Reaction mass of meso-2-{[4-(2-{4-[(oxiran-2-yl)methoxy]phenyl}-1,1-dichloroethylidene-2 yl)phenoxy]methyl}oxirane and 2RS)-2-({4-[2-(4-{[(2RS)-oxiran-2-yl]methoxy}phenyl)-1,1-dichloroethylidene-2-yl]phenoxy}methyl)oxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Testing in an OECD guideline, GLP in vitro AMES study gave negative results

Testing in an OECD guideline, GLP, in vitro chromosome aberration study gave positive results.

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:

15t March 2018 to 30th April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE

Description:
Whitish yellow solid
Storage Conditions:
Room temperature, protected from light
Receipt Date:
09 February 2018

Purity:
Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials

Target gene:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 3880, Exp. Date: 31 Oct 2019) was purchased commercially from MolTox (Boone, NC).

Concentrations in the preparations can be found in Table 1.

Test concentrations with justification for top dose:

In the initial toxicity-mutation screen, a maximum dose of 5000 μg per plate was achieved using a concentration of 100mg/mL and a 50.0 μL plating aliquot. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed. No positive mutagenic responses were observed.

Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Evaluation criteria:

All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
Based on historical control data (95% control limits), all tester strain cultures must exhibit characteristic numbers of spontaneous revertants per plate with the vehicle controls.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Based upon the results of the initial toxicity-mutation assay, the dose levels selected for the confirmatory mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed.
BioReliance Study No. AF23LZ.503.BTL 17
Positive mutagenic responses were observed with tester strains TA100 and TA1535 in the presence and absence of S9 activation and WP2 uvrA in the absence of S9 activation.

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE did cause a positive mutagenic response with tester strain TA100 and TA1535 in the presence or absence of S9 activation and WP2 uvrA in the absence of S9 activation.

Executive summary:

The test substance, REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. The tester strain WP2 uvrA in the presence of S9 activation was not evaluated as the top three dose levels were plated without tester strains due to technician error. This condition was repeated. Precipitate was observed beginning at 1500 μg per plate with all conditions (except WP2 uvrA in the presence of S9 activation). No toxicity was observed (except WP2 uvrA in the presence of S9 activation). Positive mutagenic responses were observed with tester strains TA100 and TA1535 in the presence and absence of S9 activation and WP2 uvrA in the absence of S9 activation. Based upon these results, the maximum dose tested in the retest of initial toxicity-mutation assay and confirmatory mutagenicity assay was 5000 μg per plate.

In the retest of initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate with WP2 uvrA in the presence of S9 activation. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed. No positive mutagenic responses were observed.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 1500 μg per plate with all conditions. No toxicity was observed. Positive mutagenic responses were observed with tester strains TA100 and TA1535 in the presence and absence of S9 activation and WP2 uvrA in the absence of S9 activation.

These results indicate REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE was positive for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2018 to April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification:
REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE
Purity / Molecular Weight:
Substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB)
Description:
Whitish yellow solid
Storage Conditions:
Room Temperature, protected from light
Receipt Date:
09 February 2018

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA.

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:

Cytotoxicity results of the preliminary toxicity assay conducted at doses ranging from 0.5 to 5000 μg/mL in DMS.
Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 50 μg/mL in all three exposure groups. Based upon the results of the preliminary toxicity assay, the doses selected for the chromosome aberration assay were 5, 10,20,40,45,50,55,60,70 for both activated and non-activated groups.

Vehicle / solvent:

Solubility Determination
DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. In a 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.
Preparation of Target Cells
Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium containing 10% fetal bovine serum, 1.5 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B) for each treatment condition at a target of 5 x 105 cells/culture. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 16-24 hours.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The in vitro mammalian chromosomal aberration assay was conducted by exposing CHO cells to appropriate concentrations of the test substance as well as the concurrent positive and vehicle controls, in the presence and absence of an exogenous metabolic activation system. Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).

Evaluation criteria:

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:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the conditions of the assay described in this report, REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE was concluded to be positive for the induction of structural chromosomal aberrations and negative for the induction of numerical chromosomal aberrations in the presence and absence of the exogenous metabolic activation systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

The test substance, REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE, 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 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. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 μg/mL, which was the limit dose for this assay (test substance is a UVCB). Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 50 μg/mL in all three exposure groups. 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 chromosome aberration assay ranged from 5 to 70 μg/mL for all three exposure groups.

