N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A reverse mutation tests on bacteria (on S. typhimurium and on E. coli) is available on N-methoxy-1- (2,4,6 -trichlorophenyl)propan-2 -amine (Harlan 2013).

The study was realised according to the OECD 471 guideline and in compliance with GLP, using the plate incorporation method (experiment I) and the pre-incubation methode (experiment II). No evidence of mutagenicity was observed with and without metabolic activation.

Two in vitro gene mutation test on mammalian cells were conducted with n-methoxy-1 -(2,4,6 -trichlorophenyl)propan-2 -amine at different level of purity (Harlan 2014 and 2016).

N-methoxy-1 -(2,4,6 -trichlorophenyl)propan-2 -amine was tested in to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y (Harlan 2014) Mutation Assays in the absence and presence of metabolic activation, according to the OECD 476 guideline and in compliance with GLP and give homogeneous negative results.

One in vitro Chromosome Aberration Test in Human Lymphocytes was also performed with the substance (Harlan 2014). The study was conducted according to the OECD 473 guideline and in compliance with GLP. No clastogenic and aneugenic effects were noted at the dose tested and the substance was considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

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:

13 July 2015 to 29 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - 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:

This in vitro test is an assay for the detection of forward gene mutations at the autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK+/- cells to TKP-/- mutants.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Prior to mutagenicity testing the amount of spontaneous mutants was reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
Hypoxanthine 5.0 x 10-3 M
Aminopterin 2.0 x 10-5 M
Thymidine 1.6 x 10-3 M
Glycine 5.0 x 10-3 M

The incubation of the cells in HAT-medium was followed by a recovery period of 2 days in RPMI 1640 medium containing:
Hypoxanthine 1.0 x10-4 M
Thymidine 1.6 x 10-3 M

After this incubation the L5178Y cells were returned to normal RPMI 1640 medium (complete culture medium).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I
without metabolic activation: 4.1; 8.2; 16.4; 32.9; 65.8, 132.0 µg/mL
with metabolic activation: 4.1, 8.2; 16.4; 32.9; 65.8, 132.0 µg/mL

Experiment II
without metabolic activation: 11.0; 22.0; 44.0, 66.0, 88.0, 110.0 µg/mL
with metabolic activation: 22.0; 44.0; 66.0, 88.0, 132.0, 176.0 µg/mL

Due to phase separation visible at the end of treatment only the following concentrations were evaluated:

Experiment I
without metabolic activation: 4.1; 8.2; 16.4; 32.9 and 65.8 µg/mL
with metabolic activation: 8.2; 16.4; 32.9; 65.8 and 132.0 µg/mL

Experiment II
without metabolic activation: 11.0; 22.0; 44.0 and 66.0 µg/mL
with metabolic activation: 22.0; 44.0; 66.0 and 88.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix 19.5 µg/mL = 0.18 mM

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix 3.0 µg/mL = 10.7 µM, 4.5 µg/mL = 16.1 µM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-15 days

SELECTION AGENT (mutation assays): TFT (SERVA, 69042 Heidelberg, Germany)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4x10^3 cells in selective medium. The viability (cloning efficiency 2) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37°± 1.5°C in 4.5% CO2/95.5% water saturated air for 10 - 15 days. Then the plates were evaluated. Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test substance. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the large colonies).

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (main expt), relative suspension growth (pre-expt)

Evaluation criteria:

A test substance is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively.

A relevant increase of the mutation frequency should be concentration-dependent.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.

However, in the evaluation of the test results the historical variability of the mutation rates in solvent controls and the mutation rates of all solvent controls of this study are taken into consideration.

Results of test groups are generally rejected if the relative total growth and the cloning efficiency 1 (survival) is less than 10% of the solvent control unless the exception criteria specified by the IWGT recommendations are fulfilled.

Whenever a test substance is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and concentration dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

A test substance is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10^6 cells above the corresponding solvent control or negative control, respectively.

A test substance not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) was performed to assess a possible concentration dependent increase of mutant frequencies using the validated R Script LM.Rnw statistics software. The number of mutant colonies obtained for the groups treated with the test substance was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Expt I at 65.8 µg/mL (+/- S9 mix). Expt II at 66.0 µg/mL (- S9 mix) and ≥ 44.0 µg/mL (+ S9 mix). Phase separation noted +/- S9 mix in Expt I and -S9 mix in Expt II.

Vehicle controls validity:

valid

Positive controls validity:

valid

No substantial or reproducible concentration-dependent increase of the mutation frequency was observed in the main experiments with and without metabolic activation. The threshold was exceeded at 44.0 and 88.0 µg/mL in culture II of the second experiment with metabolic activation. However, this increase was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions and no increase in mutation frequency was seen in the first experiment with metabolic activation.

 

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using R Script LM.Rnw statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in the first culture of the first experiment with metabolic activation. This trend however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. Another significant dose dependent trend was determined in the second culture of the second experiment with metabolic activation. This trend was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

During the mutagenicity test described and under the experimental conditions reported the test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test substance is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

 

The assay was performed in two independent experiments, using two parallel cultures each. Experiments I and II were performed with and without liver microsomal activation and a treatment period of 4 hours

          

 The maximum concentration of the pre-experiment was 2819 µg/mL, equal to approximately 10 mM, based on the molecular weight (268.6 g/mol) and the purity (95.3%) of the test substance. The concentration range of the main experiments was limited by cytotoxicity and solubility of the test item.

