Butanedioic acid, sulfo-, C-[2-[(1-oxododecyl)amino]ethyl] ester, monosodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a key Ames test no increase in mutations were observed in different Salmonella typhimureum strains with and without metabolic activation up to 5.0 µL/plate.

In a key mammalian gene mutation test ( HPRT) in V79 cells, the test item did not induce mutations in the absence and presence of metabolic activation when tested up to cytotoxic concentrations of 300 and 900 µg/mL, respectively. 

Furthermore, the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes in an in vitro Micronucleus Test (according to OECD 487).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr. - July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

Histidine (his- → his+)
Tryptophane (trp- → trp+)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA98, TA1535 and E. coli are obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA100 and TA1537 are obtained from Xenometrix AG, Switzerland. Stored as stock cultures with nutrient broth (OXOID)/DMSO (approx. 8% v/v) over liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 :
The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Gießen, Germany.

- method of preparation of S9 mix:
Male Wistar rats are induced with phenobarbital (80 mg/kg bw) and β- naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins) or male Sprague Dawley rats are induced with phenobarbital/ β-naphthoflavone (Trinova).
A stock of the supernatant containing the microsomes is frozen in aliquots between 2 and 5 mL and stored at
- concentration of S9 mix:
The protein concentration in the S9 preparation of Eurofins Munich was 34.4 mg/mL (Lot: 070521), and in the S9 preparation of Trinova, 37.8 mg/mL (Lot: 4423). The protein concentration of Lot No.: 4423 was adjusted to 30 mg/mL

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
at Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test

at Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and mutation induction with each three plates:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate.
The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test).
As no precipitation nor limiting toxicity of the test item was observed in either tester strain used in the pre-experiment, 5.0 μL/plate was selected as the maximum concentration to be used in experiment I.

Vehicle / solvent:

- Justification for choice of solvent/vehicle:
solubility

The test item was dissolved in A. dest. and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity. A correction factor of 2.55 was applied to consider the active components of the test item.

Untreated negative controls:

yes

Remarks:

Aqua dest.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD, 4-nitro-o-phenylene-diamine, 10 µg/plate; 2-AA, 2-aminoanthracene, 2.5 µg/plate (S. thyphimurium) or 10 µg/plate (E. coli)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicate
- Number of independent experiments : 2, if first experiment negative or equivocal

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
Samples of each tester strain are grown by culturing for 12 h at 37 °C in S. typhimurium medium (Nutrient Broth) and E. coli medium (Luria Bertani), respectively, to the late exponential or early stationary phase of growth (approx. 109 cells/mL).
- Test substance added:
1. experiment - in agar (plate incorporation)
2. experiment - preincubation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 60 min.
- Incubation after exposure: 48 h at 37°C on selective medium

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

according to current OECD guideline

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 0.316 μL/plate and higher (with and without metabolic activation)

Vehicle 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

Remarks:

at concentrations of 0.316 μL/plate and higher (with and without metabolic activation)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 0.316 μL/plate and higher (with and without metabolic activation)

Vehicle 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:

cytotoxicity

Remarks:

at concentrations of 0.316 μL/plate and higher (with and without metabolic activation)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In tester strain E. coli WP2 uvrA (pKM101), toxic effects of the test item were noted at concentrations of 2.5 μL/plate and higher (without metabolic activation).

Vehicle controls validity:

valid

Positive controls validity:

valid

please refer to attachment

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 μL/plate
(TA98, TA100, TA1535, TA1537)
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
(E. coli WP2 uvrA (pKM101))

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
The microbial contamination observed in one plate (experiment II, TA98, 0.0316 μL/plate, with metabolic activation) did not affect the quality or integrity of the results as the microbial contamination could be clearly distinguished from the Salmonella typhimurium revertants and thus did not affect the evaluation.
Toxic effects of the test item were observed in experiment I in tester strains TA98 and TA100 at concentrations of 2.5 μL/plate and higher (without metabolic activation). In tester strain TA1535, toxic effects of the test item were noted at concentrations of 1.0 μL/plate and higher (without metabolic activation) and at a concentration of 5.0 μL/plate (with metabolic activation). In tester strain TA1537 toxic effects of the test item were observed at a concentration of 5.0 μL/plate (without metabolic activation).
In experiment II, toxic effects of the test item were noted in tester strains TA98, TA100, TA1535 and TA1537 at concentrations of 0.316 μL/plate and higher (with and without metabolic activation). In tester strain E. coli WP2 uvrA (pKM101), toxic effects of the test item were noted at concentrations of 2.5 μL/plate and higher (without metabolic activation).
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr - Juyl 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt locus at the X-chromosome

