Fenuron

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A battery of key in vitro genotoxicity assays including bacterial mutation, mammalian gene mutation and micronucleus assay was conducted with Fenuron according to most recent standards. Based on these well conducted studies, Fenuron does not have a genotoxic potential. Older published in vitro chromosome aberration and sister chromatid studies were also reported showing equivocal results. The reliability of these studies was questionable, therefore they were disregarded.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.l: 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 05/oct /2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (+10°C to +25°C) in a tightly closed container in a dry, cool and well-ventilated place, avoiding exposure to sunlight and moisture.

FORM AS APPLIED IN THE TEST: IsoQure UR 300 was completely dissolved in dimethyl sulfoxide (DMSO)

OTHER SPECIFICS: Tradename: IsoQure UR 300

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES (1983) was obtained from Trinova Biochem . S9 was collected from male rats.

Test concentrations with justification for top dose:

Preliminary test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 μg/plate
Main test: 31.6, 100, 316, 1000, 3160 and 5000 µg of IsoQure UR 300 per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Batch no. STBG7748; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA100 (10 µg/plate): without metabolic ativation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 (10µg/plate): without metabolic ativation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 (10µg/plate): without metabolic ativation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA 102 (10µg/plate): without metabolic ativation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

TA98, TA102, TA1537 (10 µg/plate): with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA100, TA1535 (2 µg/plate): with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1st independent experiment - Plate Incorporation Method
2nd independent experiment - Preincubation Method
- Cell density at seeding (if applicable): 0.1 mL of Salmonella cell suspension (containing approximately 10E8 viable cells in the late exponential or early stationary phase)

DURATION
- Preincubation period: 20 min (2nd independent experiment)
- Exposure duration: 48 h to 72 h (1st independent experiment and 2nd independent experiment)

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn

Evaluation criteria:

Bacteria colonies were counted employing the Biosys Biocount 5000 system. Print outs of the colony counts were filed with the raw data. Occurrence of test item precipitation would have been documented after visual inspection of the cultures with the unaided eye. Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.
The results of the negative and positive control cultures should be within the range of the historical data generated by LPT.
The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20
Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.

Statistics:

A test item is considered to show a positive response if:
- the number of revertants is significantly increased (p< 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- Biological relevance of the results should be considered first.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Species / strain:

other: TA1535, TA100, TA98, TA1537 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Citotoxicity
The reductions of the number of revertants by more than 50% in test strain TA1537 in the plate incorporation test without metabolic activation at 31.6 and 3160 µg/plate are considered to be caused by the high variation in individual counts and not due to cytotoxicity above all as no concentration response relationship was noted.
Mutagenicity
No increase in revertant colony numbers as compared with control counts was observed for IsoQure UR 300, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test and preincubation test).
The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures are within the range of the historical data generated by LPT

Conclusions:

Under the present test conditions, IsoQure UR 300 tested up to a concentration of 5000 µg/plate (cytotoxic in the preincubation test) caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in DMSO. The vehicle DMSO was employed as the negative control.

Preliminary test

The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000μg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000μg/plate. Hence, 5000μg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations of 31.6, 100, 316, 1000, 3160 and 5000μg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted up to the top concentration of 5000 µg IsoQure UR 300/plate in both experiments.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system

 In conclusion, under the present test conditions, IsoQure UR 300 tested up to a concentration of 5000 µg/plate (cytotoxic in the preincubation test) caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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:

adopted 29 July, 2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 30 May, 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Isochem Kautschuk GmbH; Batch no. 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 05 October, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10 to +25°C, in a tightly closed container in a dry, cool and well-ventilated place, avoid exposure to sunlight and moisture.