 

The main experiments I and II were evaluated at the following concentrations:

Experiment I

without metabolic activation:               4.1; 8.2; 16.4; 32.9; and 65.8 µg/mL
with metabolic activation:                8.2; 16.4; 32.9; 65.8; and 132.0 µg/mL

 

Experiment II

without metabolic activation:                    11.0; 22.0; 44.0; and 66.0 µg/mL
with metabolic activation:                         22.0; 44.0; 66.0; and 88.0 µg/mL

 

Relevant cytotoxic effects indicated by a relative total growth of less than 50% in both parallel cultures occurred in experiment I at 65.8 µg/mL without metabolic activation and at 132.0 µg/mL with metabolic activation. In experiment II cytotoxic effects as described above were noted at 66.0 µg/mL without metabolic activation and at 44.0 µg/mL and above with metabolic activation.

 

No substantial or reproducible concentration-dependent increase of the mutation frequency was observed in the main experiments with and without metabolic activation. The threshold was exceeded at 44.0 and 88.0 µg/mL in culture II of the second experiment with metabolic activation. However, this increase was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions and no increase in mutation frequency was seen in the first experiment with metabolic activation.

 

A linear regression (least squares) was performed to assess a possible concentration dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in the first culture of the first experiment with metabolic activation. This trend however, was judged as irrelevant as it actually was reciprocal, going down versus increasing concentrations. Another significant dose dependent trend was determined in the second culture of the second experiment with metabolic activation. This trend was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical experimental conditions.

In conclusion, it can be stated that during the mutagenicity test described and under the experimental conditions reported the test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test substance is considered to be non-mutagenic in this mouse lymphoma assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-11 to 2014-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study under GLP and according to OECD TG 471 (1997), OPPTS 870.5100 (1998) and EC method B13/14 (2008)

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

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Remarks:

S. typhimurum: histidine locus, faulty lipopolysaccharide envelope, excision repair system, nitrat reductase, biotin synthesis, ampicillin resistance marker; Strain WP2: tryptophan biosynthesis, DNA repair process

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 – 12 w old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands).

Test concentrations with justification for top dose:

In the pre-experiment (experiment I): 3 - 5000 μg/plate
experiment II:
Without S9 mix: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate
With S9 mix:
Salmonella strains: 1, 3, 10, 33, 100, 333, 1000, and 2500 μg/plate
E.coli strains: 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubilisation properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

N-dimethylnitrosamine

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Precultures
The thawed bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 50 mL nutrient medium. A solution of 50 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, WP2 uvrA pKM101, and WP2 pKM101. This nutrient medium contains per litre:
8 g Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10^8-10^9 cells/mL).
Selective agar: The plates with the selective agar were obtained from E. Merck, D-64293 Darmstadt.
Overlay agar: The overlay agar contains per litre:
for Salmonella strains (all constituents from MERCK, D-64293 Darmstadt):
7.0 g Agar Agar
6.0 g NaCl
10.5 mg L-Histidine×HCl×H2O
12.2 mg Biotin
for Escherichia coli (all constituents from MERCK, D-64293 Darmstadt):
7.0 g Agar Agar
6.0 g NaCl
10.2 mg Tryptophan
Sterilisations were performed at 121 °C in an autoclave.

DURATION
- Preincubation period:
The bacterial strains TA1535, TA1537, TA98, TA100, WP2 uvrA pKM101, and WP2pKM101 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany). The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
For each strain and concentration level including the controls, three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each concentration level, solvent (negative control) or reference mutagen solution (positive control),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (Substitution buffer: 7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M; for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains; OD = 1.0 - 1.5, wavelength = 500 nm; approx. 8x108 cells/mL),
2000 μL Overlay agar
For the pre-incubation method 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer (Substitution buffer: 7 parts of the 100 mM sodium-ortho-phosphate-buffer pH 7.4 with 3 parts of KCl solution 0.15 M) and 100 μL bacteria suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After preincubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
- Exposure duration:
After solidification the plates were incubated upside down for 72 hours at 37°C in the dark, plates were then stored at 4°C until counted.
- Expression time (cells in growth medium): 60 min pre-incubation + 72 hours incubation
- Selection time (if incubation with a selection agent): 60 min pre-incubation + 72 hours incubation
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): yes
SPINDLE INHIBITOR (cytogenetic assays): no
STAIN (for cytogenetic assays): no

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 100 μL Bacteria suspension (test system, pre-culture of the strains; OD = 1.0 - 1.5, wavelength = 500 nm; approx. 8x10 8 cells/mL), i.e. 8x10 7 cells/100 µL

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

Data recording
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to precipitation of the test item, reduced background growth and wide spread bacteria colony growth some plates were counted manually.