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
* Freshly thawed V79 cells from stock cultures are maintained in plastic culture flasks in minimal essential medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature.
* For purifying the cell population of pre-existing HPRT- mutants cells are exposed to HAT medium containing 10 μM hypoxanthine, 3.2 μM aminopterin, 5 μM thymidine and 10 μM glycine for several cell doublings (2-3 days) with a subsequent recovery period in medium supplemented with 10 μM hypoxanthine and 5 μM thymidine
* Treatment Medium:
MEM supplemented with pnicillin/streptomycin, L-glutamine, HEPES and amphotericin B (FBS for long-term exposure)
* Selective Medium:
MEM supplemented with FBS, penicillin/Streptomycin, L-glutamine, HEPES, amphotericin B, thioguanine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, via PCR
- The spontaneous mutation rate was continuously monitored.

Metabolic activation:

with and without

Metabolic activation system:

S9-mixType and composition of metabolic activation system:
- source of S9 :
The S9 liver microsomal fraction is prepared at Eurofins Munich or obtained from Trinova Biochem GmbH, Giessen, Germany. Male Wistar rats are induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins Munich) or male Sprague Dawley rats are induced with phenobarbital/ β-naphthoflavone (Trinova).
- method of preparation of S9 mix :
An appropriate quantity of the S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors are added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix is stored on ice.
- concentration or volume of S9 mix and S9 in the final culture medium:
The S9 fraction will be used at concentrations between 36% and 50% in the S9 mix. The percentage of S9 mix in the final treatment medium is 5% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
The following quality control determinations are performed:

A. Eurofins Munich-prepared S9 Homogenate:
Quality control determinations performed by Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test

B. Trinova Biochem GmbH-prepared S9 Homogenate:
Quality control determinations performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2- aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

The toxicity of the test item was determined in a pre-experiment. Eight concentrations [25, 50, 100, 250, 500, 1000, 1500 and 2000 μg/mL] were tested with and without metabolic activation for the 4 h short-term exposure assay.
Toxicity of the test item was evaluated using the relative survival (RS). A cytotoxic effect is considered when the relative survival decreases below 70%. The relative survival was calculated based on the cloning efficiency of the cells plated immediately after treatment adjusted by any loss of cells during treatment.
RS[%] = (adjusted Cloning Efficiency in treated culture)/(adjusted Cloning Efficiency in negative control) x 100
The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476:

-without metabolic activation: 150, 200, 230, 250, 260, 280, 300 µg/mL
- with metabolic activation: 500, 600, 800, 850, 900 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (MEM medium)
- Justification for choice of solvent/vehicle: based on the result of the solubility experiment
The medium was compatible with the survival of the cells and the activity of the S9 mix. The pH-value of 7.25 was within the physiological range. A correction factor of 2.55 was applied to consider the purity of the test item. Osmolality of the highest test item concentration of 5.1 mg/mL was 293 mOsm/kg (negative control: 285 mOsm/kg).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
4 hours (with and without metabolic activation) if necessary
- Expression time (cells in growth medium): 7-9 days in which the cells will be subcultured
in complete culture medium (MEM supplemented with 10% FBS) in a sufficient number of cells (at least 2 x 10(e)6 cells per treatment group).
- Selection time (if incubation with a selection agent): 9-11 days
SELECTION AGENT (mutation assays): 6-thioguanine (11 µg/mL)

NUMBER OF REPLICATIONS:
cytotoxicity: two
mutagenicity: for selection of mutants 5 replicate plates

DETERMINATION OF CYTOTOXICITY
- Method: relative survival

Rationale for test conditions:

according to OECD 476

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the
concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with
the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutant frequency in the corresponding negative and
solvent controls a concentration related increase of the mutations within their range has to be
discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the concentration groups for any significant difference in mutant frequency compared to the solvent controls. Mutant frequencies of the solvent controls were used as reference.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation:
No precipitation of the test item was noted in any of the experiments.