OTHER SPECIFICS: IsoQure UR 300

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

heterozygous at the TK locus (+/-); 3.7.2C clone

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (American Type Culture Collection), 0801 University Blvd., Manassas, VA 20110-2209, USA.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Growth medium RPMI 1640 with glutamaxTM medium supplemented with Pluronic® F68 , gentamycin , amphotericin B1 and horse serum1 (10% by volume).
* Treatment medium is growth medium with a reduced horse serum content (5% by volume).
* Plating medium is growth medium with increased horse serum content (approx. 20% by volume) but without Pluronic® F68.
* Selection medium is growth medium that contains 3 µg/mL of TFT
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver, Aroclor 1254 induced)

Test concentrations with justification for top dose:

-preliminary cytotoxicity study without and with S9 metabolic activation: 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium.
-mutagenicity tests experiments without and with S9 metabolic activation: 125, 250, 500, 1000 and 2000 µg/mL medium.
The preliminary cytotoxicity information was evaluated by the determination of the relative survival (relative plating efficiency). The relative plating efficiency was then used to select concentration levels for the mutation assay. Concentrations were selected using the following criteria:
At least four analysable concentrations are used. Where there is cytotoxicity, these concentrations cover a range from the maximum to little or no toxicity and are separated by no more than a factor between 2 and 3.16 (half log dilution). If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10%) relative survival or relative total growth (RTG). For relatively non-cytotoxic compounds, the maximum concentration is 2 mg/mL, 2 µL/mL, or 10 mM. In addition, among the criteria to be considered when determining the highest concentration are cytotoxicity and solubility in the test system and changes in pH or osmolality.
Although more concentrations may be used to initiate a mutation experiment, the objective is to carry at least 5 concentrations through the entire experiment. This procedure compensates for normal variations in cellular toxicity and helps to ensure the choice of at least 4 concentrations appropriately spaced in the relative total growth range of approximately 10% to 100%.
The separation factor of 2 was used. No increase in the mutant frequency was observed. Hence, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: IsoQure UR 300 was completely dissolved in dimethyl sulfoxide (DMSO).

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

13 or 12 µg/mL medium (3-h or 24-h exposure, respectively) without metabolic activation

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

1.0 µg/mL with S9 metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): The final cell density at start of treatment was 0.5 x 10E6 cells/mL.

DURATION
- Exposure duration:
first experiment: 3 hours with and without S9;
second experiment: 3 hours with S9, 24 hours without S9
- Expression time (cells in growth medium): 48 hours (incubation for 24 hours and, after adjustment to 2 x 10E5 cells/mL, the cells were incubated for another 24 hours.)
- Selection time (if incubation with a selection agent): 10 to 12 days with TFT

SELECTION AGENT (mutation assays): 5 trifluorothymidine (TFT) final concentration 3 μg/mL

NUMBER OF REPLICATIONS: none

NUMBER OF CELLS EVALUATED: 10E6 cells for mutant frequency

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as the Relative Total Growth (RTG) which includes the Relative Suspension Growth (RSG) during the 2 day expression period and the Relative Plating Efficiency (RPE) obtained at the time of mutant selection. RTG, RSG and RPE are all expressed as a percentage and were calculated with the following equations: RTG = RSG x RPE

RSG = (SG value (treatment) / (Mean SG value (control)) x 100

With SG being the suspension growth, the measure of the growth in suspension during treatment and the expression period. SG was calculated as follows:
SG = a x b x c

Where a = D0 post-treatment cell count / Pre-treatment cell density

Where b = D1 cell count / Cell count set up on D0 post-treatment

Where c = D2 cell count / Cell count set up on D1

RPE = (PE value (treatment) / Mean PE value (control)) x 100

With PE being the plating efficiency obtained at the time of mutant selection. The calculations are based on P, the probable number of clones per well:

PE = P / Number of cells plated per well

Where P = - ln (Number of wells with no colony / Total number of wells)
The cloning efficiency (CE) is defined as the plating efficiency in percent (CE=PE x 100).
- Any supplementary information relevant to cytotoxicity: For the evaluation of the cytotoxicity in the preliminary experiment the relative plating efficiency RPE obtained at the time after treatment is sufficient and was evaluated. The RPE was calculated as described above for the main experiment.

