aluminium-magnesium-carbonate-hydroxide-perchlorate-hydrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG 471): negative

In vitro chromosome aberration test (OECDTG 473): negative

Genemutation in mammalian cells (OECDTG 490): negative

Link to relevant study records

Reference

Reference 1

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:

March 31, 1995 - April, 13, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.14: "Other Effects-Mutagenicity: Salmonella typhimurium Reverse Mutation Assay". (1992)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw).

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2: All 4 strains
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: daunomycine 4 µg/plate in saline for TA98

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine 60 µg/plate in saline for TA1537

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: sodium azide 1 µg/plate in saline for TA1535

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 5 µg/plate in DMSO for TA1535 and TA1537, and 0.5 µg/plate in DMSO for TA98 and TA100.

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 any tester strain at any concentration is not greater than two (2) times the concurrent control, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) It induces at least a two-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant. If in the first test the test substance showed only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) 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:

cytotoxicity

Remarks:

In both experiments a clear decrease in the number of revertants in strains TA100 and TA1537 in the absence of S9-mix was observed.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed in the top agar at dose levels of 1000 µg/plate and upwards but precipitation was not observed at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
- No reduction of the bacterial background lawn was observed. A dose-related decrease was observed in the number of revertants.

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:
The bacterial background lawn was not reduced at all concentrations tested. In both experiments a clear decrease in the number of revertants in strains TA100 and TA1537 in the absence of S9-mix was observed.

Conclusions:

Alcamizer 5 is not mutagenic in the Salmonella typhimurium reverse mutation assay, performed according to OECD 471 guideline and GLP principles with or without metabolic activation.

Executive summary:

The genetic toxicity of Alcamizer 5 was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, in accordance with OECD 471 guideline and GLP principles. No precipitation was observed at the end of the incubation period. The bacterial background lawn was not reduced at all concentrations tested. In both experiments a clear decrease in the number of revertants in strains TA100 and TA1537 in the absence of S9-mix was observed. All bacterial strains showed negative responses up to 5000 µg/plate, i.e. no biologically significant dose-related increase in the number of revertants with or without metabolic activation was seen in two independently repeated experiments. 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. Based on the results of this study it is concluded that Alcamizer 5 is not mutagenic in the Salmonella typhimurium reverse mutation assay with or without metabolic activation.

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:

30 January, 1990 - 17 February, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1983)

Deviations:

yes

Remarks:

No scientifically justification for plating in duplicate in stead of triplicate.

Qualifier:

according to guideline

Guideline:

other: "Standards to be observed Mutagenicity Testing Institutions", published by the Japanese Ministry of Labour in 1988.

Qualifier:

according to guideline

Guideline:

other: "Standards to be Observed by Test Relating Facilities in the Order Prescribing the Item 4 of the Testing Relating to New Chemical Substances" of Japan in 1984.

GLP compliance:

no

Remarks:

but Quality Assurance Certificate included.

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 a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose-finding test, experiment 1, all 5 strains:
Without and with S9-mix: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate
Experiment 2, all 5 strains:
Without and with S9-mix: 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: Distilled water.
- Justification for choice of solvent: Test compound was suspended in distilled water and distilled water has been accepted and approved by authorities and international guidelines. There was not any bacterial growth in the test substance solution.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide in DMSO, 0.01 µg/plate for TA100 and WP2uvrA, and 0.1 µg/plate for TA98

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

other: sodium azide 0.5 µg/plate in distilled water for TA1535

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

other: ICR-191 1 µg/plate in DMSO for TA1537

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 2 µg/plate in DMSO for TA1535, and 20 µg/plate in DMSO for WP2uvrA.

Remarks:

with S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

Positive controls:

yes

Positive control substance:

other: benzo[a]pyrene 5 µg/plate in DMSO for TA98, TA100 and TA1537.

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

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

NUMBER OF CELLS EVALUATED: No data (up to early stationary phase of growth)

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:

The criteria for determining a positive result are a significant dose-related increase in the number of revertants and the detection of a reproducible and significant positive response (increase over twice in number of revertants compared with solvent control) for at least one of the test points.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

of 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

of 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

of 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

of 5000 µg/plate

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

of 5000 µg/plate

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 (experiment 1):
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertant colonies in the positive controls increased over twice compared to the solvent controls, 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:

Alcamizer 5 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, performed similar to OECD 471 guideline with or without metabolic activation.