In the chromosomal aberration 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 and 20-hour exposure groups, and at 70 μg/mL in the S9-activated 4-hour exposure group. The doses selected for evaluation of chromosome aberrations were 20, 40, and 50 μg/mL for the non-activated 4-hour exposure group; 10, 40, and 70 μg/mL for the S9-activated 4-hour exposure group; and 10, 40, and 50 μg/mL for the non-activated 20-hour exposure group.

In the non-activated 4-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (14.0% and 23.3%) were observed at doses 40 and 50 μg/mL, respectively (p ≤ 0.01; Fisher’s Exact test and p ≤ 0.05; Cochran-Armitage test).

In the S9-activated 4-hour exposure group, a statistically significant and dose-dependent increase in structural aberrations (4.0%) was observed at doses 70 μg/mL (p ≤ 0.05; Fisher’s Exact and Cochran-Armitage tests). Statistically significant increases in numerical (polyploid or endoreduplicated cells) aberrations were observed at doses 10 and 70 μg/mL (p ≤ 0.05; Fisher’s Exact test). However, due to subjectivity in scoring between the scorers, the slides were rescored by a third scorer. The statistically significant increase observed in the initial scoring was not observed upon rescoring. The Study Director has accepted the results from the rescoring and the initial observation was considered irrelevant. Data from the initial scoring are maintained in the study file, but not reported.

In the non-activated 20-hour exposure group, statistically significant and dose-dependent increases in structural aberrations (16.0% and 27.3%) were observed at doses 40 and 50 μg/mL, respectively (p ≤ 0.01; Fisher’s Exact test and p ≤ 0.05; Cochran-Armitage test).

No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose in the non-activated 4 and 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate that REACTION PRODUCTS OF 4,4'- (DICHLOROVINYLIDENE) DIPHENOL, FORMALDEHYDE, OLIGOMERIC REACTION PRODUCTS WITH PHENOL, FORMALDEHYDE, POLYMER WITH 2-METHYLPHENOL and 1-CHLORO-2, 3-EPOXYPROPANE was positive for the induction of structural chromosomal aberrations and negative for the induction of numerical chromosomal aberrations in the presence and absence of the exogenous metabolic activation system.

Genetic toxicity in vivo

Description of key information

A read across approach was utilised to fulfil the need for in vivo testing information in the light on inconclusive in vitro testing. Full details of this can be found in the Read Across justification document enclosed in Chapter 13.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

05 December 2017 - 11 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please refer to the Read Across justification document enclosed in Chapter 13 for further details.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

transgenic rodent mutagenicity assay

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Rats have been used historically in safety evaluation and genotoxicity studies and are recommended by regulatory agencies. Because this study was conducted in accordance with regulatory guidelines, alternatives could not be considered.
The Big Blue® in vivo mutation assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (OECD, 2013). TGR assays in general, and the Big Blue® assay in particular, have been reviewed by OECD (OECD, 2009 and 2011a) and are identified in OECD Test Guideline 488 (OECD, 2011b and OECD, 2013) as being appropriate to investigate in vivo mutagenicity in any tissue of interest. In addition, the TGR assays are recommended to investigate a potential mutagenic mode of action in the etiology of rodent tumors.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BioReliance colony housed at Taconic Biosciences, Inc., Germantown, NY
- Age at study initiation: 9-10 weeks (initial cohort), 13-14 weeks (extended cohort)
- Weight at study initiation: 213.1 to 249.2 grams (initial cohort), 236.3 to 333.6 grams (extended cohort)
- Assigned to test groups randomly: yes, under following basis: by body weight
- Housing: multiple-housed during acclimation and following randomization in polycarbonate cages
- Diet (e.g. ad libitum): TEKLAD Global Diet #2018C (Certified 18% Protein Rodent Diet, Envigo, Madison, WI) in pellet form, in stainless steel rodent feeders, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 or 40 days prior to the first dose administration, for the initial or extended cohorts, respectively

