Dimethyl (p-methoxybenzylidene)malonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenicity of the test item was observed in a reliable in vitro gene mutation test in bacteria.

The test item did also not induce gene mutations in a reliable study using CHO cells in vitro with and without metabolic activation.

In an in vitro clastogenicity test in CHO-K1 cells, the test item induced chromosome aberrations, excluding gaps, both in the presence and absence of metabolic activation.

 

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:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

On the day of the experiment, the test item was dissolved in DMSO (MERCK, D-64293 Darmstadt; purity > 99 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
No precipitation of the test item occurred up to the highest investigated dose.

Target gene:

Salmonella typhimurium; STRAINS TA 1535, TA1537, TA 98, TA 100, and E. coli WP2 uvrA

Test concentrations with justification for top dose:

To establish a dose response effect 6 dose levels with adequately spaced intervals were tested: 33, 100, 333, 1000, 2500 and 5000 µg/plate. The maximum dose level was 5000 µg/plate.

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

Negative controls:
Concurrent untreated and solvent controls were performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Sanduvor PR-25 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Distinct toxic effects, evident as a reduction in the number of revertants, occurred in the Salmonella strains with and without metabolic activation at higher concentrations.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.
Batch No. 3 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark and certified free of mycoplasma contamination on 08 August 2014.
Cells were grown in T-75 cm² flasks at 37 +/- 1 °C in a carbon dioxide incubator (5 +/- 0.2 % CO2 in air).

Test concentrations with justification for top dose:

PRELIMINARY CYTOTOXICITY TEST FOR SELECTION OF TEST CONCENTRATIONS:
The preliminary cytotoxicity test was conducted twice.
In the first cytotoxicity test, CHO-K1 cells were exposed to the test item at 30, 60, 120, 240, 480, 960 and 2000 µg/mL along with a DMSO control.
Since the cell counts were negligible because of cytotoxicity even at the lowest concentration of 30 µg/mL, cytotoxicity test was repeated with lower concentrations.
Test item precipitation, initial pH and osmolality determinations carried out in the first preliminary cytotoxicity test are presented in this report.

The following concetrations of the test item were used in the repeat cytotoxicity test:
a) 0.9, b) 1.9, c) 3.75, d) 7.5, e) 15, f) 30 and g) 60 µg/mL

Vehicle / solvent:

DMSO was used as the vehicle control.

Details on test system and experimental conditions:

OUTLINE OF THE METHOD:
Cell cultures were exposed to the test item for 3 hours both in the presence and absence of exogenous metabolic activation and for 21 hours in the absence of metabolic activation. Following exposure of cell cultures to the test item, they were treated with Colchicine to arrest the cells in a metaphase-like stage of mitosis (c-metaphase). Cells were then harvested and chromosome preparations made. Preparations were stained and metaphases were analyzed for chromosomal aberrations.

Statistics:

Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations, numerical aberration (Poly) were analyzed. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fisher exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

CHROMOSOMAL ABERRATION ASSAY

Experiment 1

At the highest concentrations tested (6.5 µg/mL), the reduction in the cell growth was 43 % compared to the vehicle control.

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

The test item showed a statistically significant dose related increase in the incidence of aberrant metaphases excluding gaps compared to the vehicle control at all the concentrations tested.

The positive control, cyclophsophamide monohydrate caused a statistically significant increase in the aberrant metaphases excluding gaps.

Experiment 2

At the highest concentration tested (1.5 µg/mL), the reduction in the cell growth was 54 % compared to the vehicle control.

The incidence of aberrations in the vehicle control was within the range of the in-house historical data.

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at the lowest concentration of 0.17 µg/mL. There was an incidence of Endoreduplication at this test concentration.

However, at 0.5 and 1.5 µg/mL test concentrations, there was a significant increase in the incidence of aberrant metaphases excluding gaps compared to the vehicle control.

Experiment 3

At the highest concentration tested (1 µg/mL), the reduction in the cell growth was 50 % compared to the respective vehicle control.

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

The test item showed s statistically significant dose related increase in the incidence of aberrant metaphases excluding gaps compared to the vehicle control at all the concentrations tested.

Ethyl methanesulphonate caused a statistically significant increase in the aberrant metaphases excluding gaps.

Conclusions:

The study indicated that the test item is clastogenic at the concentrations tested and under the conditions of testing.