Executive summary:

The genetic toxicity of Alcamizer 5 was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains and Escherichia coli WP2uvrA strain, similar to OECD 471 guideline. No precipitation was observed up to and including the top dose of 5000 µg/plate. The bacterial background lawn was not reduced at all concentrations tested. All bacterial strains showed negative responses up to 5000µg/plate, i.e. no biologically significant dose-related increase in the number of revertants with or without metabolic activation was seen in two independently repeated experiments. The number of revertant colonies in the positive controls increased over twice compared to the solvent controls, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Alcamizer 5 is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay with or without metabolic activation.

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:

29 March, 1995 - 20 May, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1983)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1992)

Deviations:

no

GLP compliance:

yes

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 from adult male volunteers 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.
- Average Generation Time: 14.9 - 16.7 h.
- Culture medium
Culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine, 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), sodium bicarbonate (1.2 g/L) 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 Aroclor 1254 (500 mg/kg bw).

Test concentrations with justification for top dose:

Dose range finding test:
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1, 3, 10, 33 and 100 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 1, 3, 10, 33 and 100 µg/mL
First cytogenetic test:
With and without S9-mix, 3 h exposure time, 24 h fixation time: 10, 33 and 100 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 24, 33 and 42 µg/mL
With S9-mix, 3 hr exposure; 24 hr fixation: 24, 33 and 42 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Mitomycin C in Hank's Balanced Salt Solution: 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period.

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide in Hank's Balanced Salt Solution: 15 µg/mL.

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with 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, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant increase in the frequency of 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, 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, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 42 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES: No toxicity was observed up to and including the highest precipitating tested dose.

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:
- No toxicity was reached up to the highest precipitating tested dose levels selected for scoring.

Conclusions:

A chromosome aberration study with Alcamizer 5 was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Alcamizer 5 is not clastogenic in cultured human lymphocytes.

Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of Alcamizer 5 (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles. In the first cytogenetic assay, Alcamizer 5 was tested up to and including precipitating concentration of 100 μg/mL, with and without S9 -mix,

for a 3, 24 and 48 h exposure time with a 24 and 48 h fixation time. In the second cytogenetic assay, Alcamizer 5 was tested up to precipitating concentrations of 42 µg/mL for a 24 h continuous exposure time with a 24 h fixation time in the absence of S9 -mix and for 3 h exposure time with a 24 h fixation time in the presence of S9 -mix. In the first experment, Alcamizer 5 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 the second experiment in the absence of S9 -mix, Alcamizer 5 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. In the presence of S9 -mix the lowest dose level of 24 µg/mL induced a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations only when gaps were included. Since this increase is observed only when gaps were included and this increase is within the historical control data range, this increase is not considered biologically relevant. No effects of Alcamizer 5 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 Alcamizer 5 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

 

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2017 - 27 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

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, 3 hours treatment: 1.57, 3.13, 6.25, 12.5 and 25 μg/mL
Without S9-mix, 24 hours treatment: 1.57, 3.13, 6.25, 12.6 and 25 μg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.1, 0.2, 0.39, 0.79, 1.57, 3.13, 6.25, 12.5 and 25 μg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus:
0.1, 0.2, 0.39, 0.79, 1.57, 3.13, 6.25 and 12.5 μg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.1, 0.2, 0.39, 0.79, 1.57, 3.13, 6.25, 12.5 and 25 μg/mL
The following dose levels were selected to measure mutation frequencies at the TK-locus:
0.2, 0.39, 0.79, 1.57, 3.13, 6.25, 12.5 and 25 μg/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item was suspended in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a
homogeneous suspension. Test item concentrations were used within 1 hour of preparation.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY CRITERIA
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the exposure medium at the test item concentrations of 12.5 and 25 μg/ml.

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 1.57 to 25 μg/ml in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 25 μg/ml compared to the suspension growth of the solvent control at the 3 hour treatment period. The test item precipitated in the exposure at concentrations of 12.5 and 25 μg/ml.
No toxicity in the relative suspension growth was observed up to the test item concentration of 25 μg/ml compared to the solvent control at the 24 hour treatment period. The test item precipitated in the exposure at concentrations of 12.5 and 25 μg/ml.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 857 688 1710
SD 246 187 815
n 110 102 139
Upper control limit
(95% control limits) 1425 1124 4214
Lower control limit
(95% control limits) 289 253 -793

SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between January 2013 and November 2016.

