Phosphoric acid, iron(2+) lithium salt (1:1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation = negative (with and without metabolic activation), OECD 471, OECD B.13/14, van Sikkelerus (2006)

In vitro mammalian chromosome aberration = negative (with and without metabolic activation), OECD 473, OECD B.10, Buskens (2006)

In vitro mammalian chromosome aberration = negative (with and without metabolic activation), PECD 473, Fangfang (2016)

In vitro mammalian cell gene mutation = negative (with and without metabolic activation), OECD 476, OECD B.17 Verspeek-Rip (2009)

Endpoint Conclusion: No adverse effect observed (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:

19 July 2006 to 27 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine
Escherichia coli: tryptophan

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix induced by phenobarbital/ ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1: 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)

Experiment 2: 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)

Vehicle / solvent:

Solvent: Dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 5000 µg/plate

Evaluation criteria:

Acceptability of the assay: The assay was considered acceptable if the following criteria were met:
a) The negative control data (number of spontaneous revertants per plate) were within the laboratory historical range for each tester strain
b) The positive control chemicals produced responses in all tester strains, which were within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count was at least three times the concurrent vehicle control group mean
c) The selected dose range included a clearly toxic concentration or exhibited limited solubility as demonstrated by a preliminary toxicity range-finding test or extended to 5 mg/plate

Data evaluation and statistical procedures:

A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is not greater than 2 times the concurrent control, and the total number of revertants in any tester strain is not greater than 3 times the concurrent control
b) The negative response should be reproducible in at least one independently repeated experiment

A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is greater than 2 times the concurrent control, or the total number of revertants in any tester strain is greater than 3 times the concurrent control
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:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

EXPERIMENT 1
Precipitate
Test material precipitate was observed on the plates at the start of the incubation period at the concentrations of 3330 and 5000 µg/plate and at the end of the incubation period at the concentration of 5000 µg/plate.

Toxicity
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity
No biologically relevant increase in the number of revertants was observed upon treatment witht the test material under all conditions tested.

EXPERIMENT 2
Precipitate
Test material precipitate was observed on the plates at the start of the incubation period at the concentrations of 3330 and 5000 µg/plate and at the end of the incubation period at the concentration of 5000 µg/plate.

Toxicity
The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Mutagenicity
No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: (Experiment 1)

Controls

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

Conclusions:

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

Under the conditions of the study all bacterial strains showed negative responses over the entire dose range since there were no significant dose-related increase in the number of revertants in two independently repeated experiments.
Based on the findings of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenic activity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.13/14.

During the study the test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the

presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The test material was suspended in dimethyl sulfoxide at concentrations of 0.1 mg/mL and above. In the first mutation assay, the test material was tested up to the concentration of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. Test material precipitated on the plates at the top dose level of 5000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test material was tested up to the concentration of 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix. The test material precipitated on the plates at the top dose of 5000 µg/plate. The bacterial background lawn was

not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test material 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 our 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 the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2009 to 6 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

- The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

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: 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).
- 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: 0.3, 1, 1, 10 and 100 µg/mL
Without S9-mix, 24 hours treatment: 0.3, 1, 1, 10 and 100 µg/mL

Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 µg/mL

Experiment 2:
Without S9-mix, 24 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 100 µg/mL
With S9-mix, 3 hours treatment: 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Accepted and approved by authorities and international guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix

Migrated to IUCLID6: : 15 µg/ml for the 3 hours treatment period and 5 µg/ml for the 24 hours treatment period

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Migrated to IUCLID6: : 7.5 µg/ml

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 10^5 cells/concentration

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

RANGE-FINDING/SCREENING STUDIES:
-The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWTG 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.

A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more then MF(controls) + 126 in a dose-dependent manner; or
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.
An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance 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 substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

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

Untreated negative controls validity:

not applicable

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 10 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest tested dose level of 100 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence and presence of S9-mix, no toxicity was observed up to and including the highest tested dose level in both experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

The test material was found not mutagenic in the TK mutation test system.

Executive summary:

The mutagenic activity of the test material was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476 and EU Method B.17.

In the first experiment, the test material was tested up to concentrations of 10 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. Appropriate toxicity was observed at this dose level in the absence and presence of S9-mix. The test material was tested up to the precipitating dose level of 10 µg/mL and no toxicity was observed at this dose level in the absence and presence of S9-mix.