Cytotoxicity:
In experiment I without metabolic activation, a concentration-related reduction of the relative survival was noted from 100 μg/mL (119%) to 300 μg/mL (11% rel. survival).
With metabolic activation a concentration-dependent reduction in survival was seen in the concentration range from 500 μg/mL (100%) to 900 μg/mL (9% rel. survival).

Mutagenicity:

- Controls
In the main experiment I without and with metabolic activation, the mean mutant values of the negative controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%.
The positive controls, DMBA (1.0 μg/mL) and EMS (300 μg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.

- Experiment I without metabolic activation
In the experiment I without metabolic activation the mutant values of one of the negative controls and some mutant values found for the applied concentrations of the test item were within the historical control data of the test facility (about 8.6 - 46.6 mutants per 10e6 cells). The individual mutant value of one of the negative controls was increased to 50.78 mutants per 10e6 cells and outside the historic control range. However, the mean value of this control was within the historical data range of the test facility and therefore considered acceptable.
Increased mutant frequencies were observed at concentrations of 150 μg/mL (52.04 mutants per 10e6 cells), 200 μg/mL (48.80 mutants per 10e6 cells), 230 μg/mL (61.39 mutants per 10e6 cells) and 260 μg/mL (49.04 mutants per 10e6 cells).
A statistical analysis displayed that none of the mutant frequencies were significantly increased over those of the negative controls. The chi² test for trend revealed a concentration related increase in mutant frequency. However, a closer examination of the individual values does not
show any upward trend. The mutation frequencies rather vary within the concentration range. If the elevated value of one of the negative control is taken as a basis, the mutant frequencies induced by the test item are comparable to the negative control, so that no mutagenic effect is evident.

- Experiment I with metabolic activation
In experiment I with metabolic activation, the individual mutant value of the negative controls and all test item concentrations were within the historic control range (about 8.4 – 50.1 mutants per 10e6 cells).

please refer to attachment

Conclusions:

In the described mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.
The selection of the concentrations was based on data from the pre-experiments. The experiment I with and without metabolic activation were performed as a 4 h short-term exposure assay.
The test item was investigated at the following concentrations:

Experiment I
without metabolic activation:
150, 200, 230, 250, 260, 280 and 300 μg/mL

and with metabolic activation:
500, 600, 800, 850 und 900 μg/mL

No precipitation of the test item was noted in any of the experiments.
A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment I with and without metabolic activation.
In the experiment I without metabolic activation the mutant frequency values of one of the negative controls and some mutant frequency values found for the applied concentrations of the test item were within the historical control data of the test facility (about 8.6 - 46.6 mutants per 10e6 cells). The individual mutant frequency value of one of the negative controls was increased to 50.78 mutants per 10e6 cells and was outside the historic control range. However, the mean value of this control was within the historical data range of the test facility and therefore considered acceptable. If the elevated value of one of the negative control is taken as a basis, the mutant frequencies induced by the test item are comparable to the negative control. Additionally, a statistical analysis displayed that none of the mutant frequencies were significantly increased over those of the negative controls. The chi² test for trend revealed a concentration-related increase in mutant frequency. However, a closer examination of the individual values does not show any upward trend. The mutation frequencies rather vary within the concentration range. Therefore, the increased mutant
frequencies were not regarded as biologically relevant.
In experiment I with metabolic activation, the individual mutant value of the negative controls and all test item concentrations up to a concentration of 850 μg/mL were within the historic controlrange (about 8.4 – 50.1 mutants per 10e6 cells). The concentration of 900 μg/mL was included into discussion because relative survival was only slightly below 10%.
DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - Dec 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: Human peripheral blood was obtained by venipuncture from a healthy, non-smoking donor with no known recent exposures to genotoxic chemicals and radiation, and collected in heparinised vessels.