Acceptability of the assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation of results
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

Statistics:

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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: the solvent DMSO was used due to limited water solubility of the test substance.
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 μg/plate up to the highest investigated concentration in experiment I with and without S9 mix and in experiment II with S9 mix. In experiment II without S9 mix precipitation was observed at 2500 up to 5000 μg/plate. The undissolved particles had no influence on the data recording
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
To evaluate the cytotoxicity of the test substance a pre-experiment was performed with all strains. Eight concentrations were tested for cytotoxicity and mutation induction each with three replicate plates. The experimental conditions in this pre-experiment were the same as described below for experiment I (plate incorporation test). Cytotoxicity of the test substance results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. The pre-experiment is reported as the main experiment I since the criteria mentioned under Acceptability of the assay were met..

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment II, the number of colonies did not quite reach the lower limit of our historical control data in strain TA1535 (solvent control, wit S9 mix), in strain TA100 (solvent control, with S9 mix) and in strain WP2 uvrA pKM101(solvent control without S9 mix, solvent and untreated control with S9 mix)). Since these deviations are rather small, these effects are judged to be based upon statistical fluctuations and have no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed reduced background growth at the following concentrations (μg/plate):
Strain --- Exp. I (without S9 mix) --- Exp. I (with S9 mix) --- Exp. II (without S9 mix) --- Exp. II (with S9 mix)
TA 1535 --- 5000 --- 333 - 5000 --- 1000 - 5000 --- 1000 - 2500
TA 1537 --- 1000 - 5000 --- 1000 - 5000 --- 100 - 5000 --- 100 - 2500
TA 98 --- 1000 - 5000 --- 1000 - 5000--- 333 - 5000 --- 333 - 2500
TA 100 --- 333 - 5000 --- 333 - 5000 --- 100 - 5000 --- 333 - 2500
WP2 pKM101 --- / --- / --- / --- /
WP2uvrA pKM101 --- / --- / --- / --- /
/ = no reduced background growth

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (μg/plate):
Strain --- Exp. I (without S9 mix) --- Exp. I (with S9 mix) --- Exp. II (without S9 mix) --- Exp. II (with S9 mix)
TA 1535 --- / --- 1000 - 5000 --- 5000 2500
TA 1537 --- 1000 - 5000 --- 1000 - 5000 --- 2500 - 5000 --- 333 - 2500
TA 98 --- 2500 - 5000 --- 2500 - 5000 --- 5000 --- 1000 - 2500
TA 100 --- 1000 - 5000 --- 1000 - 5000 --- 333 - 5000 --- 1000 - 2500
WP2 pKM101 --- 2500 - 5000 --- 2500 - 5000 --- 1000 - 5000 --- 2500 - 5000
WP2 uvrA pKM101 --- / --- / --- / --- /
/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 Summary of Results Pre-Experiment/Experiment I

Metabolic Activation

Test Group

Concentration Level (per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 pKM101