In the second experiment, the test material was tested up to concentrations of 100 and 10 µg/mL, but in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. No severe toxicity was observed at this dose level in the absence and presence of S9-mix. The test material was tested up to the cytotoxic levels of 40% in the absence of S9-mix and no toxicity was observed at the precipitating dose level of 10 µg/mL in the presence of S9-mix.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 22- and 11-fold for MMS in the absence of S9-mix, and by 16- and 13-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9 -mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeated experiment with modifications in duration of treatment time.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is therefore concluded that the test material is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions that were applied in this study.

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:

2 August 2006 to 11 October 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system.
Blood was collected from healthy adult, non-smoking male volunteers. Blood samples were taken 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: consisting of RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated foetal calf serum, L-glutamine, penicillin/steptomycin and 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 respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.

Environmental conditions: all incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 77 - 90%, containing 5.0 ± 0.5% CO2 in air at a temperature of 36.3 - 37.5 °C.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix induced by phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding study (3 h exposure): 0.3, 1, 3, 10, 33 µg/mL (with and without metabolic activation)
Dose range finding study (24 h and 48 h continuous exposure): 0.3, 1, 3, 10, 33, 100 µg/mL (without metabolic activation)

Cytogenetic assay 1 (3 h exposure, 24 h fixation): 3*, 10*, 33* µg/mL (with and without metabolic activation)

Cytogenetic assay 2 (24 and 48 h exposure; 24 h and 48 h fixation): 3*, 10*, 33*, 100 µg/mL (without metabolic activation)
Cytogenetic assay 2 (3 h exposure; 48 h fixation): 3*, 10*, 33* µg/mL (with metabolic activation)

* doses scored for chromosome aberrations

Vehicle / solvent:

- Vehicle: dimethyl sulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(dimethyl sulfoxide)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cytogenetic assay 1
Lymphocytes were cultured for 48 hours and thereafter exposed in duplicate to selected doses of test material for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20-22 hours (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.

Cytogenetic assay 2
Lymphocytes were cultured for 48 h and thereafter exposed in duplicate to selected doses of test material for 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix.
After 3 h exposure, the cells exposed to test material in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS and incubates in 5 mL culture medium for another 44-46 h (48 h fixation time).
The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).

Chromosome preparation
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

Preparation of slides
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least 2 slides were prepared per culture. Slides were allowed to dry before being stained with 5% (v/v) Giemsa solution in tap water.
Thereafter slides were rinsed in water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic Index/ dose selection for scoring of the cytogenetic assay
The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay. The highest concentration analysed was based on the solubility data of the test material in culture medium.

Analysis of slides for chromosome aberrations
100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. Only metaphases containing 46 ± 2 chromosomes were analysed.

Evaluation criteria:

A chromosome aberration test is considered acceptable if the following criteria are met:
- the number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range
- the positive control substances should produce a statistically significant increase in the number of cells with chromosome aberrations
- a homogeneous response between the replicate cultures is observed
- a possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

A test material is considered clastogenic in the chromosome aberration test if:
a) it induces a dose-related statistically significant 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 is observed in the absence of a clear dose-response relationship.

Statistics:

The incidence of aberrant cells 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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding test
Test material preciptated in the culture medium at a concentration of 33 µg/mL. The test material was tested beyond the limit of solubility to obtain adequate toxicity data.

Cytogenetic assay 1
Both in the absebce and presence of S9-mix the test materil did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Both in the absence and presence of S9-mix the test material did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Cytogenetic assay 2
Both in the absebce and presence of S9-mix the test materil did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Both in the absence and presence of S9-mix the test material did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: (Dose range finding test)

Controls

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:

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

Under the conditions of the study the test material did not disturb mitotic processes or cell cycle progression and did not induce numerical chromosome aberrations. It is therefore concluded that the test material is not clastogenic in human lymphocytes.

Executive summary:

The ability of the test material to induce chromosome aberrations in cultured peripheral human lymphocytes, in the presence and absence of a metabolic activation system (phenobarbitol and ß-naphthoflavone induced rat liver S9 -mix), was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 473 and EU Method B.10.

In the first cytogenetic assay, the test material was tested up to 33 µg/mL for a 3 hour exposure time with a 24 hour fixation time in the absence and presence of 1.8% (v/v) S9 -fraction. The test material precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test material was tested up to 33 µg/mL for a 24 hour and 48 hour continuous exposure time with a 24 hour and 48 hour fixation time in the absence of S9 -mix. In the presence of S9 -mix the test material was also tested up to 33 µg/mL for a 3 hour exposure time with a 48 hour fixation time. The test material precipitated in the culture medium at this dose level.

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.

The test material 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 two independently repeated experiments.

No effects 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 the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions.