Details on mammalian cell type (if applicable):

- Type and identity of media:
* RPMI 1640 medium supplemented with 15% fetal calf serum (FCS) and Penicillin/Streptomycin
- Treament mdieum (short-term):
* Medium without FBS
-After treatment/treatment medium (long term):
* complete medium with 15% FBS and 6µg/mL cytochalasin B

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

S9-mixType and composition of metabolic activation system:
- source of S9
The S9 liver microsomal fraction is obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats were induced with phenobarbital / β naphthoflavone.
- method of preparation of S9 mix
An appropriate quantity of the S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors are added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix is stored on ice.
The final concentration of S9 mix in the cultures is 5%.
- concentration or volume of S9 mix and S9 in the final culture medium
The protein concentration in the S9 preparation is usually between 20 and 45 mg/mL.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-0-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

A pre-experiment was conducted under identical conditions as described for the main experiment I (4 h incubation) in order to determine the toxicity of the test item. The CBPI was used for the quantification of cytotoxicity. The following concentrations were tested without and with S9 mix:
3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL
The concentration of 2000 μg/mL was considered to be the highest test concentration used in this test system following the recommendation of the corresponding OECD testing guideline 487.

Duplicate cultures were treated at each concentration. The selection of the concentrations used in experiment I and II based on data from the pre-experiment. The following concentrations were used in the main experiments:
Experiment I:
without and with metabolic activation: 25, 50, 100, 250, 500, 1000 and 2000 μg/mL
Experiment II:
without metabolic activation: 25, 50, 100, 250, 500, 1000 and 2000 μg/mL

The following concentrations were selected in the main experiments for the microscopic analyses. For experiment I the maximum concentration of 2000 μg/mL was chosen as maximum concentration for microscopic evaluation. For experiment II the selection was based on cytotoxicity.
Experiment I with short-term exposure (4 h):
without and with metabolic activation: 500, 1000 and 2000 μg/mL
Experiment II with long-term exposure (44 h):
without metabolic activation: 100, 250 and 500 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
The test item was dissolved and diluted in cell culture medium (RPMI) within 1 hour prior to treatment.

- Vehicle(s)/solvent(s) used: RPMI culture medium

- Justification for choice of solvent/vehicle: solubility
The solvent was compatible with the survival of the cells and the S9 activity.
A correction factor of 2.55 was applied to consider the active components of the test item.

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

other: Aneugenic Control: Colchicine, 0.01 - 0.8 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4h (with and without S9)
Experiment 2: 4h (with S9) and 20 h (without S9)
Experiment 3: 4h (with and without S9)
- Fixation time (start of exposure up to fixation or harvest of cells):
28 h (Experiment 1with and without S9; Experiment 2 with S9 and Experiment 3 with and without S9)
44h (Eperiment 2 without S9)

SPINDLE INHIBITOR (cytogenetic assays): 5µg/mL Cytochalasin B
STAIN (for cytogenetic assays): 10% Giemsa.

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000/concentration

DETERMINATION OF CYTOTOXICITY
- Method: other: Treatment of cultures with cytoB, and measurement of the relative frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture, provides an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and ensures that only cells that divided during or after treatment are scored.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : at least duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
Experiment I
Whole blood samples will be treated with anti-coagulant (Heparin) and pre-cultured (44 to 48 h) in presence of PHA prior to exposure to the test item. It is recommended to test human lymphocytes 44 to 48 h after PHA stimulation, when the cell cycle synchronisation disappears. The cells will be incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium will be removed and the cells will be washed twice with PBS + 10% FBS. Subsequently the cells will be incubated in complete culture medium + 6 µg/mL cytochalasin B for 40 h to 42 h at 37°C and 5% CO2.
Experiment II
If negative or equivocal results are obtained, they should be confirmed using continuous treatment (long-term treatment) without metabolic activation. The whole blood cultures will be pre-cultured in the presence of PHA for 44 to 48 h prior to exposure to the test item. Then the test item will be added in complete culture medium. 1 h later 6 µg/mL cytochalasin B will be added and the cells will be incubated for further 43 h at 37 °C. At the end of the treatment the cell culture medium is removed and the cells are prepared for microscopic analysis.

