chapter 8.6 (page 15). The scored experimental points and the results are summarised in

Table 1 (page 22).

In the absence and the presence of S9 mix, no cytotoxicity was observed up to the highest

applied concentration.

No clastogenicity was observed at the concentrations evaluated, either with or without

metabolic activation.

No relevant increase in the frequencies of polyploid metaphases was found after treatment

with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant

increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - November 2007

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:

Identity: SALACOS 222
Batch No.: 7-2-D
Aggregate state at
room temperature: Liquid
Colour: Colourless to pale yellow
Purity: > 98 %
Stability in solvent: Not indicated by the sponsor
Storage: At room temperature
Expiration Date: April 30, 2010

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:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Pre-Experiment/ Experiment I: 3; 10, 33; 100; 333; 1000; 2500; and 5000 Jg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 Jg/plate

Vehicle / solvent:

On the day of the experiment, the test item SALACOS 222 was dissolved in DMF
(MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was chosen because of its
solubility properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD, 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 60 min
- Exposure duration = Expression time = 48 h

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: evaluation of number of colonies and bacterial background

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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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.

Executive summary:

This study was performed to investigate the potential of SALACOS 222 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. Due to contamination of strain TA 1537 in the pre-experiment, this

part was repeated (reported as part of experiment I). Each concentration, including the

controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/ Experiment I: 3; 10, 33; 100; 333; 1000; 2500; and 5000 Jg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 Jg/plate

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

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

No toxic effects, evident as a reduction in the number of revertants, occurred in the test

groups with and without metabolic activation.

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

observed following treatment with SALACOS 222 at 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)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

as no genotoxic effects are observed, no mode of action can be established

Justification for classification or non-classification

The available information is conclusive but not sufficient for classification.