OTHER EXAMINATIONS:
-the determination of large and small colonies, i.e. number of wells containing large colonies and number of wells containing small colonies in the negative control, the positive controls, and the different test item concentrations.
- the percentage of large colonies and of small colonies, and the small : large ratio

Evaluation criteria:

To define positive and negative results and to assure that the increased MF is biologically relevant instead of a statistical analysis (generally used for other tests), the interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10E-6) is based on the analysis of the distribution of the negative control MF data from participating laboratories (M.M. Moore et al., 2006). Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system. In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data is evaluated by expert judgement and/or further investigations.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

was noted at the top concentration of 2000 µg/mL medium in the absence (3- or 24 hour exposure) and presence (second experiment) of metabolic activation and, in addition, at 1000 µg/mL in the 24-hour exposure experiment

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 and effects of osmolality: No changes in pH or osmolality of the test item formulations at concentrations of 10.0 to 2000 µg/mL medium compared to the negative control were noted. The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter (SevenCompact™ pH/Ion S210)
Osmolality: with a semi-micro osmometer

Conclusions:

Under the present test conditions, IsoQure UR 300, tested up to the cytotoxic concentration of 2000 µg/mL medium, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions, the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, IsoQure UR 300 also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that IsoQure UR 300, tested up to the cytotoxic concentration of 2000 µg/mL medium, neither induced mutations nor had any chromosomal aberration potential.

Executive summary:

In order to investigate the mutagenic potential on mammalian cells, the IsoQure UR 300 was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/‑) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254‑induced rats. The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours, the experiment with S9 mix was carried out in two independent assays.

IsoQure UR 300 was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle dimethyl sulfoxide (DMSO) served as the negative control.

A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation. A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000 and 2000 µg IsoQure UR 300/mL medium were tested for cytotoxicity. Pronounced cytotoxicity (decreased survival and a relative plating efficiency (RPE) of 13%) was noted at the top concentration of 2000 µg/mL in the absence of metabolic activation (24-hour exposure). No signs of cytotoxicity were noted in the presence of metabolic activation (3-hour exposure). No changes in pH or osmolality were noted in the test item formulations compared to the control.

Hence, in the main study the highest concentrations employed were 2000 µg IsoQure UR 300/mL medium in the experiments without and with metabolic activation.

Methylmethanesulfonate (13 or 12 µg/mL for a 3- and 24-hour exposure, respectively) was employed as a positive control in the absence of exogenous metabolic activation and 3‑Methylcholanthrene (1.0 µg/mL) in the presence of exogenous metabolic activation.

In the main study, cytotoxicity (decreased relative total growth) was noted at the top concentration of 2000 µg/mL medium in the absence (3- or 24‑hour exposure) and presence (second experiment) of metabolic activation and, in addition, at 1000 µg/mL in the 24-hour exposure experiment.

The negative controls had mutation frequencies of 62.88 or 84.06 mutant colonies per 10E6 cells in the experiments without metabolic activation (3- or 24-hour exposure, respectively), and 74.77 or 77.65 mutant colonies per 10E6 cells in the experiments with metabolic activation and, hence, were all well within the historical data-range.

The mutation frequencies of the cultures treated with IsoQure UR 300 ranged from 51.71 to 165.87 mutant colonies per 10E6 cells (3 hours exposure) and from 63.98 to 105.71 mutant colonies per 10E6 cells (24 hours exposure) in the experiments without metabolic activation. In the experiments with metabolic activation, mutation frequencies ranged from 81.25 to 131.28 mutant colonies per 10E6 cells (3 hours exposure, first assay) and from 51.45 to 67.51 mutant colonies per 10E6 cells (3 hours exposure, second assay). The highest concentrations of 2000 µg/mL medium in the second experiments in the absence or presence of metabolic activation were not evaluated because of the almost complete cytotoxicity of the test item (RTG: 2 or 14, respectively). These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10E6 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation. In addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.21 to 1.33 for IsoQure UR 300-treated cells and ratios of 0.46 to 1.19 for the negative controls.

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3‑MC) caused pronounced increases in the mutation frequency of 427.34 and 533.09 mutant colonies per 106 cells in the case of MMS and of 444.65 and 609.46 mutant colonies per 10E6 cells in the case of 3‑MC. All positive controls showed an increase in the small colony MF of at least 150 x 10E-6 above that seen in the concurrent solvent control and an absolute increase in total mutation frequency of at least 300 x 10E-6. Furthermore, the mean relative total growth (RTG) for the positive controls was greater than or equal to 10%. All acceptance criteria were met.