TREATMENT AND HARVEST SCHEDULE:
At the end of the cultivation, the complete culture medium is removed. Subsequently, the cells are treated with cold hypotonic solution (0.075 M KCl) for some minutes at room temperature and immediately centrifuged. The pellet is resuspended with a solution consisting of fixation solution + NaCl 0.9% (1+1) and centrifuged. After that the cells are fixed with methanol + glacial acetic acid (3+1). The cells are resuspended gently and the suspension is dropped onto clean glass slides. Consecutively, the cells are dried on a heating plate. The cells are stained with acridine orange solution.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure/ If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure:
6 µg/mL cytochalasin for 43 h at 37°C
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays)/ Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
All slides, including those of positive and negative/solvent controls are independently coded before microscopic analysis. At least 2000 binucleated cells per concentration (1000 binucleated cells per slide) are analysed for micronuclei according to the criteria of Fenech [7], i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated cells, multinucleated cells and cells with more than six micronuclei are not considered
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
according to the criteria of Fenech

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method/ Any supplementary information relevant to cytotoxicity:
Evaluation of toxicity is based on the cytokinesis block proliferation index (CBPI), which is determined from 500 cells according to the following formula:
CBPI= (c1 x 1) + (c2 x 2) + (cx x 3)
n

c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells
The CBPI can be used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures:

% Cytostasis= 100 – 100 x ((CBPIT – 1) / (CBPIC – 1))

CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures

Rationale for test conditions:

According to OECD guideline

Evaluation criteria:

Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and solvent control cultures were determined. Individual culture data were provided.
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.
Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response.
A positive result from the in vitro micronucleus test indicates that the test item induces chromosome damage or damage to the cell division apparatus.
Negative results indicate that, under the test conditions used, the test substance does not induce chromosome breaks and/or gain or loss in cultured mammalian cells.
There is no requirement for verification by additional testing of a clear positive or negative response.
Equivocal results may be clarified by analysis of another 1000 cells from all the cultures to avoid loss of blinding. If this approach does not resolve the result, further testing would be necessary. Modification of study parameters over an extended or narrowed range of conditions, as appropriate, would be considered in follow-up experiments. Study parameters that might be modified include the test concentration spacing, the timing of treatment and cell harvest, and/or the metabolic activation conditions.
Although most experiments give clearly positive or negative results, in some cases the data set would preclude making a definite judgement about the activity of the test item. These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated.

Statistics:

The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971[3]) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data).

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 100 μg/mL. At 250 μg/mL a cytostasis of 33% and at 500 μg/mL a cytostasis of 52% was observed.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation
The test item was dissolved in cell culture medium (RPMI medium). No precipitate of the test item in the cultures at the end of treatment was noted in any concentration evaluated in experiment I and II.

Cytotoxicity
If cytotoxicity is observed the highest concentration evaluated should not exceed the limit of 55% ± 5% cytotoxicity according to the OECD Guideline 487. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. The other concentrations evaluated should exhibit intermediate and little or no toxicity. However, OECD 487 does not define the limit for discriminating between cytotoxic and non-cytotoxic effects. According to laboratory experience this limit is a value of the relative cell growth of 70% compared to the negative/solvent control which corresponds to 30% of cytostasis.
In experiment I with and without metabolic activation no increase of the cytostasis above 30% was
noted.
In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 100 μg/mL. At 250 μg/mL a cytostasis of 33% and at 500 μg/mL a cytostasis of 52% was observed.

Clastogenicity / Aneugenicity
In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.60%) was within the historical control limits of the negative control (0.17% - 1.17%). The mean values of micronucleated cells found after treatment with the test item were 0.55% (500 μg/mL),
0.90% (1000 μg/mL) and 0.70% (2000 μg/mL). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

In experiment I with metabolic activation the micronucleated cell frequency of the negative control (0.40%) was within the historical control limits of the negative control (0.21% - 1.19%). The mean values of micronucleated cells found after treatment with the test item were 0.65% (500 μg/mL), 1.00% (1000 μg/mL) and 0.80% (2000 μg/mL). For concentrations of 500 and 2000 μg/mL the numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control. At 1000 μg/mL a statistically significant increase was observed. Since the corresponding value of the micronuclei frequency was within the historical control limits of the negative control and no concentration-related increase was observed, this effect was considered as not biologically relevant.