Executive summary:

The clastogenic potential of the test item to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

The study consisted of a preliminary cytotoxicity test and a chromosomal aberration assay. Chromosomal aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3 -hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21 -hour exposure.

The test item formed a solution in DMSO at 200 mg/mL and was found to be stable in DMSO for 24 hours at room temperature, at the fortification levels of 10 µg/mL and 5000 µg/mL.

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, toxicity of the test item as cell growth inhibition based on RICC was observed between 3.75 and 7.5 µg/mL, in the presence of metabolic activation, with 3 -hour exposure. In the absence of metabolic activation, with 3 -hour exposure, toxicity was observed between 0.9 and 1.9 µg/mL whereas, with 21 -hour exposure, toxicity was observed at 0.9 µg/mL and there were no cells at and above 1.9 µg/mL. The test item slightly precipitated test medium at and above 480 µg/mL but did not cause any appreciable change in the pH and Osmolality of test medium. Based on these observations, in the chromosomal aberration assay, a maximum of 6.5, 1.5 and 1 µg/mL was tested in Experiments 1, 2 and 3, respectively.

In the chromosomal aberration assay, CHO-K1 cells were exposed to test item in duplicate for 3 hours at concentrations of 0.7, 2.17 and 6.5 µg/mL and at 0.17, 0.5 and 1.5 µg/mL in Experiments 1 and 2 (presence and absence of metabolic activation), respectively. Similarly, in Experiment 3, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 0.11, 0.33 and 1 µg/mL in the absence of metabolic activation for 21 hours. Concurrent vehicle (DMSO) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

At the highest concentration tested, the reduction in cell growth as RICC was 43, 54 and 50 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control.

A total of 300 metaphases each from the DMSO control, each treatment level and the positive control were evaluated for chromosomal aberrations.

There was evidence of induction of chromosome aberrations, excluding gaps, both in the presence and absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases. The results of the concentration analysis of the dose formulation samples were within the acceptable specification range and confirmed that the targeted top dose level (650 µg/mL) was achieved and the results support the validity of the study conclusion.

The study indicated that the test item is clastogenic at the concentration tested and under the conditions of testing.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

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)

GLP compliance:

yes

Type of assay:

other: in vitro Mammalian cell gene mutation test in CHO cells

Target gene:

Chinese Hamster (Criteculus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.
This cell line is capable of developing resistance to 6-thioguanine (6TG) resulting from lack of hypoxanthine phosphoribosyl transferase (HGPRT) enzyme activity as a results of mutation at the X chromosomes.
This cell line was screened for the absence of mycoplasma contamination at the Mycoplasma Laboratory, Statens Serum Institut, Copenhagen, Denmark and was certified free of mycoplasma contamination on 08 August 2014.
Cells were grown in tissue culture flasks at 37 +/- 1 °C in a carbon dioxide incubator (5 +/- 0.2 % CO2 in air).

Test concentrations with justification for top dose:

CHO cells were exposed to the test item at 10, 20, 40, 80, 160, 320, 640, 1280 and 2500 µg/mL along with a DMSO control.

Test concentrations:
Based on the results of thecytotoxicity test and in consultation with the study sponsor the following test concentrations were selected for testing in the gene mutation assay:
- Experiment No. Initial Gene Mutation Assay: Experiments 1 and 2 (Presence and Absence of Metabolic activation, respectively): Test concentrations a)5 b)10 c)20 and d)40 µg/mL (factor of 2)
- Experiment No. Confirmatory Gene Mutation Assay: Experiments 3 and 4 (Presence and Absence of Metabolic Activation, respectively): Test concentrations a)1.5 b)4 c)13 d)26 and e)40 µg/mL (factor of 3)
Note: Due to excessive cytotoxic effect of the test item, additional test concentrations of 30 µg/mL in experiments 1 and 2 and 26 µg/mL in experiments 3 and 4 were maintained.

