Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

EC Number:

944-271-1

Cas Number:

2305048-54-6

Molecular formula:

not applicable for multi-constituent.

IUPAC Name:

Reaction mass of (((1,3-phenylenebis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) and (1,3-phenylenebis(oxy))bis(propane-3,1-diyl) bis(2-methylacrylate) and 3-(3-(2-(2-(methacryloyloxy)ethoxy)ethoxy)phenoxy)propyl methacrylaterate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M Company, Batch 653940
- Purity, including information on contaminants, isomers, etc.: 100% (per Protocol)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: No data
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: No data
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: No data
- Reactivity of the test material with the incubation material used (e.g. plastic ware): No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): None, test article was administered unchanged.
- Preliminary purification step (if any): No data

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Unchanged

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Test system recommended per OECD 474.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 7 weeks
- Weight at study initiation: 30.6-35.3 g
- Assigned to test groups randomly: Yes, randomization procedure within Microsoft Excel
- Fasting period before study: No data
- Housing: See below
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72°F
- Humidity (%): 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): No data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

None

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: None, dosed neat.

Duration of treatment / exposure:

Exposed once

Frequency of treatment:

Exposed once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg diet

Doses / concentrations 2

Dose / conc.:

500 mg/kg diet

Remarks:

equivalent to 0.35 mL/kg

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg diet

Remarks:

equivalent to 0.69 mL/kg

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg diet

Remarks:

equivalent to 1.39 mL/kg

No. of animals per sex per dose:

6

Control animals:

yes, concurrent no treatment

Positive control(s):

Cyclophosphamide (CP)
- Justification for choice of positive control(s): Per OECD 474
- Route of administration: Oral gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on a dose range-finding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 and 48 hours after treatment

DETAILS OF SLIDE PREPARATION: Cells were re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide. Four slides were prepared from each animal, air dried and fixed by dipping in methanol. One set of two slides (including 5 positive control slides) were stained with acridine orange for microscopic evaluation. The other set of slides was kept as backup and were archived. Each slide was identified by the harvest date, study number, and animal number (or slide number for positive control slides). Slides were coded using a random number table by an individual not involved with the scoring process.

METHOD OF ANALYSIS: The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that generally are round and that generally are between 1/20 and 1/5 the size of the PCE. Scoring was based upon the micronucleated cell, not the micronucleus; thus, occasional cells with more than one micronucleus were counted as one micronucleated PCE (MnPCE), not two (or more) micronuclei.

OTHER: 4000 PCE/animal

Evaluation criteria:

A test article was considered to have induced a positive response if:
a) at least one of the test article doses exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01 and R2≥70%), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.
A test article was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the bone marrow was exposed to the test article (unless intravenous administration was used).

Statistics:

Statistical analysis was performed on the micronucleus frequency (%MnPCE) and %PCE
using the animal as the unit. The mean and standard deviation of %MnPCE and %PCE were
presented for each treatment group.
The use of parametric or non-parametric statistical methods in the evaluation of data was
based on the variation between groups. The group variances for micronucleus frequency for
the untreated and test article groups at the respective sampling time were compared using
Levene’s test (significant level of p less than or equal to 0.05). Since the variation between groups was found
not to be significant; a parametric one-way ANOVA was performed followed by a Dunnett’s
post-hoc analysis to compare each dose group to the concurrent untreated control.
A linear regression analysis was conducted to assess dose responsiveness in the test article
treated groups (p less than or equal to 0.01 and R2 ≥ 70%).
A pair-wise comparison (Student’s T-test; p less than or equal to 0.05) was used to compare the positive control
group to the concurrent untreated control group.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Rationale for exposure: limit dose as recommended by guideline

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically significant increase in the incidence of MnPCEs was observed in the test article treated groups relative to the untreated group at either time point (24 or 48 hours).
- Ratio of PCE/NCE (for Micronucleus assay): No appreciable reductions in the PCE/EC ratio were observed in the test article groups compared to the untreated group, indicating the test article did not induce cytotoxicity.
- Appropriateness of dose levels and route: As recommended by guideline
- Statistical evaluation: Statistical analysis was performed on the micronucleus frequency (%MnPCE) and %PCE using the animal as the unit. The mean and standard deviation of %MnPCE and %PCE were presented for each treatment group. The use of parametric or non-parametric statistical methods in the evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the untreated and test article groups at the respective sampling time were compared using Levene’s test (significant level of p < 0.05). Since the variation between groups was found not to be significant; a parametric one-way ANOVA was performed followed by a Dunnett’s post-hoc analysis to compare each dose group to the concurrent untreated control. A linear regression analysis was conducted to assess dose responsiveness in the test article treated groups (p< 0.01 and R2 ≥ 70%). A pair-wise comparison (Student’s T-test; p < 0.05) was used to compare the positive control group to the concurrent untreated control group.

Applicant's summary and conclusion

Conclusions:

Based on the increased plasma bromine concentrations following dosing, the test article was absorbed and distributed to the body and therefore the bone marrow was properly exposed. No significant increase in the presence of micronuleated polychromatic erythrocytes (MnPCEs) was observed in any group indicating that DiBrORMA is negative in the mouse micronucleus assay.

Executive summary:

The test article, DiBrorma, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocytes (PCEs) in mouse bone marrow in a GLP-compliant study conducted according to OECD Guideline 474 (2016). CD-1 mice (6 males/group at 24 hoursand an additional 6 males in the high dose and control groups at 48 hours) received 0 (untreated), 500, 1000, or 2000 mg/kg of undiluted DiBrORMA via single oral gavage. Blood was collected from 3 animals/timepoint at 1 hour (control only), 2, 4, 8, and 24 hours post-dose.The mice were euthanized at 24 hours or 48 hours post-dose as indicated above.The collected plasma samples were evaluated for the presence of bromine following a digestion step to release any covalently bound bromine (i.e., bromine bound in the test article compound or its metabolites). No significant increase in the presence of micronuleated polychromatic erythrocytes (MnPCEs) was observed in any group. Plasma bromine concentrations were increased following treatment with DiBrORMA while the concentration in the control group was approximately 2.4 ug/mL.The highest individual peak concentrations (Cmax) were 36.2, 58.9, and 57.6 ug/mL for the 500, 1000, and 2000 mg/kg groups, respectively, at 2, 4, and 2 hours post-dose, respectively (Tmax). At 24 hours post-dose,the mean plasma bromine concentrations had decreased to 6.1, 5.1, and 10.4 ug/mL for the 500, 1000, and 2000 mg/kg groups, respectively. Based on the increased plasma bromine concentrations following dosing, the test article was absorbed and distributed to the body and therefore the bone marrow was properly exposed.