Reaction mass of bis[4-(vinyloxy)butyl] hexane-1,6-diylbiscarbamate and 9,18-dioxo-3,8,19-trioxa-10,17-diazapentacos-1-en-25-yl [6-({[4-(vinyloxy)butoxy]carbonyl}amino)hexyl]carbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three reliable in vitro studies are available. The results of these studies indicate that the registered substance has no genotoxic properties.

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:

10 June, 2013 - 08 July, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Experiment 1: TA1535, TA1537, TA98, TA100 and WP2uvrA
Without and with S9-mix: 10, 33, 100, 333, 1000 and 3330 µg/plate
Experiment 2: TA1535, TA1537, TA98, TA100 and WP2uvrA
Without and with S9-mix: 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
A solution or a homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines
Test substance concentrations were used within 2.5 hrs. after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene 10 µg/plate in DMSO for TA98

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191 2.5 µg/plate in DMSO for TA1537

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

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

NUMBER OF CELLS EVALUATED: 10E8 per plate

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

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

Evaluation criteria:

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

A test substance is considered positive if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
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.

Statistics:

Not performed.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation was observed at the dose level of 1000 µg/plate and moderate precipitation was observed at the dose level of 3330 µg/plate.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY and MUTAGENICITY:
- In strain TA100 in the first experiment, a 2.2-fold increase in the number of revertant colonies was observed at the highest dose level of 3330 μg/plate in the absence of S9-mix. Since the increase was below the maximum historical solvent control data range, no dose response was observed and the increase was only observed at the moderate precipitating top dose, this increase is considered to be not biologically relevant.
In strain TA1535 (second experiment), a fluctuation in the mean number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix at the dose level of 3330 μg/plate. However, since the increase was not three-fold (a maximum of 1.1-fold was reached), this increase was not considered to be relevant.
- In all other strains, no toxicity or mutagenicity was observed up to and including the top dose of 1000 and 3330 µg/plate.

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, performed according to OECD 471 and GLP principles.

Executive summary:

The genetic toxicity of the substance was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, and Escherichia coli WP2uvrA strain, in accordance with OECD guideline 471 and GLP principles. All bacterial strains showed negative responses over the entire dose range, i.e. no biologically significant dose-related increase in the number of revertants in two independently repeated experiments with and without metabolic activation. Precipitation was observed at the highest concentration tested.

Based on the results it is concluded that the substance 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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 September, 2013 - 05 November, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

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

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: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/ml
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL
With S9-mix, 3 hours treatment: 0.1, 0.3, 1, 3, 10, 33, 100 and 333 µg/mL

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: cyclophosphamide 7.5 µg/mL at 4% S9-mix and 10 µg/mL at 8% S9-mix

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

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

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

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

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

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

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

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

A test 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.

Statistics:

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

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 33 µg/mL and above

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency of one solvent control (second experiment; in the absence of S9-mix) was recorded to be outside the range of ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
Evaluation: The value of 49 per 10^6 survivors was just below the lower limit of the range (50 per 10^6 survivors). Clear negative results were obtained. Therefore this deviation in the mutation frequency had no effect on the results of the study.

INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to the precipitating dose levels of 333 µg/mL in the absence and presence of S9-mix at the 3 hours exposure time. In the absence of S9-mix at the 24 hours exposure time, toxicity was observed at the precipitating dose level of 333 µg/mL.

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Remarks:

Migrated from field 'Test system'.

Conclusions:

A mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. It is concluded that URACROSS ZW7672P, Product ID 021116/000 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions in the absence and presence of S9-mix.

Executive summary:

A mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence and presence of S9-mix, URACROSS ZW7672P, Product ID 021116/000 did not induce a significant increase in the mutation frequency in the first experiment up to precipitating concentrations. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time in the absence of S9 -mix and modifications in S9 concentration in the presence of S9 -mix.

It is concluded that URACROSS ZW7672P, Product ID 021116/000 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the absence and presence of S9-mix.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 February 2015 - 18 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 September 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

d.d. 6 May 2013

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED:
- Source of cells: blood samples were collected by venipuncture 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.
-Suitability of cells: Cells were collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age).
- Average Generation Time (AGT) : the cells and the age of the donor at the time the AGT was determined are presented below:

Dose range finding study and first cytogenetic assay: age 32, AGT = 12.8 h
Second cytogenetic assay: age 23, AGT = 12.9 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

The highest concentration analyzed was selected based on the solubility of the test item in the culture medium.