It is therefore concluded that this test is valid and that the test material is not clastogenic in human lymphocytes under the experimental conditions utilised.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus study = negative, OECD 474, Hofman-Hüther (2012)

In vivo micronucleus study = negative, OECD 474, Yali (2016)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2012 to 10 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River. Sulzfeld Germany
- Age at study initiation: 6-12 weeks
- Weight at study initiation: male: 25.1-31.7g ; female: 20.3-25.0 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: 5 animals were housed (by sex) in IVC cages furnished with Altromin saw fiber bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice (ad libitum)
- Water: ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: cotton seed oil;
-Volume: 10 mL/kg bw

Details on exposure:

PREPARATION OF THE TEST MATERIAL
The test material was thoroughly ground in a mortar and diluted in cottonseed oil within 1 hour before treatment. All animals received a single volume ip of 10 mL/kg bw.

Duration of treatment / exposure:

Single ip administration.

Frequency of treatment:

Single test material administration.

Post exposure period:

Sampling of the peripheral blood was carried out on animals 44 hours (all groups) and 68 hours (negative control and highest dose groups) after treatment.

Remarks:

Doses / Concentrations:
0 (negative control), 400, 1000, 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 males and 5 females per dose (0, 400, and 1000 mg/kg bw);
7 males and 7 females (2000 mg/kg bw) - 2 animals per sex were reserve animals

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Justification for choice of positive control(s):
- Route of administration: ip
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

peripheral blood erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Doses were selected on the basis of the results obtained from the preliminary toxicity test.

TREATMENT AND SAMPLING TIMES
At the beginning of the experiment the animals were individually weighed and the administered volume adjusted to the animal's body weight. The animals received the test material once ip. Sampling of the peripheral blood was carried out on animals 44 and 68 hours after treatment.

DETAILS OF SLIDE PREPARATION
Blood was obtained from the animal's tail vein following incision. Blood cells were immediately fixed in ultracold methanol. Before analysis (at least 16 hours after fixation), fixed blood cells were washed in Hank's balanced salt solution, centrifuged at 600 x g for 5 minutes and the supernatant discarded. Blood cell populations were discriminated using specific antibodies against CD71 (expressed only at the surface of immature erythrocytes) and CD61 (expressed at the surface of platelets) and DNA content of micronuclei was determined by the use of a DNA specific stain.

METHOD OF ANALYSIS
Evaluation of all samples, including those of positive and negative controls, was performed using a flow cytometer. Anti-CD71 antibodies were labelled with fluorescein-isothiocyanate, anti-CD61 antibodies were labelled with phycoerythrin. Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensity were recorded on the FL1, FL2 and FL3 channels for FITC, PE and PI, respectively. At least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test material the ratio between immature and mature erythrocytes was determined.

Evaluation criteria:

The criteria for determining a positive result are as follows:
- dose related increase of micronucleated cells and/or
- biologically relevant increase in the number of micronucleataed cells for at least one of the dose groups

The criteria for a negative result are as follows:
- no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level

Acceptance criteria
The data generated are considered acceptable if:
- at commencement of the study, the weight variation of animals does not exceed ± 20% of the mean weight of each sex
- the background frequency of micronucleated cells is in the normal range as reported in literature or within the laboratory's historical range
- the test system is sensitive to the known mutagen as judged by the results in the concurrent positive control animals.

Statistics:

The non-parametric Mann-Whitney Test was performed to verify the results.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

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: After receiving a single ip dose of test material, three male and three female animals showed signs of toxicity, such as reduction in spontaneous activity, constricted abdomen, piloerection, bradykinesia, opisthotonos and half eyelid closure. Due to the results obtained, 2000 mg/kg bw was chosen as the maximum tolerable dose in the main experiment.

RESULTS OF DEFINITIVE STUDY
- Toxicity:
The animals treated with 2000 mg/kg bw showed slight to moderate signs of systemic toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, eye closure, kyphosis and opisthotonos. The animals treated with 1000 and 400 mg/kg bw showed increasingly less toxicity, respectively.
- Body weights:
The weight variation of the animals did not exceed ± 20% of the mean weight of each sex.
- Relative PCE:
The negative controls (44 h, 68 h) were within the range of the historical control data of the negative control (1.19 - 3.97). The mean values noted for the 44 hour negative control were 3.40 for the male and 1.81 for the female mice. The mean values detected for the 68 hour negative control were 3.26 for the male and 2.02 for the female mice.
Mice treated with 400 mg/kg bw test material showed mean values of the relative PCE of 2.96 (males) and 1.64 (females). These values are decreased from the negative controls, but this decrease is not statistically significant.
Mice treated with 100 mg/kg bw showed mean values of relative PCE of 2.70 (males) and 1.78 (females). These values are decreased from the negative controls, but the values were within the range of historical control data.
Mice treated with 2000 mg/kg bw (44 hour treatment) showed mean values of 2.74 (males) and 1.41 (females). These values are decreased from the negative controls.
Mice treated with 2000 mg/kg bw (68 hour treatment) showed mean values of 1.56 (males) and 0.48 (females). These values are decreased from the negative controls. Additionally, the value observed in the female group was significantly reduced compared to the corresponding control and to the historical control.