WP2 uvrA pKM101

Without Activation

DMSO

---

16 ± 4

---

13 ± 3

---

27 ± 5

---

105 ± 6

---

258 ± 12

---

395 ± 28

---

Untreated

---

16 ± 5

---

8 ± 3

---

27 ± 3

---

111 ± 5

---

266 ± 11

---

411 ± 53

---

CA5204A

3 µg

16 ± 4

---

13 ± 3

---

28 ± 5

---

104 ± 6

---

246 ± 19

---

403 ± 23

---

10 µg

15 ± 6

---

9 ± 1

---

26 ± 1

---

91 ± 6

---

247 ± 11

---

381 ± 12

---

33 µg

18 ± 4

---

15 ± 1

---

26 ± 7

---

90 ± 7

---

224 ± 25

---

362 ± 27

---

100 µg

18 ± 3

---

9 ± 1

---

25 ± 4

---

90 ± 15

---

199 ± 18

---

399 ± 23

---

333 µg

15 ± 0

---

9 ± 4

---

26 ± 6

---

54 ± 10

RM

158 ± 16

---

317 ± 20

---

1000 µg

18 ± 4

P

6 ± 1

RP

21 ± 7

PR

30 ± 5

PMR

137 ± 9

P

277 ± 36

P

2500 µg

22 ± 1

P

4 ± 1

PMR

8 ± 2

PMR

25 ± 5

PMR

94 ± 3

P

298 ± 25

P

5000 µg

11 ± 2

PMR

3 ± 1

PMR

5 ± 1

PMR

17 ± 5

PMR

72 ± 10

P

252 ± 15

P

NaN3

10 µg

2482 ± 79

---

---

---

---

---

1942 ± 52

---

---

---

---

---

4-NOPD

10 µg

---

---

---

---

274 ± 16

---

---

---

---

---

---

---

4-NOPD

50 µg

---

---

68 ± 7

---

---

---

---

---

---

---

---

---

MMS

2.0 µL

---

---

---

---

---

---

---

---

3656 ± 209

---

3847 ± 455

---

With Activation

DMSO

---

31 ± 3

---

18 ± 3

---

39 ± 5

---

114 ± 9

---

250 ± 22

---

407 ± 23

---

Untreated

---

29 ± 4

---

22 ± 8

---

46 ± 5

---

158 ± 13

---

282 ± 3

---

406 ± 23

---

CA5204A

3 µg

31 ± 5

---

25 ± 8

---

42 ± 9

---

123 ± 14

---

243 ± 10

---

393 ± 23

---

10 µg

25 ± 3

---

19 ± 7

---

38 ± 4

---

105 ± 3

---

204 ± 15

---

381 ± 12

---

33 µg

23 ± 7

---

16 ± 7

---

37 ± 6

---

106 ± 10

---

222 ± 8

---

385 ± 19

---

100 µg

22 ± 7

---

20 ± 4

---

36 ± 1

---

115 ± 9

---

211 ± 43

---

360 ± 21

---

333 µg

15 ± 1

R

15 ± 6

---

31 ± 3

---

64 ± 9

R

199 ± 11

---

370 ± 33

---

1000 µg

14 ± 4

RP

7 ± 0

RP

28 ± 5

PR

33 ± 6

PMR

132 ± 8

P

272 ± 6

P

2500 µg

7 ± 2

PMR

7 ± 2

PMR

8 ± 1

PMR

24 ± 3

PMR

97 ± 6

P

251 ± 13

P

5000 µg

4 ± 2

PMR

6 ± 1

PMR

7 ± 2

PMR

16 ± 4

PMR

42 ± 12

P

224 ± 13

P

2-AA

2.5 µg

494 ± 24

---

321 ± 30

---

2425 ± 352

---

3139 ± 156

---

---

---

---

---

2-AA

10.0 µg

---

---

---

---

---

---

---

---

3713 ± 139

---

2383 ± 214

---

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

R: Reduced background growth

M: Manual count

P: Precipitate

Table 2 Summary of Results Pre-Experiment/Experiment II

Metabolic Activation

Test Group

Concentration Level (per plate)

Revertant Colony Counts (Mean ±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 pKM101

WP2 uvrA pKM101

Without Activation

DMSO

---

22 ± 7

---

7 ± 1

---

18 ± 2

---

113 ± 32

---

201 ± 17

---

281 ± 17

---

Untreated

---

22 ± 7

---

10 ± 2

---

27 ± 8

---

93 ± 2

---

213 ± 14

---

289 ± 22

---

CA5204A

3 µg

18 ± 5

---

5 ± 3

---

20 ± 4

---

84 ± 10

---

197 ± 17

---

270 ± 28

---

10 µg

18 ± 7

---

7 ± 2

---

18 ± 2

---

78 ± 6

---

173 ± 32

---

326 ± 61

---

33 µg

22 ± 1

---

8 ± 1

---

16 ± 6

---

72 ± 12

---

174 ± 14

---

289 ± 17

---

100 µg

21 ± 6

---

5 ± 4

R

18 ± 5

---

71 ± 10

R

117 ± 2

---

213 ± 6

---

333 µg

30 ± 3

---

5 ± 2

RM

18 ± 7

R

44 ± 15

MR

93 ± 12

---

190 ± 8

---

1000 µg

27 ± 1

R

5 ± 1

MR

17 ± 3

R

12 ± 5

MR

68 ± 9

---

164 ± 13

---

2500 µg

12 ± 7

MRP

2 ± 2

PMR

14 ± 2

PMR

15 ± 8

PMR

35 ± 7

PM

164 ± 11

P

5000 µg

8 ± 2

MRP

2 ± 1

PMR

4 ± 2

PMR

15 ± 4

PMR

34 ± 9

PM

146 ± 3

P

NaN3

10 µg

2472 ± 308

---

---

---

---

---

1507 ± 273

---

---

---

---

---

4-NOPD

10 µg

---

---

---

---

341 ± 31

---

---

---

---

---

---

---

4-NOPD

50 µg

---

---

68 ± 1

---

---

---

---

---

---

---

---

---

MMS

2.0 µL

---

---

---

---

---

---

---

---

3740 ± 284

---

2871 ± 473

---

With Activation

DMSO

---

10 ± 1

BM

18 ± 3

---

38 ± 2

---

84 ± 6

---

199 ± 16

---

270 ± 15

---

Untreated

---

12 ± 2

BM

27 ± 2

---

51 ± 3

---

130 ± 9

---

266 ± 17

---

314 ± 27

---

CA5204A

1µg

10 ± 1

BM

24 ± 6

---

36 ± 9

---

93 ± 2

---

---

---

---

---

3 µg

8 ± 2

BM

21 ± 6

---

45 ± 13

---

92 ± 7

---

197 ± 18

---

275 ± 15

---

10 µg

8 ± 2

BM

16 ± 3

---

31 ± 4

---

90 ± 6

---

191 ± 30

---

249 ± 23

---

33 µg

8 ± 2

BM

17 ± 5

---

37 ± 9

---

97 ± 7

---

176 ± 11

---

256 ± 8

---

100 µg

6 ± 1

BM

18 ± 4

R

33 ± 5

---

78 ± 12

---

185 ± 15

---

246 ± 36

---

333 µg

5 ± 2

BM

6 ± 4

MRP

27 ± 5

R

64 ± 11

R

150 ± 16

---

233 ± 4

---

1000 µg

5 ± 0

BMRP

5 ± 2

MRP

6 ± 3

PMR

19 ± 3

PMR

104 ± 8

P

174 ± 14

P

2500 µg

4 ± 1

BMRP

5 ± 1

MRP

3 ± 2

PMR

14 ± 2

PMR

71 ± 8

P

163 ± 7

P

5000 µg

---

---

---

---

---

---

---

---

56 ± 6

P

165 ± 22

P

2-AA

2.5 µg

465 ± 81

---

184 ± 15

---

2374 ± 61

---

1633 ± 147

---

---

---

---

---

2-AA

10.0 µg

---

---

---

---

---

---

---

---

4085 ± 4

---

1858 ± 277

---

NaN3: sodium azide

2-AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

R: Reduced background growth

M: Manual count

P: Precipitate

B: Extensive bacterial growth

Conclusions:

Interpretation of results (migrated information):
negative

In a valid, reliable and conclusive study according to OECD TG 471 (1997), OPPTS 870.5100 (1998) and EC Method B13/14 (2008), the test item CA5204A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used during the described mutagenicity tests and under the experimental conditions reported, CA5204A is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

Study Design

This study was performed to investigate the potential of CA5204A to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101.

Results

The plates incubated with the test item CA5204A showed reduced background growth in nearly all strains. Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in almost all strains. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with CA5204A at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutationn rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, CA5204A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 3

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

Study period:

2013-10-15 to 2014-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study under GLP and according to the following guidelines: OECD 473 (1997), EPA OPPTS 870.5375 (1998), EC 440/2008 B. 10 (2008)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood cultures were established by preparing a 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), the mitogen PHA (phytohemagglutinin) (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
The following volumes were added to the flasks (per 10 mL):
7.60 mL culture medium
1.00 mL fetal bovine serum
0.10 mL antibiotic solution
0.05 mL phytohemagglutinin
0.05 mL heparin
0.10 mL HEPES
1.10 mL whole blood
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix: Phenobarbital/beta-naphthoflavone induced rat liver S9. Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

Concentrations Applied in the Chromosome Aberration Assay with CA5204A (Concentrations in µg/mL):
Without S9 mix
IA --- prep. interval 22 hrs --- exposure 4 hrs --- 18.7 --- 32.7 --- 57.3PS --- 100.3PS --- 175.5PS --- 307.1PS --- 537.4PS --- 940.4PS --- 1645.7PS --- 2880.0PS
IIA --- prep. interval 22 hrs --- exposure 22 hrs --- 0.6 --- 1.1 --- 1.9 --- 3.4 --- 6.0 --- 10.4 --- 18.3 --- 32.0 --- 56.0 --- 98.0 --- 171.4PS --- 300.0PS
IIB --- prep. interval 22 hrs --- exposure 22 hrs --- 5.0 --- 10.0 --- 20.0 --- 40.0 --- 50.0 --- 60.0 --- 70.0 --- 80.0 --- 90.0 --- 100.0 --- 120.0 --- 140.0 --- 200.0PS
With S9 mix
IA --- prep. interval 22 hrs --- expsoure 4 hrs --- 18.7 --- 32.7 --- 57.3 --- 100.3PS --- 175.5PS --- 307.1PS --- 537.4PS --- 940.4PS --- 1645.7PS --- 2880.0PS
IB --- prep. interval 22 hrs --- exposure 4 hrs --- 18.7 --- 32.7 --- 57.3 --- 100.3PS --- 175.5PS --- 307.1PS --- 537.4PS --- 940.4PS --- 1645.7PS --- 2880.0PS
IIA --- prep. interval 22 hrs --- exposure 4 hrs --- 25.0 --- 50.0 --- 75.0 --- 100.0 --- 125.0PS --- 150.0PS --- 175.0PS --- 200.0PS --- 225.0PS --- 300.0PS --- 400.0PS --- 500.0PS

PS: Phase separation was observed at the end of treatment

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, the final concentration of DMSO in the culture medium was 0.5 % (v/v).
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubilisation properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

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:
On the day of the experiment (immediately before treatment), the test substance was dissolved in DMSO. The final concentration of DMSO in the culture medium was 0.5 % (v/v). The culture medium was replaced with serum-free medium containing the test substance.

DURATION
- Preincubation period: About 48 h after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks (Nunc GmbH & Co. KG, 65203 Wiesbaden, Germany).
- Exposure duration:
Exposure time 4 hours: The culture medium was replaced with serum-free medium containing the test substance. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 h the cells were spun down by gentle centrifugation for 5 minutes (approx. 900 x g).
Exposure time 22 hours (without S9 mix): The culture medium was replaced with complete medium (with 10 % FBS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells.
- Fixation time (start of exposure up to fixation or harvest of cells): The cultures were treated with the metaphase-arresting substance Colcemid (final concentration: 0.2 µg/mL) approximately three hours before the requested harvest time. The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g) the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concentration: 0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment IIB without S9 mix, where only 50 metaphases were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis)

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates.

Evaluation criteria:

A test substance is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated concentration groups is in the range of the laboratory historical control data range.
- no significant increase of the number of structural chromosome aberrations is observed.
A test substance is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory historical control data
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
A test substance not meeting the criteria for classification as non-mutagenic or mutagenic may be considered equivocal in this assay and may be subject to further investigation.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test substance 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.