In experiment II without metabolic activation the micronucleated cell frequency of the negative control (0.60%) was within the historical control limits of the negative control (0.17% - 1.17%). The mean values of micronucleated cells found after treatment with the test item were 0.40% (100 μg/mL), 0.22% (250 μg/mL) and 0.40% (500 μg/mL). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

The nonparametric ² Test was performed to verify the results in both experiments. No statistically significant increase (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II without metabolic activation. In experiment I with metabolic activation a statistically significant increase (p = 0.0229) of cells with micronuclei was noted at a concentration of 1000 μg/mL. Since the corresponding value of the micronuclei frequency was within the historical control limits of the negative control and no concentration-related increase was observed, this effect was considered as not biologically relevant.

The ² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.

MMS (50 and 65 μg/mL) and CPA (12.5 μg/mL) were used as clastogenic controls and colchicine as aneugenic control (0.04 and 0.4 μg/mL). They induced distinct and statistically significant increases of the micronucleus frequency, demonstrating the validity of the assay.

please refer to attachment

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes.
Therefore, the test substance is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In order to investigate a possible potential of the test substance to induce micronuclei in human lymphocytes an in vitro micronucleus assay was carried out.

The selection of the concentrations was based on data from the pre-experiment. In the main experiment I without and with metabolic activation 2000 μg/mL test item and in experiment II without metabolic activation 500 μg/mL was selected as the highest concentration for microscopic evaluation.
The following concentrations were evaluated for micronuclei frequencies:

Experiment I with short-term exposure (4 h):
without and with metabolic activation: 500, 1000 and 2000 μg/mL

Experiment II with long-term exposure (44 h):
without metabolic activation: 100, 250 and 500 μg/mL

No precipitate of the test item in the cultures at the end of treatment was noted in any concentration
evaluated in experiment I and II.

In experiment I with and without metabolic activation no increase of the cytostasis above 30% was noted.
In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 100 μg/mL. At 250 μg/mL a cytostasis of 33% and at 500 μg/mL a cytostasis of 52% was observed.
In experiment I and II without and with metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

The nonparametric chi² Test was performed to verify the results in both experiments. No statistically significant increase (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II without metabolic activation. In experiment I with metabolic activation a statistically significant increase (p = 0.0229) of cells with micronuclei was noted at a concentration of 1000 μg/mL. Since the corresponding value of the micronuclei frequency was within the historical control limits of the negative control and no concentration-related increase was observed, this effect was considered as not biologically relevant.

The chi² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.
Methylmethanesulfonate (MMS, 50 and 65 μg/mL) and cyclophosphamide (CPA, 12.5 μg/mL) were used as clastogenic controls. Colchicine (Colc, 0.04 and 0.4 μg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

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

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro studies are regarded as relevant for humans.

Additional information

Additional information from genetic toxicity in vitro:  

 

Bacterial mutagenicity

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
Experiment II:
0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 μL/plate
(TA98, TA100, TA1535, TA1537)
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
(E. coli WP2 uvrA (pKM101))

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
The microbial contamination observed in one plate (experiment II, TA98, 0.0316 μL/plate, with metabolic activation) did not affect the quality or integrity of the results as the microbial contamination could be clearly distinguished from the Salmonella typhimurium revertants and thus did not affect the evaluation.
Toxic effects of the test item were observed in experiment I in tester strains TA98 and TA100 at concentrations of 2.5 μL/plate and higher (without metabolic activation). In tester strain TA1535, toxic effects of the test item were noted at concentrations of 1.0 μL/plate and higher (without metabolic activation) and at a concentration of 5.0 μL/plate (with metabolic activation). In tester strain TA1537 toxic effects of the test item were observed at a concentration of 5.0 μL/plate (without metabolic activation).
In experiment II, toxic effects of the test item were noted in tester strains TA98, TA100, TA1535 and TA1537 at concentrations of 0.316 μL/plate and higher (with and without metabolic activation). In tester strain E. coli WP2 uvrA (pKM101), toxic effects of the test item were noted at concentrations of 2.5 μL/plate and higher (without metabolic activation).
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