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

CYTOTOXICITY TEST AND JUSTIFICATION FOR THE SELECTION OF TEST DOSES:
At the end of 3-hour exposure, slight precipitation of the test item was observed in the test medium at 640, 1280 and 2500 µg/mL. At the end of 3-hour exposure, the pH of the test medium in the presence of metabolic activation ranged from 7.02 to 7.14 with 7.13 in the DMSO control while in the absence of metabolic activation it was between 7.11 and 7.19 with 7.12 in the DMSO control. At the end of 3-hour exposure, in the presence of metabolic activation, osmolality of the test medium at the highest test item treatment level (2500 µg/mL) was 0.426 OSMOL/kg, at the lowest precipitating test item level (640 µg/mL) was 0.451 OSMOL/kg, and at the highest soluble test item level (320 µg/mL) was 0.453 OSMOL/kg whereas the corresponding osmolality in the DMSO control was 0.445 OSMOL/kg.
Similarly, at the end of 3-hour exposure, in the absence of metabolic activation, osmolality of the test medium at the highest test item treatment level (2500 µg/mL) was 0.428 OSMOL/kg, at the lowest precipitating test item level (640 µg/mL) was 0.448 OSMOL/kg, and at the highest soluble test item level (320 µg/mL) was 0.448 OSMOL/kg whereas the corresponding osmolality in the DMSO control was 0.434 OSMOL/kg.
The test item did not show evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20 % RCE compared to vehicle control) up to 40 µg/mL in the presence and absence of metabolic activation with 3-hour exposure. However, there was absence of cell monolayer at and above 80 µg/mL.
Based on these observations, a maximum of 40 µg/mL was tested in the gene mutation assay.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

GENE MUTATION ASSAY

Initial Gene Mutation Assay: Experiment 1 & 2 - Presence and Absence of Metabolic Activation

There was no evidence of excessive cytotoxicity (i.e., < 10 % RCE) at any of the tested concentrations either in the presence or absence of metabolic activation. The RCE values in the presence of metabolic activation, ranged from 24.2 to 98.1 % while in the absence of metabolic activation, ranged from 22.5 to 88.0 % compared to the vehicle control.

The frequency of mutants in the DMSO control was within the range of the in-house historical control data.

The test item did not cause a significant increase in the frequencies of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations.

Under similar conditions the positive controls 3 -methylcholanthrene (3 -MCA) and ethyl methanesulfonate (EMS) both induced statistically significant increases in the mutant frequency as compared with the vehicle control.

Confirmatory Gene Mutation Assay: Experiment 3 & 4 - Absence and Presence of Metabolic Activation

There was no evidence of excessive cytotoxicity (< 10 % RCE) at any of the tested concentrations either in the presence or absence of metabolic activation. The RCE values in the presence of metabolic activation, ranged from 20.2 to 101 % while in the absence of metabolic activation, ranged from 22.2 to 100.5 % compared to the vehicle control.

The frequency of mutants in the DMSO control was within the range of the in-house historical control data.

The test item did not cause a significant increase in the frequencies of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations. Under similar conditions the positive control 3 -methylcholanthrene (3 -MCA) and ethyl methanesulfonate (EMS) both induced a statistically significant increase in the mutant frequency as compared with the vehicle control.

Conclusions:

It is concluded that the test item does not have the potential to induce gene mutation in CHO cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The assay conditions fulfilled all the specifications as per the OECD 476 guideline. Since, the test item exhibited cytotoxicity, a maximum of 40 µg/mL test concentrations were expsoed in the assay in the presence and absence of metabolic activation, which met the maximum limit of exposure in case the test item is cytotoxic. Similarly, all the acceptance criteria for a valid test were met.

The Cloning Efficiency of the vehicle controls was more than 60 %. The mean mutant frequency of the vehicle controls in each experiment was between 6.2 and 7.3 mutants per 1000000 clonable cells. The positive controls induced a statistically significant response.

No evidence for the induction of gene mutation was observed in any of the concentrations of the test item either in the presence or in the absence of metabolic activation. Taken together, the results of the initial and confirmatory assays indicated that the test item does not have the potential to induce gene mutation either in the presence or in the absence of metabolic activation. In each of these assays, the positive control chemicals produced a statistically significant increase in the mutant frequencies, demonstrating that the system was able to detect the effect of known mutagens.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A GLP-compliant in vivo mammalian erythrocyte micronucleus test according to OECD 474 guideline was made in Swiss Albino mice at dose levels of 250, 500 and 1000 mg/kg body weight. The test item did not trigger induction of micronuclei in the bone marrow cells under the presented test conditions.