Species / strain:

lymphocytes:

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: use of the solvent DMSO
- Precipitation: Phase separation was observed at the end of treatment in the absence of S9 mix at 57.3 µg/mL and above (Exp. IA), at 171.4 µg/mL and above (Exp. IIA) and at 200.0 µg/mL (Exp. IIB). In the presence of S9 mix phase separation was observed at the end of treatment at 100.3 µg/mL and above (Exp. IB) and at 125.0 µg/mL and above (Exp. IIA).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test substance and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 22 h after start of the exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical Control Data available as percentage of aberrant cells in human lymphocyte cultures (2011-2012)


ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment IA and IIB in the absence of S9 mix and in Experiment IB in the presence of S9 mix cytotoxicity was observed at the highest evaluated concentration (51.8, 39.9 and 46.9 % of control, respectively). In Experiment IIA in the absence and presence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1 Summary of results of the chromosomal aberration study with CA5204A without S9 mix

Exp.	Preparation interval	Test item concentration	Mitotic indices	Aberrant cells (in %)
		in µg/mL	% of control	incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs without S9 mix
IA	22 hrs	Solvent control^1	100	1	0.5	0
		Positive control^2	65.8	13.5	13.0^S	4
		32.7	118.9	3	2.5	0
		57.3^PS	105.7	1	1	0
		100.3^PS	51.8	1.5	1	0
Exposure period 22 hrs without S9 mix
IIA	22 hrs	Solvent control^1	100	2.5	1	0.5
		Positive control^3	31.2	8.5	8.5^S	2
		18.3	121.9	2	2	0
		32	119.3	1.5	1.5	0
		56	73.6	0.5	0.5	0
IIB	22 hrs	Solvent control^1	100	2	2	0
		Positive control^4#	35.4	51	48.0^S	10
		50	87.8	2.5	2.5	0
		60	112.5	3	2.5	0
		70	39.9	1	1	0

*: Including cells carrying exchanges

PS: Phase separation occurred at the end of treatment

S: Aberration frequency statistically significant higher than corresponding control values

#: Evaluation of 50 metaphases per culture

1: DMSO 0.5 % (v/v)

2: EMS 770.0 µg/mL

3: EMS 660.0 µg/mL

4: EMS 700.0 µg/mL

Table 2 Summary of results of the chromosomal aberration study with CA5204A with S9 mix

 

Exp.	Preparation interval	Test item concentration	Mitotic indices	Aberrant cells (in %)
		in µg/mL	% of control	incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs with S9 mix
IB	22 hrs	Solvent control^1	100	1.5	1	0
		Positive control^2	39.8	15.5	15.0^S	3.5
		57.3	103.5	3	2.5	0
		100.3^PS	101.3	1	1	0
		175.5^PS	46.9	2.5	2.5	0.5
IIA	22 hrs	Solvent control^1	100	2	2	0
		Positive control^2	42.2	8.5	8.5^S	1.5
		100	95	1	1	0
		125.0^PS	107.6	1	0.5	0
		150.0^PS	100.3	3.5	3.5	0.5

*: Including cells carrying exchanges

PS: Phase separation occurred at the end of treatment

S: Aberration frequency statistically significant higher than corresponding control values

1: DMSO 0.5 % (v/v)

2: CPA 7.5 µg/mL

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In a valid, conclusive and reliable study according to OECD TG 473 (1997), EPA OPPTS 870.5375 (1998) and EC 440/2008 B.10 (2008), the test substance CA5204A did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, CA5204A is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest evaluable concentrations.

Executive summary:

Study Design

This in vitro assay was performed to assess the potential of CA5204A to induce structural chromosomal aberrations in cultured human lymphocytes in the absence and presence of an exogenous metabolic activation system (liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats).

In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive controls in Experiment IIB without S9 mix, where only 50 metaphases were evaluated.

The highest applied concentration in this study (2880.0 µg/mL of the test substance, approx. 10 mM) was chosen with regard to the molecular weight and the purity (93.2 %) of the test substance and with respect to the current OECD Guideline 473.

Concentration selection for the cytogenetic experiments was performed considering the toxicity data in accordance with OECD Guideline 473.

Results

In Experiment IA and IIB in the absence of S9 mix and in Experiment IB in the presence of S9 mix cytotoxicity was observed at the highest evaluated concentration. In Experiment IIA in the absence and presence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage.

In the absence and presence of S9 mix, no clastogenicity was observed at the concentrations evaluated.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test substance as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, CA5204A is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest evaluable concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

N-methoxy-1 -(2,4,6 -trichlorophenyl)propan-2 -amine was tested in the rat bone marrow micronucleus test according to OECD 474 (Sequani 2015).