 

Mammalian mutagenicity

The test item was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.
The selection of the concentrations was based on data from the pre-experiments. The experiment I with and without metabolic activation were performed as a 4 h short-term exposure assay.
The test item was investigated at the following concentrations:

Experiment I
without metabolic activation:
150, 200, 230, 250, 260, 280 and 300 μg/mL

and with metabolic activation:
500, 600, 800, 850 und 900 μg/mL

No precipitation of the test item was noted in any of the experiments.
A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in experiment I with and without metabolic activation.
In the experiment I without metabolic activation the mutant frequency values of one of the negative controls and some mutant frequency values found for the applied concentrations of the test item were within the historical control data of the test facility (about 8.6 - 46.6 mutants per 10e6 cells). The individual mutant frequency value of one of the negative controls was increased to 50.78 mutants per 10e6 cells and was outside the historic control range. However, the mean value of this control was within the historical data range of the test facility and therefore considered acceptable. If the elevated value of one of the negative control is taken as a basis, the mutant frequencies induced by the test item are comparable to the negative control. Additionally, a statistical analysis displayed that none of the mutant frequencies were significantly increased over those of the negative controls. The chi² test for trend revealed a concentration-related increase in mutant frequency. However, a closer examination of the individual values does not show any upward trend. The mutation frequencies rather vary within the concentration range. Therefore, the increased mutant
frequencies were not regarded as biologically relevant.
In experiment I with metabolic activation, the individual mutant value of the negative controls and all test item concentrations up to a concentration of 850 μg/mL were within the historic controlrange (about 8.4 – 50.1 mutants per 10e6 cells). The concentration of 900 μg/mL was included into discussion because relative survival was only slightly below 10%.
DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

 

Cytogenicity

In order to investigate a possible potential of the test substance to induce micronuclei in human lymphocytes an in vitro micronucleus assay was carried out.

The selection of the concentrations was based on data from the pre-experiment. In the main experiment I without and with metabolic activation 2000 μg/mL test item and in experiment II without metabolic activation 500 μg/mL was selected as the highest concentration for microscopic evaluation.
The following concentrations were evaluated for micronuclei frequencies:

Experiment I with short-term exposure (4 h):
without and with metabolic activation: 500, 1000 and 2000 μg/mL

Experiment II with long-term exposure (44 h):
without metabolic activation: 100, 250 and 500 μg/mL

No precipitate of the test item in the cultures at the end of treatment was noted in any concentration
evaluated in experiment I and II.

In experiment I with and without metabolic activation no increase of the cytostasis above 30% was noted.
In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 100 μg/mL. At 250 μg/mL a cytostasis of 33% and at 500 μg/mL a cytostasis of 52% was observed.
In experiment I and II without and with metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

The nonparametric chi² Test was performed to verify the results in both experiments. No statistically significant increase (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II without metabolic activation. In experiment I with metabolic activation a statistically significant increase (p = 0.0229) of cells with micronuclei was noted at a concentration of 1000 μg/mL. Since the corresponding value of the micronuclei frequency was within the historical control limits of the negative control and no concentration-related increase was observed, this effect was considered as not biologically relevant.

The chi² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.
Methylmethanesulfonate (MMS, 50 and 65 μg/mL) and cyclophosphamide (CPA, 12.5 μg/mL) were used as clastogenic controls. Colchicine (Colc, 0.04 and 0.4 μg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

 

Conclusion

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial and mammalian gene mutation and cytogenicity. Based on the available results, there were no indications of mutagenicity or genotoxicity, and no further testing is needed. The substance is considered to have no mutagenic or genotoxic potential.

 

Justification for classification or non-classification

Based on these results and CLP (No. 1272/2008 of 16 December 2008), the test item does not have to be classified and has no obligatory labelling requirement for genetic toxicity.