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:

2021-08-18 to 2021-12-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 29, July 2016

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Clariant Plastic and Coatings GmbH, Germany
- Lot/batch number of test material: CHA0122531
- Purity: 99.96%
- Expiry date: 2022-03-15

Species:

mouse

Strain:

Swiss

Remarks:

Albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: VAB Biosciences/GLP Test Facility, Vimta Labs Ltd., India.(CPCSEA Registration No.: 156/PO/RcBi-S/Rc-L/1999/CPCSEA)
- Age at study initiation: 13 weeks
- Number of animals: 66 (33M+ 33F); DRF: 2M/2F per group
- Weight at study initiation: 32.16g - 34.55g (males), 26.12g - 29.46g (females)
- Acclimation period: 6 days prior to DRF study and 22 days for the main study.
- Assigned to test groups randomly: yes
- Fasting period before study: n.a.
- Housing: in groups of 2 and 3 in clean, autoclaved conventional polycarbonate cages (size: approximately Length 425 mm x Breadth 266 mm x Height 185 mm) with stainless steel top grill having provision for holding pellet food and drinking water in polycarbonate bottle with stainless steel sipper tube. Autoclaved corn cobs were used as the bedding material. Cages and water bottles were changed at least 2 times a week. Bedding material was analysed batch wise for any microbial and chemical contaminants.
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3°C - 23.1°C
- Humidity (%): 56.0 – 69.0%
- Air changes (per hr): n.a.
- Photoperiod (hrs dark / hrs light): 12 h light & 12 h dark cycle

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: based on feasibility study
- Concentration of test material in vehicle: 62.5, 125 and 250 mg/mL
- Amount of vehicle (if gavage or dermal): 4 mL/kg bw
- Lot/batch no. (if required): 12CR5L
- Purity: n.a.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared based on stability of test item in the formulation. The test item was formulated into a suspension in Corn oil. The dose volume is 4 mL /kg b.w. The formulations were prepared separately for each group as per the below table.
Table 1: Dose Formulation Preparation
Group 1 (G1 Vehicle): Dose 0 (mg/kg bw); Concentration 0 (mg/mL); Final Volume 20 (mL)
Group 2 (G2 Low Dose): Dose 250 (mg/kg bw); Concentration 62.5 (mg/mL); Final Volume 20 (mL)
Group 3 (G3 Mid Dose): Dose 500 (mg/kg bw); Concentration 125 (mg/mL); Final Volume 20 (mL)
Group 4 (G4 High Dose): Dose 1000 (mg/kg bw); Concentration 250 (mg/mL); Final Volume 20 (mL)
Group 5 (G5 Positive Control ): Dose 30 (mg/kg bw); Concentration 3 (mg/mL); Final Volume 10 (mL)
*Equivalents weights and volumes were substituted without altering the final concentration on two consecutive dosing days.
Formulation of positive control was prepared by dissolving the 30 mg of Cyclophosphamide monohydrate in sterile water for injection and used at a final concentration of 3 mg/ml (Dose: 30 mg/kg body weight)
Formulation preparation:
Test item was transferred into a volumetric flask. Vehicle corn oil was added in the test item and was mixed well (vortexed if required) and made up to final volume in a volumetric flask. The dose formulation was not adjusted to account for its potency.
Formulation of positive control was prepared by dissolving the required quantity of Cyclophosphamide monohydrate in purified water.
Test Item formulation was analysed using validated analytical method along with the study; covering system suitability, specificity, precision, linearity, homogeneity and formulation stability up to 48 hrs. Accuracy and recovery was computed from precision experiment.

Duration of treatment / exposure:

2 days

Frequency of treatment:

Once daily for two consecutive days at 24 h intervals.
The positive control (Cyclophosphamide monohydrate) was administered on day 2 by Intraperitoneal route as a single dose at a dose volume of 10 ml/kg body weight.

Post exposure period:

On day 3, approximately 24 h after treatment, all animals from G1 to G5 groups were euthanized by CO2 asphyxiation.

Dose / conc.:

0 mg/kg bw/day

Remarks:

G1 (Control)

Dose / conc.:

250 mg/kg bw/day

Remarks:

G2 (Low dose)

Dose / conc.:

500 mg/kg bw/day

Remarks:

G3 (Mid dose)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

G4 (High dose)

No. of animals per sex per dose:

5/sex/dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate
- Justification for choice of positive control(s): n.a.
- Route of administration: intraperitoneal route as single dose (dose volume of 10 mL/kg bw)
- Doses / concentrations: Dose: 30 mg/kg bw (concentration 3 mg/mL)

Tissues and cell types examined:

Bone marrow cells were collected approximately 24h after last dose administration by flushing collected and prepared femur bones with foetal bovine serum. The collected cells were evaluated for the Poly Chromatic Erythrocytes (PCE) to Total Erythrocytes (TE) ratio and % cytotoxicity in the Total Erythrocytes.