- Micronucleated polychromatic erythrocytes:
The negative controls (44 h and 68 h) evaluated were within the range of the historical control data of the negative control (0.08 - 0.43%). The mean values of micronuclei observed for the negative control (44 h) were 0.24% for male and for female mice. The mean values for the 68 h negative control were 0.32% for the male and 0.20% for the female mice.
The mean values of micronuclei observed after treatment with 400 mg/kg bw test material were 0.26% for male and 0.19% for female mice. The value observed in male mice was slightly increased compared to the corresponding negative control. The value observed in female mice was slightly reduced as compared to the corresponding negative control but this increase/decrease was not statistically significant. Additionally, both values were within the historical negative control data.
The mean values for the animals treated with 1000 mg/kg bw dose group were 0.19% for male and 0.25% for female mice. The value observed in the female group was within the range of the corresponding negative control. The value observed in the male group was slightly decreased compared to the corresponding negative control. However, both values were within the range of the historical negative control data.
The dose group treated with 2000 mg/kg bw test material (44 h treatment) showed mean values of 0.35% for the male and 0.38% for the female mice. The values observed in the male and female groups were increased compared to the corresponding negative control, but these increases were not statistically significant. Additionally, both values were within the range of the historical negative control data.
The mean values observed for the 2000 mg/kg bw animals for the 68 hour treatment were 0.32% for the male and 0.29% for the female mice. These values were within the range of the corresponding negative control.
No biologically relevant increase of micronuclei was found after treatment with the test material in any of the dose groups evaluated.
No statistically significant increases (p<0.05) of cells with micronuclei were noted in the dose groups of mice treated with the test material.

Positive control:
Cyclophosphamide (40 mg/kg bw) induced a statistically significant increase of induced micronucleus frequency (mean percentage of cells with micronuclei was 2.19% for male and 1.88% for female mice. These results demonstrate the validity of the assay.

Dose Group	Concentration [mg/kgbw]	Preparation (h)	Male [%]	Female [%]
Negative Control	0	44	0.24	024
Cyclophosphamide	40	44	2.19	1.88
MTD	2000	44	0.35	0.38
MTD/2	1000	44	0.19	0.25
MTD/5	400	44	0.26	0.19
Negative Control	0	68	0.32	0.20
MTD	2000	68	0.32	0.29

MTD = maximum tolerable dose

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the study, the test material did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. The test material is therefore considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

Executive summary:

The potential of the test material to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 474.

During the study the test material was prepared and diluted in cottonseed oil. The volume administered ip was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after a single administration of the test material.

A pre-experiment and a dose of 2000 mg/kg bw was subsequently selected as maximum tolerable dose (MTD). The signs of toxicity were noted .

In the main experiment three dose levels were used covering a range from the maximum tolerable dose to no toxicity. The following dose groups were selected based on the toxicity observed in the pre-experiment:

Doses	Concentration (mg/kg bw)
MTD	2000
MTD/2	1000
MTD/5	400

The animals treated with doses of 400 and 1000 mg/kg bw showed only slight signs of systemic toxicity. The animals treated with a dose of 2000 mg/kg bw showed slight and moderate signs of systemic toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, eye closure, kyphosis and opisthotonos.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (44 h, 68 h) were within the range of the laboratory control data. The mean values noted for the dose groups which were treated with the test item (44 h, 68 h) were within the historical negative control data range.

No biologically relevant increase of micronuclei was found after treatment with the test mateial in any of the dose groups evaluated.

The non-parametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p <0.05) of cells with micronuclei were noted in the dose groups.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control which induced a significant increase in micronucleus frequency thereby demonstrating the validity of the assay.

The test material is therefore considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

Additional information

In vitro gene mutation

The mutagenic activity of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Method B.13/14.

During the study the test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the

presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The test material was suspended in dimethyl sulfoxide at concentrations of 0.1 mg/mL and above. In the first mutation assay, the test material was tested up to the concentration of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. Test material precipitated on the plates at the top dose level of 5000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test material was tested up to the concentration of 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix. The test material precipitated on the plates at the top dose of 5000 µg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test material 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 our 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 the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In vitro mammalian chromosome aberration

The ability of the test material to induce chromosome aberrations in cultured peripheral human lymphocytes, in the presence and absence of a metabolic activation system (phenobarbitol and ß-naphthoflavone induced rat liver S9 -mix), was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 473 and EU Method B.10.

