Reaction products of amines, polyethylenepoly-, triethylenetetramine fraction and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 2016 to 31 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 473, updated and adopted 26 September 2014

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

EDA-BADGE-EDA

Cas Number:

854009-15-7

Molecular formula:

C25H40N4O4

IUPAC Name:

EDA-BADGE-EDA

Constituent 2

Reference substance name:

EDA-BADGE-EDA-BADGE-EDA

Molecular formula:

C48H72N6O8

IUPAC Name:

EDA-BADGE-EDA-BADGE-EDA

Constituent 3

Reference substance name:

EDA-BADGE α-glycol

Molecular formula:

C23H34N2O5

IUPAC Name:

EDA-BADGE α-glycol

Constituent 4

Reference substance name:

reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

IUPAC Name:

reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

Constituent 5

Reference substance name:

Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

IUPAC Name:

Unidentified reaction product of 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane and ethylene diamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: BBF01102V1 (provided by Sponsor)- Expiration date of the lot/batch: 01 January 2021 (per Sample Label)STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light- Solubility and stability of the test substance in the solvent/vehicle: Water was initially used as the vehicle based on results from the solubility test conducted at BioReliance. However, two preliminary toxicity assays were terminated due to poor solubility of the test substance in water. Therefore, dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance, and compatibility with the target cells. In the solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Final preparation of a solid: To achieve solutions, the most concentrated dilution was vortexed for approximately 35 minutes in the preliminary toxicity assay, for approximately 15 minutes in the initial chromosomal aberration assay, and for approximately 20 minutes in the repeat chromosomal aberration assay. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Method

Target gene:

chromosomes

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the doses tested were: 0.5, 1.5, 5, 15. 50, 150, 500, 1500, and 5000 µg/mL. The top dose tested, 5000 µg/mL, was the limit dose for this assay.Doses tested in the initial chromosome aberration assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control) and were: 15, 30, 60, 70, 80, 90, 100, 125, and 150 µg/mL for the non activated and S9-activated 4-hour exposure groups; and 2.5, 5, 15, 20, 25, 30, 35, 40 µg/mL for the non-activated 20-hour exposure group.Doses tested in the repeat chromosome aberration assay were based upon post-treatment toxicity (reduction in cell growth index relative to the vehicle control) and were: 5, 10, 15, 20, 30, 40, 50, 60, 80, 90, 100, 125, and 150 µg/mL for the non activated and S9-activated 4-hour exposure groups.

Vehicle / solvent:

Water was initially used as the vehicle based on results from the solubility test conducted at BioReliance. However, two preliminary toxicity assays were terminated due to poor solubility of the test substance in water. Therefore, dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance, and compatibility with the target cells. In the solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in mediumDURATION- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed. Cells were harvested 20 hours (±30 minutes) after initiation of treatment, which corresponds to 1.5 normal cell cycles.STAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assayNUMBER OF CELLS EVALUATED: a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture), whenever possibleDETERMINATION OF CYTOTOXICITY- Method: mitotic indexOTHER EXAMINATIONS:- Determination of polyploidy: yes- Determination of endoreplication: yes

Evaluation criteria:

The mitotic index was recorded as the percentage of cells in mitosis per 500 cells counted. A minimum of 300 metaphase spreads containing 20 ± 2 centromeres from each dose (150 per duplicate treatment) were examined and scored for chromatid-type and chromosome-type aberrations. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but were considered part of the incomplete exchange. Pulverized cells and severely damaged cells (counted as 10 aberrations) were also recorded. The XY vernier for each cell with a structural aberration was recorded. The percentage of cells with numerical aberrations (polyploid and endoreduplicated cells) was evaluated for 150 cells per culture (a total of 300 per dose level). The test substance was considered to have induced a positive response if •at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and•the increase is concentration-related (p ≤ 0.05), and•results are outside the 95% control limit of the historical negative control data.The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the initial chromosome aberration assay, at doses ≥ 25 µg/mL in the non activated 20 hr group. In the repeat chromosome aberration assay, at doses ≥ 50 µg/mL in the non activated 4 hr group and at doses ≥ 60 µg/mL in the S9 activated 4 hr group

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: pH results were acceptable.- Effects of osmolality: Osmolality results were acceptable.- Water solubility: poor solubility of the test substance in water- Precipitation: In the preliminary toxicity assay, at the conclusion of the treatment period, visible precipitate was observed at doses ≥ 150 µg/mL in all three exposure groups.- Definition of acceptable cells for analysis: metaphase spreads containing 20 ± 2 centromeresHISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data: Refer to main study report- Negative (solvent/vehicle) historical control data: Refer to main study reportADDITIONAL INFORMATION ON CYTOTOXICITY:- Measurement of cytotoxicity used: Cell growth and mitotic inhibition- Other observations when applicable: Degree of monolayer confluency

Applicant's summary and conclusion

Conclusions:

The results of the assay indicate that 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediaminewas negative for the induction of structural and numerical chromosomal aberrations in the presence and absence of the exogenous metabolic activation system.

Executive summary:

The test substance,4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an exogenous metabolic activation system. CHO cells were treated for 4 hours in the absence and presence of S9, and for 20 hours in the absence of S9. 

Water was initially used as the vehicle based on results from the solubility test conducted at BioReliance. However, two preliminary toxicity assays were terminated due to poor solubility of the test substance in water. Therefore, dimethyl sulfoxide (DMSO)was used as the vehicle. Data from the two initial preliminary toxicity assays are maintained in the raw data, but not reported.

In the third preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the limit dose for this assay. Cytotoxicity ( ≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 150 µg/mL in the non-activated and S9-activated 4-hour exposure groups; and at doses ≥ 50 µg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 150 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the chromosomal aberration assay ranged from 15 to 150 µg/mL for the non-activated and S9-activated 4-hour exposure groups; and from 2.5 to 40 µg/mL for the non-activated 20-hour exposure group.

In the initial chromosomal aberration assay, cytotoxicity ( ≥ 50% reduction in cell growth index relative to the vehicle control), was observed at doses ≥ 25 µg/mL in the non-activated 20-hour exposure group. In the non-activated and S9-activated 4-hour exposure groups, due to excessive cytotoxicity, the chromosomal aberration assay was repeated at doses ranging from 5 to 150 µg/mL. 

In the repeat assay, cytotoxicity ( ≥ 50% reduction in cell growth index relative to the vehicle control), was observed at doses ≥ 50 µg/mL in the non-activated 4-hour exposure group and at doses ≥ 60 µg/mL in the S9-activated 4-hour exposure group.

The doses selected for evaluation of chromosomal aberrations were 5, 15, and 25 µg/mL for the non-activated 20-hour exposure group; 10, 40, and 50 µg/mL for the non-activated 4-hour exposure group; and 10, 40, and 60 µg/mL for the S9-activated 4-hour exposure group. 

No significant or dose-dependent increases in structural or numerical chromosomal aberrations were observed at any dose in any of the treatment groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate that 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediaminewas negative for the induction of structural and numerical chromosomal aberrations in the presence and absence of the exogenous metabolic activation system.