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 to 24.0ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% Methocel A4M methyl cellulose ethers with 0.1% Tween 80 in deionized water
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required): Methocel A4M: BCBR9701V; Tween 80: MKBQ9736V

Justification for vehicle:
Justification for the use of 0.5% Methocel A4M methyl cellulose ethers with 0.1% Tween 80 in deionized water as the vehicle for formulation of suspensions of BADGE includes the following considerations:
- Oral gavage in a vehicle was specified in the ECHA decision and is consistent with previous repeated-dose animal testing
- 0.5% Methocel A4M methyl cellulose ethers with 0.1% Tween 80 in deionized water as vehicle for dose formulation was used for the following repeated-dose animal testing: 28- and 90-day oral toxicity, 24-month chronic toxicity and carcinogenicity testing, 1- and 2-Generation Reproductive Toxicity, and OECD 414 Developmental toxicity testing.
- The ability, as demonstrated in the prior toxicity studies and as demonstrated in this study, to prepare stable dose formulations at the targeted dose concentrations (Text Table 1 summarizes the analytical results for concentration verification, homogeneity, and stability conducted on the current study). Additional details on dose formulation and analysis including chromatograms are provided in other sections of this dossier and in attachments.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An appropriate amount of test substance (no correction factor was used) along with approximately 70% of the required amount of vehicle were weighed and added to the beaker; the contents were stirred until homogeneous in appearance.
The remaining amount of vehicle was added and the contents were again stirred until homogeneous in appearance. The contents were homogenized using a polytron (if needed); the mix was further homogenized by sonication. After sonication, the formulations were heated between 35-45°C, while stirring. BADGE formulations were stored at 37°C, protected from light, prior to delivering to the animal facility for dosing, or when not in use in the animal facility. Dose formulations were stirred for at least 30 minutes prior to use for dosing as well as during dose administration. This process is standard methodology to ensure resuspension and homogeneity.

Justification for the 3-day dose formulation preparation and use schedule.
BADGE formulations were prepared at least once every 3 days. Concentration verification for each dose level was performed for the first and last dose formulations used in each of the two study phases. Dose formulation homogeneity was evaluated for each dose level for the first dose formulation of each study phase and the high and low dose level for the last dose formulation of the first study phase. The 3-day stability of dose formulations at 37C for the low and high dose levels were demonstrated once during the study. The results are presented in Text Table 1 and support that the methods for dose formulation preparation/maintenance and administration yielded the intended exposure of animals in each dose group to BADGE.
Justification for utilization of prepared dose formulations over the 3-day period is derived from the stability that was demonstrated on this study as well as from the known chemical/physical properties of BADGE and the stability of such dose formulations utilized in several other repeated dose toxicity studies.
As reported in the study report (page 159) and presented herein (Text Table 2) and in the attachment (Supplemental information Regarding Dose Formulation and Analysis for the study titled: In Vivo Mutation Assay with BADGE (ERC#1) at the cII Locus in Big Blue® Transgenic F344 Rats ) the stability of formulations prepared at the high (100 mg/ml) and low (5 mg/ml) dose concentrations were analyzed directly after preparation and then again after a 3-day holding period at 37C. The concentration of BADGE after the 3-day period was essentially unchanged at both concentration levels. The concentration of BADGE in the low dose level was 4.66 mg/ml at T = 0 and 4.71 mg/ml after 3 days. The concentration of BADGE in the high dose level was 121 mg/ml at T = 0 and 120 mg/ml after 3 days. The stability demonstrated by the analysis was further supported by the lack of any emergence of additional peaks in the chromatography as would be expected if BADGE was undergoing hydrolysis during the 3-day maintenance period. Example chromatograms from the evaluation of the high dose level are presented in the attached supplemental document.
Loss of BADGE due to hydrolysis is possible however the extent of such loss in the dose formulations is severely limited by low solubility and modest hydrolysis rate. The water solubility of BADGE has been determined experimentally, 6.9 mg/L at 20⁰C. Though the vehicle for dose formulations include a low concentration of Methocel (0.5%) and Tween 80 (0.1%) the vehicle was principally distilled-deionized water. As shown in the table below the concentration of BADGE in each dose formulation was in far excess of the water solubility (833 to 16,666-fold excess). Under these conditions it is understandable that a suspension of BADGE would result and that the bulk of the test material would have limited contact with the aqueous phase.
The epoxy functionality of BADGE is susceptible to hydrolysis and the half-life of BADGE in water at pH 7 was determined at 30⁰C (73.5 hrs) and 40⁰C (28 hrs). From these data one can estimate that the hydrolysis half-life at 37⁰C, the temperature at which dose formulations were maintained, to be approximately 42 hrs. Thus, BADGE dissolved in the aqueous phase of the dose solutions would be expected to undergo hydrolysis and with a half-life of approximately 42 hrs. However, as hydrolysis would have been limited to BADGE dissolved in the aqueous vehicle, which is limited by the low water solubility, the total mass susceptible to hydrolysis over the 3 day holding period represents only a small fraction of the total mass of BADGE in any of the dose formulations (dissolved (ug/ml) + suspension (mg/ml)). As a conservative estimation, assuming a dose formulation solubility for BADGE of 10 ug/ml, and two half-lives over the 3-day dosing period, the total loss of BADGE to hydrolysis for any of the dosing solutions would be no more than 20 ug/ml. This would amount to a loss of only 0.4% of the total mass of BADGE in the 5 mg/ml dose solution.