No evidence of clastogenicity or aneugenicity following oral (gaveage) administration of the substance uo to the MTD of 175 mg/kg/day in male rats.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration The in vivo micronucleus study was conducted solely to comply with non-EU national registration requirement, and has been provided here in accordance with REACH, Article 22(1)e

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 October 2014 to 09 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-7 Weeks
- Weight at study initiation: 155-193 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: In groups of up to three, by sex, in grid-floor cages suspended over paper-lined trays.
- Diet: LabDiet 5L0S EURodent Diet (pelleted) ad libitum
- Water: Mains tap water ad libitum
- Acclimation period: 5 days (main study)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-20
- Humidity (%): 42-70
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12/12

EXPERIMENTAL DATES: From: 02 October 2014 (1st animal arrival) To: 09 December 2014 (last day of slide scoring)

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1.0 % w/v carboxymethylcellulose with 1.0 % v/v Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated for dosing as a suspension in the vehicle. Separate formulations were prepared for each dose level, with the weighed quantity of test item (adjusted for purity) being suspended in the appropriate quantity of vehicle. Formulations were prepared on the day of use. A weighed quantity of test item was added to a suitable container calibrated to the final preparation volume. Approximately 90 % of the final volume of vehicle was added gradually, whilst stirring. The formulation was then stirred and heated to a maximum of 51°C, until a homogenous mixture was formed. The formulation was then allowed to cool before adding vehicle up to the final volume and stirring until mixed. The formulation was then transferred to an amber glass bottle.

Cyclophosphamide monohydrate (CPA), the positive control item, was a 3 mg/mL solution of CPA in UHP water.

Duration of treatment / exposure:

2 days

Frequency of treatment:

once per day

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
0, 45, 90, 175 mg/kg/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CPA)
- Route of administration: Oral gavage
- Doses / concentrations: 15 mg/kg (single dose at a dose volume of 5 mL/kg)

Tissues and cell types examined:

Bone marrow cells (from femur)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A dose-sighting phase was carried out (2 males/group, dose levels 175, 200, 320 mg/kg/day, 2 doses) in order to select the highest dose of the test item that did not produce mortality or severe signs of clinical toxicity up to the MTD level. Then a dose range-finding phase was carried out (175 mg/kg/day - 3 males and 3 females, 275 and 450 mg/kg/day - 3 females/group only, 2 doses) to confirm that the MTD level identified in the dose-sighting phase was tolerated in males and females.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Three groups, each of six male rats were dosed with 45, 90 or 175 mg/kg/day test item on two successive days, separated by approximately 24 hours (Groups 2 to 4). A group of six male rats (negative control - Group 1) was dosed with the vehicle alone on two successive days, separated by approximately 24 hours and a positive control group (Group 5), also of six male rats, was given a single 15 mg/kg oral (gavage) dose of Cyclophosphamide monohydrate (CPA).

Animals were killed approximately 24 hours after their second dose (negative control and test item-treated groups) or first dose (positive control group). Bone marrow was harvested from each animal and smears prepared. The stained slides were coded, 2000 polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei and the group frequencies were statistically analysed.

DETAILS OF SLIDE PREPARATION: One femur from each animal was exposed by dissection of the surrounding muscle and connective tissues and the shank of the bone removed. The bone marrow cells from each femur were aspirated into labelled centrifuge tubes using a syringe containing foetal bovine serum. The bone marrow cells were centrifuged, the supernatant withdrawn, and the cells re-suspended in a minimal volume of foetal bovine serum. One drop of cell suspension was placed on each of two slides and spread by drawing an edge of a clean glass microscope slide along from the drop to the end of the slide. All slides were left to air dry and age overnight before fixing for 5 minutes in methanol. Fixed slides were stained for 20 to 30 minutes in 11.5 % (v/v) Giemsa in Sorensen’s buffer pH 6.0, based on the method of Gollapudi and Kamra (Gollapudi, B. and Kamra, O.P. Application of a Simple Giemsa-Staining Method in the Micronucleus Test. Mutation Research. (1979) 64 45-46.).

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCE), including micronucleated PCE (MN-PCE), were counted for each animal. The numbers of normochromatic (NCE) and micronucleated NCE (MN-NCE) erythrocytes were also recorded for the first 1000 cells scored. Only areas of slides of good technical quality and appropriate staining characteristics were scored. As NCE were only recorded for the first 1000 erythrocytes scored, the PCE/NCE ratio was calculated as follows: PCE/NCE = (1000-NCE count)/NCE count)

OTHER: A proof of exposure phase was conducted to demonstrate that the bone marrow was exposed to the test item. This was demonstrated by analysis of test item in the whole blood of treated animals. The presence of the test item was confirmed by analysis of the study samples alongside samples of blank matrix and matrix spiked with the test item.

Evaluation criteria:

The test was considered to be positive, i.e. test item was considered to induce clastogenic/aneugenic damage, if all of the following were observed:
• A statistically significant increase in the frequency of MN-PCE occurred at one or more dose levels.
• The incidence and distribution of MN-PCE in individual animals at the dose level(s) showing statistical significance exceeded the laboratory’s historical negative control data.
• A dose-related increase in the frequency of MN-PCE (where more than two dose levels are analysed) was observed.
Results which only partially satisfy the above criteria would be dealt with on a case-by-case basis. Evidence of a dose-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between dose levels.
A test was considered to be negative, i.e. non-clastogenic/aneugenic, if there was neither a dose-response curve nor any group showed statistically significant increases in the frequency of micronucleated PCEs compared to the negative controls.