The proportion of Polychromatic Erythrocytes (PCE) among Total Erythrocytes (TE = Polychromatic Erythrocytes + Normochromatic Erythrocytes) were determined for each animal by counting a total of at least 500 erythrocytes. At least 4000 PCE per animal were scored for the incidence of Micronucleated Polychromatic Erythrocytes (MNPCE).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on dose range finding study

TREATMENT AND SAMPLING TIMES: 2 consecutive days. Sampling on day 3 (24 h after last treatment)

BONE MARROW SAMPLING:
On day 3, approximately 24 h after treatment, all animals from G1 to G5 groups were euthanized by CO2 asphyxiation and following euthanasia, both femurs from each animal were removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, marrow was flushed from the bone cavity with foetal bovine serum (3 ml) into pre-labelled centrifuge tubes.

DETAILS OF SLIDE PREPARATION: Bone marrow were centrifuged, pellet gently resuspended.From suspension small drops were placedon the end of labelled glass microsope slides. A smear was made from cell suspension by drawing theendof a clean slide along the labelled slide.Minimum of 2 slides were prepared for each animal. Slides were allowed to air-dry prior to fixation (3 min) of cells in absolute ethanol. All slides wer stained with May-Grunwald stain (5 min) followed by rinsing with water (3 min). Immediatelyslides were stainded with 5 % Giemsa stain in Sorenson phosphate buffer (pH 6.8) for 5 minutes. Excess stain was removed by rinsing in water for 3 minutes, air-dried and mounted with DPX (Distrene-80 Plasticizer and Xylene). Quality check for slides was performed for uniform distribution of cells and staining. Both the slides from each animal were selected for the evaluation of the test item and selected for scoring. To control bias, all slides were coded prior to slide observation analysis for main study.

METHOD OF ANALYSIS: The proportion of Polychromatic Erythrocytes (PCE) among Total Erythrocytes (TE = Polychromatic Erythrocytes + Normochromatic Erythrocytes) were determined for each animal by counting a total of at least 500 erythrocytes. At least 4000 PCE per animal were scored for the incidence of Micronucleated Polychromatic Erythrocytes (MNPCE).
The criteria for the identification of micronuclei were those of Schmid (1976). The unit of scoring was the micronucleated cell, not the micronucleus; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei.

Evaluation criteria:

The test item is considered a positive mutagen, if at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the vehicle control or the frequency of micronucleated cells is found to be dose dependant and statistically significant in treatment groups as compared to vehicle control group. For a valid study result, the positive control group should exhibit statistically significant increase in the number of micronucleated cells as compared to control. The test item is considered negative, if it does not meet any of the criteria as described in this section.

Statistics:

Statistical analysis was performed using GraphPad Prism, version 4.0. Statistical comparisons of body weight data were carried out between treatment and control groups using non-parametric (Kruskal-Wallis test, Mann Whitney’s Test) test procedures. The homogeneity of variance was evaluated by Bartlett’s test. Statistical significance was evaluated at p ≤ 0.05. All quantitative data was summarized and expressed as Mean ± SD. Comparison between two groups was done by Student’s t-test. The data of micronucleated polychromatic cells was analysed using chi-square test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

non-clastogenic

Executive summary:

An OECD 474 study was performed to evaluate the ability of Hostavin PR-25P to induce micronuclei in polychromatic erythrocytes (PCEs) in the bone marrow of mice.

Initially, a Dose Range Finding Study (DRF) was conducted in two male and two female animals per sex per group and treated at dose level of 500, 1000 and 2000 mg/kg body weight for two consecutive days. The animals in the high dose group ejected out a few quantities of the formulated suspension immediately after oral dosing. All animals were monitored for morbidity, mortality, clinical signs of toxicity and body weight. Further bone marrow cells were collected approximately 24h after last dose administration by flushing with foetal bovine serum. The collected cells were evaluated for the Poly Chromatic Erythrocytes (PCE) to Total Erythrocytes (TE) ratio and % cytotoxicity in the Total Erythrocytes. The percentage
cytotoxicity (70.65 and 58.21); ratio of PCE to TE (0.165 and 0.275) was reduced in the male and female animals of the high dose (2000 mg/kg body weight) treated group as compared to the control group (0.562 and 0.658). Based on these results of the DRF study; the dose levels selected for the Main Study were 250, 500 and 1000 mg/kg body weight.