Under the present test conditions, IsoQure UR 300, tested up to the cytotoxic concentration of 2000 µg/mL medium, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions, the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.

In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, IsoQure UR 300 also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that IsoQure UR 300, tested up to the cytotoxic concentration of 2000 µg/mL medium, neither induced mutations nor had any chromosomal aberration potential.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July, 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Isochem Kautschuk GmbH; Batch no. 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 5 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10 to +25°C, in a tightly closed container in a dry, cool and well-ventilated place, avoid exposure to sunlight and moisture.

OTHER SPECIFICS: IsoQure UR 300

Species / strain / cell type:

lymphocytes: cultured human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral blood was obtained by venipuncture and collected in heparinised vessels.
- Sex, age and number of blood donors if applicable: young, healthy, non-smoking individuals with no known recent exposures to genotoxic chemicals or radiation.
- Whether whole blood or separated lymphocytes were used if applicable:
- Methods for maintenance in cell culture if applicable: Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes were sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin
* Ham’s F10 supplemented with 10% fetal calf serum (FCS)
* Chromosome complete medium with 5 μg/mL Cytochalasin B.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

CytoB used in a concentration 5 µg/mL

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver, Aroclor 1254 induced)

Test concentrations with justification for top dose:

-Preliminary experiment (without and with metabolic activation): 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium
-Main Test (4 h exposure with and without S9 and 24 h exposure without S9): 125, 250, 500,1000, and 2000 μg/mLmedium
In the preliminary test no signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium in the absence and presence of metabolic activation (24-hour or 4-hour exposure). No changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium. Hence, 2000 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: IsoQure UR 300 was completely dissolved in dimethyl sulfoxide (DMSO).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

mitomycin C

Remarks:

0.1 µg/mL and 0.2 µg/mL without S9

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

cyclophosphamide

Remarks:

10 µg/mL and 20 µg/mL with S9

Positive controls:

yes

Remarks:

aneugen

Positive control substance:

other: Colchicine in aqua ad iniectabilia

Remarks:

0.01 µg/mL and 0.02 µg/mL without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Exposure duration:
Without S9: 4 hours and 24 hours.
With S9: 4 hours
- After exposure: 20 h incubation with spindle inhibitor Cytochalasin B
- Fixation time (start of exposure up to fixation or harvest of cells):
- After exposure:
17 minutes in KCl (0.56%)
30 mminutes in fixative (3 parts methanol : 1 part glacial acetic acid v/v)

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: duplicate (main study), one culture per concentration in the preliminary test

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Each culture was harvested and processed separately. After the test item incubation, mitotic activity was arrested by the addition of CytoB to each culture at a final concentration of 5 µg/mL. After an additional incubation of 20 hours the cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%). After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide. The slides were then stained using 10% Giemsa and left to air-dry at room temperature.

NUMBER OF CELLS EVALUATED:
-At least 500 cells per replicate cell culture were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity.
-The micronucleus frequencies were analysed in at least 2000 binucleate cells per concentration (at least 1000 binucleate cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

DETERMINATION OF CYTOTOXICITY
- Method: other:The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
The CBPI indicates the average number of nuclei per cell during the period of exposure to CytoB, and is used to calculate cell proliferation.
The RI indicates the relative number of cell cycles in treated cultures compared to control cultures and can be used to calculate the % cytostasis:

CBPI =((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells)) / (Total number of cells)

Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Cytostasis = 100 - RI

RI = [((No. binucleate cells)+(2×No. multinucleate cells))÷(Total number of cells)T / ((No. binucleate cells)+(2×No. multinucleate cells))÷(Total number of cells)C ] × 100

T= treated cultures
C= control cultures

Thus, an RI of 53% means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53% of this number divided in the treated culture, i.e. 47% cytostasis.
- Any supplementary information relevant to cytotoxicity: Treatment of cultures with CytoB, and measurement of the relative frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture, provides an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and ensures that only cells that divided during or after treatment are scored.