Statistics:

The data analysed were the proportion of MN-PCEs and the ratio of polychromatic to normochromatic cells (PCE/NCE). The preferred approach for the MN-PCE data is to combine the data within each group and construct a 2x2 contingency table for each treated group with the negative control. The groups are then compared using a one tailed Fisher Exact test. However, this approach must first be validated by carrying out a test for between animal heterogeneity using a chi
square test. If the heterogeneity test is significant at the 1% level then an exact Wilcoxon Rank Sum test is used instead of the Fisher Exact test. The same method is used to compare the positive and negative controls. For the PCE/NCE data, the treated groups were compared with the negative control using one tailed exact Wilcoxon Rank Sum tests.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

in males at concentrations of 175 mg/kg/day

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DOSE-SIGHTING STUDY:
- Doses: 175, 200, 320 mg/kg/day (males only)
- Clinical signs of toxicity in test animals: Yes at 200 and 320 mg/kg/day (piloerection, tremors, hunched posture, vocalisation on handling, incoordination, eyes partially closed, unsteady gait). Animals killed approximately 6 hours post second dose of 320 mg/kg/day due to clinical signs.

RESULTS OF RANGE-FINDING STUDY
- Doses: 175 (males and females), 275 (females), 450 (females) mg/kg/day
- Clinical signs of toxicity in test animals: Yes in females only (abnormal sensitivity to touch or disturbance, intermittent tremors @ 175 and 275 mg/kg/day; unsteady and abnormal gait @ 275 and 450 mg/kg/day; decreased activity, tremors, intermittent twitching, piloerection, vocalisation on handling, incoordination @450 mg/kg/day). Females killed approximately 0.75 hours post second dose of 450 mg/kg/day due to clinical signs.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Yes at 175 mg/kg/day (abnormal sensitivity to touch or disturbance)
- Induction of micronuclei (for Micronucleus assay): No
- Ratio of PCE/NCE (for Micronucleus assay): 0.74, 0.98, 0.85, 0.93, 0.89 for 0, 45, 90, 175 mg/kg/day test item and 15 mg/kg CPA, respectively.
- Appropriateness of dose levels and route: Satisfactory
- Statistical evaluation: There were no statistically significant differences in any parameter between test item-treated groups and the vehicle control group. The mean MN-PCE for the positive control group was statistically significantly different (p<0.001, Fischer's Exact test) from the vehicle controls.

Table 1: Micronucleus results

	Dose level (mg/kg/day)
	0 mg/kg/day (control)	45 mg/kg/day	90 mg/kg/day	175 mg/kg/day	15 mg/kg CPA (positive control)
Mean MN-PCE	1.00±1.26	1.33±0.82	0.83±0.98	1.83±1.33	15.17±2.99***
Mean PCE/NCE ratio	0.74±0.17	0.98±0.17	0.85±0.19	0.93±0.28	0.89±0.11
*** Statistically significant p<0.001 (Fisher’s Exact test)

 

Conclusions:

Interpretation of results (migrated information): negative
There was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test item up to the MTD of 175 mg/kg/day in male rats. Therefore, the test item is considered to be neither clastogenic nor aneugenic in this bone marrow micronucleus assay.

Executive summary:

The MTD was confirmed (in dose-sighting and range-finding phases) as 175 mg/kg/day in male rats and 275 mg/kg/day in female rats. As there were no substantial inter-sex differences in toxicity, the main study was conducted in males only, with the high dose selected as 175 mg/kg/day.

 

Three groups, each of six male rats were dosed, by oral gavage, with 45, 90 or 175 mg/kg/day test item on two successive days, separated by approximately 24 hours. A group of six male rats (negative control) was dosed with the vehicle alone on two successive days, separated by approximately 24 hours and a positive control group, also of six male rats, was given a single 15 mg/kg oral (gavage) dose of Cyclophosphamide monohydrate (CPA). Animals were killed approximately 24 hours after their second dose (negative control and test item-treated groups) or first dose (positive control group). Bone marrow was harvested from each animal and smears prepared. The stained slides were coded, 2000 polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei and the group frequencies were statistically analysed.

 

There were no statistically significant increases in micronucleus frequency in male rats treated at any dose level with the test item, compared with the negative control group. There was no evidence of a statistically significant reduction in the PCE/NCE ratio in male rats treated with the test item, indicating a lack of toxicity to the bone marrow. Proof of exposure was demonstrated in the range-finding and main study phases. The animals dosed with CPA, the positive control item, had statistically significant increases in the number of micronucleated cells compared to the concurrent control group, which demonstrated that the test system was capable of detecting a known clastogen and that the scorers were capable of detecting micronuclei. There was no statistically significant decrease in the PCE/NCE ratio in the positive control group, indicating a lack of toxicity to the bone marrow.

 

In conclusion, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test item up to the MTD of 175 mg/kg/day in male rats. Therefore, the test item is considered to be neither clastogenic nor aneugenic in this bone marrow micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the available studies, the substance does not meet the criteria for classification according to CLP Regulation (EC) No 1272/2008 Of the European parliament and the Council.