- Negative (solvent/vehicle) historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 86 81 87
SD 23 26 28
n 220 202 273
Upper control limit
(95% control limits) 135 135 145
Lower control limit
(95% control limits) 37 28 28

SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between January 2013 and November 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence and presence of S9-mix , the relative total growth (RTG) was not decreased compared to the RTG of the solvent control groups up to and including the highest precipitating dose level.

Conclusions:

A mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of the study.

Executive summary:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

Based on the results of the dose-range finding test, Alcamizer 5 was tested in two mutation assays. In the first experiment, ALCAMIZER5 was tested up to concentrations of 12.5 μg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. ALCAMIZER5 precipitated in the culture medium at this dose level.

In the second experiment, ALCAMIZER5 was tested up to concentrations of 25 μg/ml in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. ALCAMIZER5 precipitated in the culture medium at this dose level. Reliable negative and positive controls were included.

In the absence of S9-mix, ALCAMIZER5 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, ALCAMIZER5 did not induce a significant increase in the mutation frequency.

In conclusion, ALCAMIZER5 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

The genetic toxicity of Alcamizer 5 was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, in accordance with OECD 471 guideline and GLP principles. No precipitation was observed at the end of the incubation period. The bacterial background lawn was not reduced at all concentrations tested. In both experiments a clear decrease in the number of revertants in strains TA100 and TA1537 in the absence of S9-mix was observed. All bacterial strains showed negative responses up to 5000 µg/plate, i.e. no biologically significant dose-related increase in the number of revertants with or without metabolic activation was seen in two independently repeated experiments. 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. Based on the results of this study it is concluded that Alcamizer 5 is not mutagenic in the Salmonella typhimurium reverse mutation assay with or without metabolic activation.

The genetic toxicity of Alcamizer 5 was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains and Escherichia coli WP2uvrA strain, similar to OECD 471 guideline. No precipitation was observed up to and including the top dose of 5000 µg/plate. The bacterial background lawn was not reduced at all concentrations tested. All bacterial strains showed negative responses up to 5000µg/plate, i.e. no biologically significant dose-related increase in the number of revertants with or without metabolic activation was seen in two independently repeated experiments. The number of revertant colonies in the positive controls increased over twice compared to the solvent controls, indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Alcamizer 5 is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay with or without metabolic activation.

Chromosome aberration test:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of Alcamizer 5 (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 473 guideline and GLP principles. In the first cytogenetic assay, Alcamizer 5 was tested up to and including precipitating concentration of 100 μg/mL, with and without S9 -mix,

for a 3, 24 and 48 h exposure time with a 24 and 48 h fixation time. In the second cytogenetic assay, Alcamizer 5 was tested up to precipitating concentrations of 42 µg/mLfor a 24 h continuous exposure time with a 24 h fixation time in the absence of S9 -mix and for 3 h exposure time with a 24 h fixation time in the presence of S9 -mix. In the first experment, Alcamizer 5 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 the second experiment in the absence of S9 -mix, Alcamizer 5 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. In the presence of S9 -mix the lowest dose level of 24 µg/mLinduced a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations only when gaps were included. Since this increase is observed only when gaps were included and this increase is within the historical control data range, this increase is not considered biologically relevant. No effects of Alcamizer 5 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 Alcamizer 5 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

Mouse lymphoma test:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

Based on the results of the dose-range finding test, Alcamizer 5 was tested in two mutation assays. In the first experiment, ALCAMIZER5 was tested up to concentrations of 12.5 μg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. ALCAMIZER5 precipitated in the culture medium at this dose level.

In the second experiment, ALCAMIZER5 was tested up to concentrations of 25 μg/ml in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. ALCAMIZER5 precipitated in the culture medium at this dose level. Reliable negative and positive controls were included.

In the absence of S9-mix, ALCAMIZER5 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, ALCAMIZER5 did not induce a significant increase in the mutation frequency.

In conclusion, ALCAMIZER5 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the results of the Ames test, in vitro chromosome aberration and mouse lymphoma studies, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its updates.