Evaluation criteria:

Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction.Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined.Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if: at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control; the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test; any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits).Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if: none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control; there is no concentration-related increase when evaluated with an appropriate trend test; all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).Equivocal results may be clarified by analysis of another 1000 cells from all the cultures to avoid loss of blinding.If this approach does not resolve the result, further testing would be necessary.Modification of study parameters over an extended or narrowed range of conditions, as appropriate, would be considered in follow-up experiments.Study parameters that might be modified include the test concentration spacing, the timing of treatment and cell harvest, and/or the metabolic activation conditions.Although most experiments give clearly positive or negative results, in some cases the data set would preclude making a definite judgement about the activity of the test item.These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated.

Statistics:

- statistically significant increase compared with the concurrent negative control;
- dose-response evaluated with an appropriate trend test;
- results outside the distribution of the historical negative control data: Poisson-based 95% control limits.

Species / strain:

lymphocytes: human peropheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 relevant changes in pH of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium. Digital pH meter type SevenCompact S210 Mettler-Toledo GmbH, 35396 Gießen, Germany was used.
- Effects of osmolality: No relevant changes in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium. Semi-micro osmometer Typ ML A0299 Knauer GmbH, 14163 Berlin, Germany was used.
- Precipitation: Precipitation of the test item was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.
- Definition of acceptable cells for analysis: . Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: For the last 27 studies (most recent background data of the years 2015 to 2017, not audited by the QAU-department):
*Mitomycin C
Mean: 26.0
SD: 10.3
Range: 12-61
*Colchicine
Mean : 31.2
SD: 20.6
Range:12-125
*Cyclophosphamide
Mean : 22.5
SD: 9.1
Range: 12-55
- Negative (solvent/vehicle) historical control data: The results for the vehicle controls were within the historical control range.
For the last 27 studies (most recent background data of the years 2015 to 2017, not audited by the QAU-department):
WITHOUT METABOLIC ACTIVATION
*Vehicle controls 4 hour exposure
Mean: 5.4
SD: 2.6
Range:
95% Confidence interval:1.0 - 16
*Vehicle controls 24 hour exposure
Mean: 5.2
SD: 2.4
Range: 1.0 - 13
95% Confidence interval: 4.6 – 5.8
WITH METABOLIC ACTIVATION
*Vehicle control
Mean: 4.7
SD: 2.1
Range: 1 - 9
95% Confidence interval: 4.2 – 5.4

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: . The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

Conclusions:

Under the present test conditions, IsoQure UR 300 tested up to the concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Executive summary:

Test samples of IsoQure UR 300 were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

IsoQure UR 300 was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle DMSO was employed as the negative control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg IsoQure UR 300/mL medium were employed. No signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium in the absence and presence of metabolic activation (24-hour or 4-hour exposure). No changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium.

Hence, 2000 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

In the main study no signs of cytotoxicity were noted up to the top concentration of 2000 µg IsoQure UR 300/mL medium in the experiments without and with metabolic activation.

Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 250, 500, 1000 and 2000 µg IsoQure UR 300/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 2.0 to 5.5 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 µg/mL medium. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test mean frequencies of 3.0 or 4.5 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively, were observed.

The vehicle result was within the historical control ranges.

In the positive control cultures the mean micronucleus frequencies were increased to 20.5 or 22.5 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 250, 500, 1000 and 2000 µg IsoQure UR 300/mL medium (4-h exposure) in the presence of metabolic activation ranged from 3.0 to 5.0 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 µg/mL medium. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 3.5 micronucleate cells per 1000 binucleate cells was observed. The vehicle result was within the historical control ranges.

In the positive control culture the mean micronucleus frequency was increased to 22.0 micronucleate cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.

