Dimethyl (p-methoxybenzylidene)malonate

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl (p-methoxybenzylidene)malonate

EC Number:

231-185-8

EC Name:

Dimethyl (p-methoxybenzylidene)malonate

Cas Number:

7443-25-6

Molecular formula:

C13H14O5

IUPAC Name:

1,3-dimethyl 2-[(4-methoxyphenyl)methylidene]propanedioate

Test material form:

solid: particulate/powder

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

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

G1 (Control)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Remarks:

G2 (Low dose)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Remarks:

G3 (Mid dose)

Doses / concentrations 4

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)

Examinations

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.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

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.