Magnesium octadecylbenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3 in vitro genetic toxicity studies on read-across materials.

OECD 471 - negative

OECD 473 - negative

OECD 476 - negative

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD guidelines and to GLP, so therefore meets the criteria of Klimisch code 1.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 cells

Test concentrations with justification for top dose:

500, 1000, 1500, 2000, 4000, and 5000 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with activation

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without activation

Details on test system and experimental conditions:

The test material was suspended in culture and palced on a restricted media containing trifluorothymidine.
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 4 hours
- Exposure duration: 10-12 days

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Number of colonies/plate.

Statistics:

Mean and standard deviation.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Significant toxicity (<90% total growth) at 500 µg/ml occurred both with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material was not genotoxic under the conditions of test.

Executive summary:

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells cultured in vitro were exposed to a petroleum derived calcium salt, at concentrations of  500, 1000, 1500, 2000, 4000 and 5000 µg/ml in the presence and absence of mammalian metabolic activation. 

 

The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD guidelines and to GLP, so therefore meets the criteria of Klimisch code 1.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 cells

Test concentrations with justification for top dose:

500, 1000, 1500, 2000, 4000, and 5000 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with activation

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without activation

Details on test system and experimental conditions:

The test material was suspended in culture and palced on a restricted media containing trifluorothymidine.
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 4 hours
- Exposure duration: 10-12 days

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Number of colonies/plate.

Statistics:

Mean and standard deviation.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Significant toxicity (<90% total growth) at 500 µg/ml occurred both with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material was not genotoxic under the conditions of test.

Executive summary:

In a mammalian cell gene mutation assay, mouse lymphoma L5178Y cells cultured in vitro were exposed to a petroleum derived calcium salt, at concentrations of  500, 1000, 1500, 2000, 4000 and 5000 µg/ml in the presence and absence of mammalian metabolic activation. 

 

The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

20-Jun-2014 to 31-Aug-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Reason / purpose for cross-reference:

read-across source

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1.7, 5.4, 17, 52, 164, 512 and 1600 µg/mL

First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 52, 164 and 512 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 75, 100 and 125 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 50, 75 and 100 µg mL

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

other:

Remarks:

without S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

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

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human pheripheral blood

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 512 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose in the absence and presence of S9, 3 hr treatment. Toxicity was observed at dose levels of 164 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test substance was tested up to precipitate or appropriate toxicity was reached at the dose levels selected for scoring.

Conclusions:

Interpretation of results (migrated information):
negative

EXP1313100 is not clastogenic in human lymphocytes under the experimental conditions described in the report

Executive summary:

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

EXP1313100 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

 

No biologically relevant effects of EXP1313100 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that EXP1313100 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Reference 4

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:

20-Jun-2014 to 31-Aug-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1.7, 5.4, 17, 52, 164, 512 and 1600 µg/mL

First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 52, 164 and 512 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 75, 100 and 125 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 50, 75 and 100 µg mL

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

other:

Remarks:

without S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

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

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human pheripheral blood

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 512 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose in the absence and presence of S9, 3 hr treatment. Toxicity was observed at dose levels of 164 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test substance was tested up to precipitate or appropriate toxicity was reached at the dose levels selected for scoring.

Conclusions:

Interpretation of results (migrated information):
negative

EXP1313100 is not clastogenic in human lymphocytes under the experimental conditions described in the report

Executive summary:

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

EXP1313100 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

 

No biologically relevant effects of EXP1313100 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that EXP1313100 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

12-Jun-2014 to 23-Jun-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Conclusions:

It is concluded that EXP1313100 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Executive summary:

EXP1313100 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Reference 6

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:

12-Jun-2014 to 23-Jun-2014

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Conclusions:

It is concluded that EXP1313100 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Executive summary:

EXP1313100 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

in vivo micronucleus study on a read-across material

OECD 474 = negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-Jun-2014 to 08-Oct-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 144 - 169 gram
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 23.0°C
- Humidity (%): 45 - 81%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Propylene glycol
- Justification for choice of solvent/vehicle: Test compound was stable in Propylene glycol (Stability for at least 6 hours at room temperature is confirmed over the concentration range 20 to 200 mg/mL)and a suspension could be obtained in Propylene glycol. Propylene glycol has been accepted and approved by authorities and international guidelines. EXP1313100 concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension


- Concentration of test material in vehicle: 100, 200 and 400 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 5 ml/kg body weight

Details on exposure:

Chemical analysis of dose preparations
The concentrations analysed in the formulations of the dose levels groups 500, 1000 and 2000 mg/kg BW were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group A formulation. It was considered not to derive from the formulation since a small response was obtained in the analytical blanks.