Conclusion

Under the present test conditions, IsoQure UR 300 tested up to the concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within the historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo Micronucleus assay with Fenuron in mice was negative. Details were only limited, therefore the study was supporting.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genotoxicity in vitro

A key study for bacterial gene mutation was performed for Fenuron in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (Spruth, 2018c). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in DMSO, which was employed as the negative control. In a preliminary test, 5000μg /plate wa schosen as top concentration for the main study. Six concentrations of 31.6, 100, 316, 1000, 3160 and 5000μg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. No cytotoxicity was noted up to 5000 µg/plate in both experiments. No increase in revertant colony numbers were observed as compared with control counts up to a concentration of 5000 μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test). Hence Fenuron tested up to a concentration of 5000 µg/plate (cytotoxic in the preincubation test) caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

A key gene mutation assay in cultured mammalian cells (L5178Y TK +/‑) was conducted with Fenuron in the presence (3h exposure) and absence of metabolic activation (3 & 24 h exposure) (Spruth, 2018d). Fenuron was co dissolved in dimethyl sulfoxide (DMSO), which also served as the negative vehicle control. A preliminary study was conducted at 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL, showing pronounced cytotoxicity at 2000 µg/mL in the absence of metabolic activation (24 h). No signs of cytotoxicity were noted in the presence of metabolic activation (3-hour exposure). No changes in pH or osmolality were noted in the test item formulations compared to the control. In the main study,125, 250, 500, 1000 and 2000 µg/mL were tested, and cytotoxicity was noted at 2000 µg/ in the absence (3- or 24‑h) and presence of metabolic activation and, in addition, at 1000 µg/mL (24 h). Fenuron, tested up to the cytotoxic concentration of 2000 µg/mL in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. In addition, no change was noted in the ratio of small to large mutant colonies.

A key in vitro micronucleus test using human peripheral lymphocytes was conducted with Fenuron both in the presence (4h exposure) and absence of metabolic activation (4 &24 h exposure) and 20 h harvest time after exposure (Spruth, 2018e). Fenuron was dissolved in dimethyl sulfoxide (DMSO), which also served as the negative vehicle control. A preliminary experiment with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL did not demonstrated signs of cytotoxicity up to 2000 µg/mL in the absence and presence of metabolic activation). No changes in pH or osmolality were noted in the test item formulations compared to the control. In the main study 125, 250, 500,1000, and 2000 μg/mL were tested, and no signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL in the experiments without and with metabolic activation. Fenuron, tested up to the concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

The chromosome aberration (CA) potential of Fenuron was examined in Chinese hamster ovary (CHO) and epithelial liver (CHEL) cell lines in the absence or the presence of metabolic activation (Frederico et al., 2011). In the same study, Sister Chromatid Exchange (SCE) potential was examined in Chinese hamster epithelial liver (CHEL) cell lines with metabolic activation. Fenuron was dissolved in acetone at test concentrations of 1.10, 11, 110 and 220 µg/mL and an amount of acetone not exceeding 0.2% (vehicle control). At the highest doses, the mitotic indices (MIs) were never reduced up to 65% of the control values. In the CHO cell line, at the lower dose levels no statistically significant increase of CAs was observed, whereas at 110- and 220 µg/mL doses a significant increase was seen in CAs. In the CHEL cell line, a statistically significant increase of CAs and SCE’s was observed also at the lower dose level, corresponding to 1.1µg/mL of Fenuron. This study was conducted in a very similar procedure to OECD 473 before the revision in 2013, revealed positive results for metabolic competent (CHEL) and incompetent (CHO) cells. The concentrations used resulted in too high cytotoxicity. The mitotic index is no longer recommended to measure cytotoxicity in the revised OECD 473. For the SCE assay, the test method is currently not applicable anymore: Following the OECD Council decision, the Test Guideline 479 ‘Genetic Toxicology: In vitro Sister Chromatid Exchange Assay in Mammalian Cells’ was deleted in 2014. The results are considered questionable and the studies are disregarded

Genotoxicity in vivo

An in vivo micronucleus test was performed (Seiler, 1978). In this study, 6 mice received 1000 mg/kg bw/day Fenuron per os. As a result, 0.17% micronucleated polychromatic erythrocytes were observed in bone marrow cells of the treated mice. Fenuron was negative under the test conditions used. The study did not provide many details, therefore it was considered supportive only.

Justification for classification or non-classification

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