In the first cytogenetic assay, the test material was tested up to 33 µg/mL for a 3 hour exposure time with a 24 hour fixation time in the absence and presence of 1.8% (v/v) S9 -fraction. The test material precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test material was tested up to 33 µg/mL for a 24 hour and 48 hour continuous exposure time with a 24 hour and 48 hour fixation time in the absence of S9 -mix. In the presence of S9 -mix the test material was also tested up to 33 µg/mL for a 3 hour exposure time with a 48 hour fixation time. The test material precipitated in the culture medium at this dose level.

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.

The test material 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 two independently repeated experiments.

No effects 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 the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions.

It is therefore concluded that this test is valid and that the test material is not clastogenic in human lymphocytes under the experimental conditions utilised.

In a supporting study, the ability of the test material to induce chromosome aberrations in Chinese hamster lung cells, in the presence and absence of a metabolic activation system (S9 -mix), was investigated in accordance with the standardised guideline OECD 473.

During the study, the cells were treated for 6 and 24 hours in the non-activated test system and for 6 hours in the S9 -activated system. Concentrations of 0 (blank control), 150, 300 and 500 µg/mL test material were included together with positive controls.

The percentage of cells with structural aberrations in the test material-treated groups was not increased relative to the solvent control at any dose level.

Under the conditions of the study the test material was concluded to be negative for the induction of structural chormosome aberrations in both non-activated and S9-activated test systems.

In vitro mammalian cell gene mutation

The mutagenic activity of the test material was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476 and EU Method B.17.

In the first experiment, the test material was tested up to concentrations of 10 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. Appropriate toxicity was observed at this dose level in the absence and presence of S9-mix. The test material was tested up to the precipitating dose level of 10 µg/mL and no toxicity was observed at this dose level in the absence and presence of S9-mix.

In the second experiment, the test material was tested up to concentrations of 100 and 10 µg/mL, but in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. No severe toxicity was observed at this dose level in the absence and presence of S9-mix. The test material was tested up to the cytotoxic levels of 40% in the absence of S9-mix and no toxicity was observed at the precipitating dose level of 10 µg/mL in the presence of S9-mix.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 22- and 11-fold for MMS in the absence of S9-mix, and by 16- and 13-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9 -mix) functioned properly.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeated experiment with modifications in duration of treatment time.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is therefore concluded that the test material is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions that were applied in this study.

In vivo micronucleus assay

The potential of the test material to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 474.

During the study the test material was prepared and diluted in cottonseed oil. The volume administered ip was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after a single administration of the test material.

A pre-experiment and a dose of 2000 mg/kg bw was subsequently selected as maximum tolerable dose (MTD). The signs of toxicity were noted. In the main experiment three dose levels were used covering a range from the maximum tolerable dose to no toxicity.

The animals treated with doses of 400 and 1000 mg/kg bw showed only slight signs of systemic toxicity. The animals treated with a dose of 2000 mg/kg bw showed slight and moderate signs of systemic toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, eye closure, kyphosis and opisthotonos.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (44 h, 68 h) were within the range of the laboratory control data. The mean values noted for the dose groups which were treated with the test item (44 h, 68 h) were within the historical negative control data range.

No biologically relevant increase of micronuclei was found after treatment with the test mateial in any of the dose groups evaluated.

The non-parametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p <0.05) of cells with micronuclei were noted in the dose groups.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control which induced a significant increase in micronucleus frequency thereby demonstrating the validity of the assay.

The test material is therefore considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

In a supporting study, the potential of the test material to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was investigated in accordance with the standardised guideline OECD 474.

During the study 30 male and 30 femal ICR mice were randomly assigned to 5 groups. The positive controls were treated with cyclophosphamide at a concentration of 40 mg/kg and the negative controls received vehicle only. The treated groups were dosed with test material at a dose of 2000 mg/kg twice, with an interval of 24 hours. Bone marrow was harvested 20 hours after the last dosing and analysed for frequency of MNPCE. A minimum of 2000 polychromatic erythrocytes (PCEs) was analysed for each animal. The ratio of PCE/NCE was determined by counting a minimum of 200 erythrocytes.

Under the conditions of the study, no effects on the induction of MNPCE were observed with the test material in the doses of 2000 mg/kg in treated mice. The test material is therefore considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

Justification for classification or non-classification

The substance does not fulfil the classification criteria for genetic toxicity according to European Regulation (EC) No 1272/2008.