The formulations of the dose levels groups 500 and 2000 mg/kg BW were homogeneous (i.e. coefficient of variation = 10%).

Formulations at the entire range were stable at room temperature under normal laboratory light conditions for at least 4 hours

Duration of treatment / exposure:

Treatment:
Solvent control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours
Positive control: 48 hours

Frequency of treatment:

Once

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/bw
Basis:
analytical conc.

No. of animals per sex per dose:

Five animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 20 mg/kg body weight

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
-It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Statistics:

Wilcoxon Rank Sum Test, one-sided, p < 0.05

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg BW
- Clinical signs of toxicity in test animals: No treatment related clinical signs of toxicity or mortality.


RESULTS OF DEFINITIVE STUDY

- Dose range: 500, 1000 and 2000 mg/kg BW
- Clinical signs of toxicity in test animals: No treatment related clinical signs of toxicity or mortality
- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with EXP1313100.
- Ratio of PCE/NCE (for Micronucleus assay):
No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis.

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that EXP1313100 is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male rats up to a dose of 2000 mg/kg the recommended dose in accordance with current regulatory guidelines under the experimental conditions described in the report.

Executive summary:

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with EXP1313100 compared to the vehicle treated animals.

 

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

 

The groups that were treated with EXP1313100 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

11-Jun-2014 to 08-Oct-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 144 - 169 gram
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 23.0°C
- Humidity (%): 45 - 81%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Propylene glycol
- Justification for choice of solvent/vehicle: Test compound was stable in Propylene glycol (Stability for at least 6 hours at room temperature is confirmed over the concentration range 20 to 200 mg/mL)and a suspension could be obtained in Propylene glycol. Propylene glycol has been accepted and approved by authorities and international guidelines. EXP1313100 concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension


- Concentration of test material in vehicle: 100, 200 and 400 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 5 ml/kg body weight

Details on exposure:

Chemical analysis of dose preparations
The concentrations analysed in the formulations of the dose levels groups 500, 1000 and 2000 mg/kg BW were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group A formulation. It was considered not to derive from the formulation since a small response was obtained in the analytical blanks.

The formulations of the dose levels groups 500 and 2000 mg/kg BW were homogeneous (i.e. coefficient of variation = 10%).

Formulations at the entire range were stable at room temperature under normal laboratory light conditions for at least 4 hours

Duration of treatment / exposure:

Treatment:
Solvent control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours
Positive control: 48 hours

Frequency of treatment:

Once

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/bw
Basis:
analytical conc.

No. of animals per sex per dose:

Five animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 20 mg/kg body weight

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
-It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Statistics:

Wilcoxon Rank Sum Test, one-sided, p < 0.05

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg BW
- Clinical signs of toxicity in test animals: No treatment related clinical signs of toxicity or mortality.


RESULTS OF DEFINITIVE STUDY

- Dose range: 500, 1000 and 2000 mg/kg BW
- Clinical signs of toxicity in test animals: No treatment related clinical signs of toxicity or mortality
- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with EXP1313100.
- Ratio of PCE/NCE (for Micronucleus assay):
No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis.

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that EXP1313100 is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male rats up to a dose of 2000 mg/kg the recommended dose in accordance with current regulatory guidelines under the experimental conditions described in the report.

Executive summary:

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with EXP1313100 compared to the vehicle treated animals.

 

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

 

The groups that were treated with EXP1313100 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No adverse effects were observed in any study on the source substances, therefore in accordance to Regulation (EC) No. 1272/2008 no classification for genetic toxicity is required.