- Dose range finding study and first cytogenetic assay:
3 h exposure, 24 h fixation, with and without S9: 5.2, 17, 52 μg/mL
24 h exposure, 24 h fixation, with and without S9: 0.54, 1.7, 5.2, 17, 52 μg/mL
48 h exposure, 48 h fixation, with and without S9: 0.54, 1.7, 5.2, 17, 52 μg/mL

- Second cytogenetic assay:
24 h exposure, 24 h fixation, without S9: 5.4, 17, 52 μg/mL
48 h exposure, 48 h fixation, without S9: 5.4, 17, 52 μg/mL



The stock solution was treated with ultrasonic waves until URALAC P 1920C had completely dissolved. URALAC P 1920C concentrations were used within 0.5 hours after preparation.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent: The test item was dissolved in DMSO, which is recommended by international guidelines as solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

Two independent cytogenetic assays were performed, preceeded by a dose-range finding assay.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration experiment 1: 3 h (with and without S9)
- Exposure duration experiment 2: 24, 48 h (without S9)
- Fixation time: 24 h (for 3 or 24 hour exposure period) and 48 h (for 48 hour exposure period)

ENVIRONMENTAL CONDITIONS:
- Humidity: 54-91%
- Temperature: 35.0-37.3 °C

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 μg/ml medium)

STAIN (for cytogenetic assays): Giemsa (5% v/v)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 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 two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene and mounted with a coverslip in an automated coverslipper.

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 in each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (the highest concentration analyzed was determined by the solubility in the culture medium)

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

Evaluation criteria:

ACCEPTANCE CRITERIA
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.


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

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

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

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

DOSE LEVELS SELECTED FOR SCORING:
- First cytogenetic assay: all dose levels tested were selected for scoring.
- Second cytogenetic assay: 5.4, 17 and 52 μg/ml were selected for scoring

RANGE-FINDING/SCREENING STUDIES:
- The test item precipitated in culture medium at a concentration of 52 μg/mL

FIRST CYTOGENETIC ASSAY
- Precipitation in the test: In the first cytogenetic test, the test item precipitated in the culture medium at 52 µg/mL.
- The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
- The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
- Precipitation in the test: In the first cytogenetic test, the test item precipitated in the culture medium at 52 µg/mL.
- The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
- The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.


COMPARISON WITH HISTORICAL CONTROL DATA AND VALIDITY:
- 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 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:

A chromosome aberration study with URALAC P 1920C was performed according to OECD 473 guideline and GLP principles. Based on the results of two independent experiments in cultured peripheral human lymphocytes, it was concluded that URALAC P 1920C is not clastogenic in human lymphocytes under these experimental conditions.

Executive summary:

A chromosome aberration study with URALAC P 1920C was performed according to OECD 473 guideline and GLP principles.

Two independent experiments in cultured peripheral human lymphocytes were performed. URALAC P 1920C was tested up to and including precipitating concentrations, with and without metabolic activation. The outcome of the solvent control and positive controls validated the experimental set-up. URALAC P 1920C did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of URALAC P 1920C on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Based on these results it was concluded that URALAC P 1920C is not clastogenic in human lymphocytes under these experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus study with URALAC P 1920C, conducted according to OECD/EC guidelines and GLP principles, was performed to fulfill data requirements for a registration in China. The result of this study indicate that the registered substance has no genotoxic properties.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 2015 - 06 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed to fulfill data requirements for a registration in China.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 06 May 2013

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Purity/composition correction factor: No correction factor required

Species:

mouse

Strain:

other: NMRI BR

Details on species / strain selection:

These mice are recommended by international guidelines (e.g. OECD, EC).

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation (mean per group): 29 - 37 g
- Assigned to test groups randomly: yes
- Housing: group-housed in labelled polycarbonate cages (maximum of 5 animals/cage).
- Diet: Free access to standard pelleted laboratory animal diet (SM RM-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except 3- 5.5 hours prior to dosing when feed was withheld
- Water: Free access to tap water
- Acclimation period: At least 7 days

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 – 22.2
- Humidity (%): 38 - 50
- Air changes (per hr): appr. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: propylene glycol
- Amount of vehicle: 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was crushed and ground in a mortar with pestle to improve the consistency. URALAC P 1920C concentrations were treated with ultra-sonic waves and heated for approximately 30 to 60 minutes up to a maximum of 80°C to obtain a homogeneous suspension. In the main study the formulation was prepared under nitrogen and the formulation was blended. URALAC P 1920C concentrations were dosed within 4 hours after preparation.

Duration of treatment / exposure:

Test substance and negative control: Two treatments (in two steps each, separated by no more than 2-3 hours) at 0 and 24 hours.
Positive control: Single treatment at start of the experiment (t=0 hours)

Frequency of treatment:

Two treatments at consecutive days. The test substance (and solvent control) was administered as a split dose, i.e., two treatments on the same day separated by no more than 2-3 hours, to facilitate administering a large volume necessary due to limited solubility of the test substance.
Positive control: single oral intubation (40 mg/kg body weight)

Post exposure period:

Test item groups and solvent control group: 24 hours;
Positive control group: 48 hours

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Pre test: 3
Main study: 5 (males only)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide in physiological saline
- Route of administration: Single oral intubation
- Dosis: 40 mg/kg body weight (10 mL/kg body weight)

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. The test procedure and conditions were similar to those applied in the main test. One dose group, comprising 3 males and 3 females received a double dose of URALAC P 1920C with a 24 hour interval. This group was dosed with the highest concentration that was used for the main study (i.e. 2000 mg/kg bw). The observation period after dosing was 3 days. During this period mortality and physical condition were recorded at least once a day.

