Reaction products of diethylenetriamine and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, BADGE-DETA did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Further information on genetic toxicity is available using a read across approach. Testing was carried out on

BADGE-EDA.

The results of the assay indicate that BADGE EDA was negative for the induction of structural and numerical chromosomal aberrations in the presence and absence of the exogenous metabolic activation system.

BADGE-EDA was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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

Justification for type of information:

Justification for the read across approach is available in the document enclosed in chapter 13

Reason / purpose for cross-reference:

read-across source

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

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.

Target gene:

chromosomes

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.

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.

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

Conclusions:

The results of the assay indicate that 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine was 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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

11th January 2017 to 15th March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

4. DATA MATRIX

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian cell forward mutation assay

Specific details on test material used for the study:

Test Substance
Identification:
4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine
Batch No.:
BBF01102V1
CAS No.:
72480-18-3
Purity:
UVCB
Molecular weight:
418.53 to 861.12 g.mol-1
Description:
Clear colorless resin
Storage Conditions:
Room temperature, protected from light
Receipt Date:
30 August 2016

Target gene:

The test substance was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Dimethyl sulfoxide (DMSO) was used as the vehicle.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.

Test concentrations with justification for top dose:

Preliminary Toxicity Test for Selection of Dose Levels
Cells were treated with 10 test substance concentrations, as well as the vehicle control, in the presence and absence of S9 using single cultures. The maximum concentration evaluated was the limit dose for this assay. Lower concentrations were prepared by 2-fold dilutions. The pH of the treatment medium was measured and adjusted at concentrations ≥9.77 μg/mL using 1N hydrochloric acid (HCl; CAS No. 7647-01-0, Lot No. RNBD4214, Exp. Date: 31 Mar 2018).
Osmolality of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured at the beginning of treatment. Precipitation was assessed at the beginning and end of treatment. Concentrations evaluated in the definitive mutation assay were based on adjusted relative survival, calculated as described below.

Vehicle / solvent:

DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

Test System
The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency generally greater than 80% (Li et al., 1987). The CHO-K1-BH4 cells used in this study were obtained from A.W. Hsie, Oak Ridge National Laboratories (Oak Ridge, TN).
Solubility Determination
DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

Evaluation criteria:

Once criteria for a valid assay were met, the responses observed in the assay were evaluated as follows.
The test substance was considered to have produced a positive response if it induced a dose-dependent increase in mutation frequency and an increase exceeding 95% historical vehicle control limits in at least one test dose level(s) as compared with concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the result were considered equivocal. If none of these criteria were met, the results were considered to be negative.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test substance, 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).
Under the conditions of the assay described, 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Executive summary:

SUMMARY

The test substance, 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥78.1 μg/mL at the beginning of treatment and at concentrations ≥156 μg/mL with S9 and ≥ 2500 μg/mL without S9 by the end of treatment. Adjusted relative survival was 74.61 and 16.78% at a concentration of 156 and 19.5 μg/mL with and without S9, respectively. Adjusted relative survival was approximated 0% at all higher concentrations. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 5, 10, 25, 75, 100 and 156 μg/mL with S9 and 1, 2.5, 5, 10, 15, 20, 25 and 40 μg/mL without S9.

In the initial definitive mutagenicity assay, visible precipitate was observed at concentrations ≥75 μg/mL at the beginning of treatment with S9 and at concentrations ≥100 μg/mL with S9 and ≥15 μg/mL without S9 by the end of treatment. The treatment condition without S9 was not scored since the average absolute cloning efficiency of vehicle controls was < 60% (at initial survival and selection). This was repeated in the retest of definitive mutagenicity assay. The average adjusted relative survival was 86.28% at a concentration of 100 μg/mL with S9. Cultures treated at concentrations of 5, 10, 25, 75 and 100 μg/mL with S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p < 0.01).

In the retest of definitive mutagenicity assay with same concentrations of dose levels, no visible precipitate was observed at the beginning of treatment and visible precipitate was observed at concentrations ≥15 μg/mL at the end of treatment. The average adjusted relative survival was 79.62 % at a concentration of 15 μg/mL without S9. Cultures treated at concentrations of 1, 2.5, 5, 10 and 15 μg/mL without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.01). The positive controls induced significant increases in mutant frequency (p < 0.01).

These results indicate 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with ethylenediamine was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Conclusive but not sufficient for classification.