The main study was conducted with five male and five female animals per sex per group. After two consecutive days of oral dosing of test item formulated with Corn oil, the animals were subjected to in-life observations, including morbidity, mortality, clinical signs of toxicity and body weight. The animals were terminally sacrificed, bone marrow cells extracted from
femur bone and slides prepared. The ratio between polychromatic erythrocytes (PCEs) and Total erythrocytes (TEs) were determined to evaluate cytotoxicity and reported as the number
of PCEs per 500 erythrocytes.

Slides were coded prior to scoring. The slides were observed to detect for any presence of micronuclei in the PCEs. A minimum of 4000 polychromatic erythrocytes (PCEs) per animal were scored. The mean numbers of PCEs in treated groups were found to be comparable to the mean value of PCEs in vehicle control indicating that the test item did not exert any
cytotoxic effects.

The mean percent micronucleated cells were 0.01, 0.02, 0.07 in male animals and 0.05, 0.04, 0.01 in female animals treated at the doses of 250, 500 and 1000 mg/kg body weight respectively. There was neither dose dependence nor statistical significance in the frequency of micronucleated PCEs in the test item treated groups when compared to the vehicle control group (0.06 for males and 0.05 for females).
The positive control, cyclophosphamide monohydrate, induced a marked increase in the incidence of micronucleated polychromatic erythrocytes along with statistical significance (p<0.05) as compared to the vehicle control (1.08 in males and females) thus demonstrating the sensitivity of the assay.
The vehicle control group displayed micronucleated PCEs, the group mean of which was comparable to the historical control.

Conclusion: Hostavin PR-25P did not trigger induction of micronuclei in the bone marrow cells of Swiss Albino Mice at all tested dose levels (250, 500 and 1000 mg/kg body weight).
Based on these findings, it is concluded that Hostavin PR-25P is “non-clastogenic” in the in vivo micronucleus assay in Swiss Albino Mice upto the highest tested concentration of 1000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the observed effects are regarded as relevant for humans.

Additional information

No gene mutagenicity in bacteria was observed in a plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA in concentrations up to 5000 μg/plate with and without metabolic activation. The study is GLP-compliant and was performed according to OECD guideline 471.

There was also no gene mutagenicity in mammalian CHO cells in vitro with and without metabolic activation, if tested in concentrations up to 40 µg/mL. Higher concentrations were cytotoxic to the cells. The study is GLP-compliant and was performed according to OECD guideline 476.

In the chromosomal aberration assay, CHO-K1 cells were exposed to test item with and without metabolic activation in concentrations up to 6.5 µg/ml, based on a cytotoxicity pre-test. There was an induction of chromosome aberrations, excluding gaps, both in the presence and absence of metabolic activation. The study is GLP-compliant and was performed according to OECD guideline 473.

Due to these positive findings in the Chromosomal aberration assay, a GLP-compliant in vivo mammalian erythrocyte micronucleus test according to OECD 474 guideline was made in Swiss Albino mice at dose levels of 250, 500 and 1000 mg/kg body weight. The test item did not trigger induction of micronuclei in the bone marrow cells under the presented test conditions. Based on the findings, it is concluded that Hostavin PR-25P was “non-clastogenic” in the in vivo micronucleus assay in Swiss Albino mice upto the highest tested concentration of 1000 mg/kg body weight.

Justification for classification or non-classification

In vitro assays on mutagenicity revealed contradicting results (negative in an Ames Assay and a gene mutation assay, positive in a chromosome aberration test). In order to clarify the contradicting results, a GLP-compliant in vivo mammalian erythrocyte micronucleus test according to OECD 474 guideline was made in Swiss Albino mice at dose levels of 250, 500 and 1000 mg/kg body weight. The test item did not trigger induction of micronuclei in the bone marrow cells under the presented test conditions.

Taken all available data on in vitro and in vivo genetic toxicity together it can be concluded that the test item is not to classify for genetic toxicity.