SAMPLING: Bone marrow was collected by flushing the femurs with fetal calf serum.

DETAILS OF SLIDE PREPARATION: A drop of the bone marrow cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Three slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" (based on Giemsa).

METHOD OF ANALYSIS: The number of micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

Acceptability of the assay
A micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the negative historical control data range.
b) The positive control substance induced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes. The positive control data was analysed by the Students t test (one-sided, p < 0.05) in case of homogeneous variances or by the Welch t test in case of inhomogeneous variances (one-sided, p < 0.05).
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range.

Statistics:

A test substance is considered positive in the micronucleus test if:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals should be above the historical control data range.

A test substance is considered negative in the micronucleus test if:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals should be within the historical control data range. In case the micronucleus data is not normally distributed the data will be transformed by using the formula y = 1/y. Thereafter the Dunnett’s test will be performed. In case the Dunnett’s test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.
In case the micronucleus data is not normally distributed the data will be transformed by using the formula y = 1/y. Thereafter the Dunnett’s test will be performed. In case the Dunnett’s test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: Ataxia and lethargy were observed in all animals within 3 hours after dosing on day 1 and day 2. All animals appeared normal on day 2 (before dosing) and on day 3. No mortality occurred.
- Since there were no differences between sexes in toxicity only male animals were used in the main study.

RESULTS OF DEFINITIVE STUDY
- No mortality occurred. Within 3.5 hour after dosing on the first and second day all animals treated with URALAC P 1920C and the vehicle control animals were lethargic and showed ataxia. Within 20 hours after dosing all animals recovered from the treatment.
- No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of URALAC P 1920C treated animals compared to the vehicle treated animals. Although a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes was observed at a concentration of 500 mg/kg body weight, the mean and all individual number of micronucleated polychromatic erythrocytes were within the historical control data range and the increase was only observed at the lowest concentration tested, not dose related and therefore not considered biologically relevant.
- The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.
- Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes.
- The animals of the groups, which were treated with URALAC P 1920C and the positive control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis.

Conclusions:

An in vivo micronucleus study with URALAC P 1920C in the mouse (5 males/dose, oral exposure) was conducted according to OECD/EC guidelines and GLP principles. It is concluded that URALAC P 1920C is not clastogenic or aneugenic in the bone marrow micronucleus test.

Executive summary:

An in vivo micronucleus study with URALAC P 1920C in the mouse was conducted according to OECD/EC guidelines and GLP principles. Five males/ dose were exposed via the oral route to 500, 1000 and 2000 mg/kg bw. No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of URALAC P 1920C treated animals compared to the vehicle treated animals. Positive and negative controls were included and the results validated the study. It is concluded that this test is valid and that URALAC P 1920C is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 48 hours post dosing of male mice up to a dose of 2000 mg/kg bw/day (the maximum recommended dose in accordance with current regulatory guidelines).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test:

The genetic toxicity of the substance was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains, and Escherichia coli WP2uvrA strain, in accordance with OECD 471 guideline and GLP principles. All bacterial strains showed negative responses over the entire dose range, i.e. no biologically significant dose-related increase in the number of revertants in two independently repeated experiments with and without metabolic activation. Precipitation was observed at the highest concentration tested. Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome aberration test in vitro:

A chromosome aberration study with URALAC P 1920C was performed according to OECD 473 guideline and GLP principles.

Two independent experiments in cultured peripheral human lymphocytes were performed. URALAC P 1920C was tested up to and including precipitating concentrations, with and without metabolic activation. The outcome of the solvent control and positive controls validated the experimental set-up. URALAC P 1920C did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of URALAC P 1920C on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Based on these results it was concluded that URALAC P 1920C is not clastogenic in human lymphocytes under these experimental conditions.

Mouse lymphoma assay:

A mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence and presence of S9-mix, URACROSS ZW7672P, Product ID 021116/000 did not induce a significant increase in the mutation frequency in the first experiment up to precipitating concentrations. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time in the absence of S9 -mix and modifications in S9 concentration in the presence of S9 -mix. It is concluded that URACROSS ZW7672P, Product ID 021116/000 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the absence and presence of S9-mix.

In vivo micronucleus test:

An in vivo micronucleus study with URALAC P 1920C in the mouse was conducted according to OECD/EC guidelines and GLP principles. Five males/ dose were exposed via the oral route to 500, 1000 and 2000 mg/kg bw. No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of URALAC P 1920C treated animals compared to the vehicle treated animals. Positive and negative controls were included and the results validated the study. It is concluded that this test is valid and that URALAC P 1920C is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines).

Justification for classification or non-classification

Based on the available data, URALAC P 1920C does not have to be classified for genotoxicity according to Regulation (EC) No. 1272/2008.