The sum total of the evidence that includes the practical demonstration of 3-day dose formulation stability under the actual use conditions within the study provide justification for the dose formulation schedule and use. The use of this 3-day dose formulation schedule as compared with daily dose preparation does not impact on the validity of the study.

Additional Notes:
Results for stability testing at room and refrigerated temperatures were reported in the study report (Study Report Appendix C). These results were included in the report because they were conducted, however, stability at room and refrigerated temperatures were not relevant to the dose formulations used to dose animals on study and were not pursued further. Additionally, the study report misstated the results of the 3-day at 37C stability evaluation. The report will be amended to correctly state that stability assessments for formulations held at room temperature and at cold temperatures did not meet acceptance criteria. The stability assessment for dose formulations held at 37C were stable and met the acceptance criteria. Acceptance criteria for formulation analysis of concentration, stability and homogeneity are defined by the laboratory's SOP. Acceptance criteria are specific to the character of the formulation. Solutions have narrower acceptance limits than suspensions. Acceptance criteria initially stated in the study protocol was set with the anticipation that dose formulations would be solutions. The protocol was amended with acceptance criteria deemed appropriate by the laboratory for suspensions.

BADGE is known to solidify at low ambient temperatures as was apparent during the days before the initial dosing of animals. As such the Sponsor recommended that the bulk test article be warmed to between 40 – 60C to acquire liquified samples for formulation. This realization occurred before preparation of dose formulations used on day 1. The laboratory observed improved ease with formulation preparation and use by incorporating warming to 37C into the process of dose formulation and maintenance.

Duration of treatment / exposure:

28 d (except 1000 mg/kg bw/d dose group: 25 d; positive control: days 1, 2, 3, 12, 19, and 26)

Frequency of treatment:

daily (except positive control: days 1, 2, 3, 12, 19, and 26)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Extended Phase

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Extended Phase

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylnitrosurea
- Route of administration: oral(gavage)
- Doses / concentrations: 20 mg/kg/day

Tissues and cell types examined:

liver, duodenum, and glandular stomach were collected for cII mutant analysis; testes and cauda were also collected but not analyzed for mutants
In addition, for Groups 6-8 (extended cohort), the median lobe of the liver (with the associated mass), approximately one third of the glandular stomach, and one of the three 1-inch sections of the duodenum, were saved in 10% neutral buffered formalin (10% NBF) for possible future staining and microscopic evaluation

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose levels were also selected based on available toxicity data in rats from a 28-day study, where 40 Ralf (SPF) rats, 5 males and 5 females per dose group, were administered BADGE daily by gavage for 28 days at doses of 0, 50, 200, or 1000 mg/kg bw per day. A no observed effect level (NOEL) of 1000 mg/kg bw was identified after 28 days of repeated, once daily oral gavage administration.

DETAILS OF DNA PREPARATION:
Isolated DNA was processed using Packaging Reaction Mix (PRM), purchased from New York University, New York, NY. This product is similar to Transpack manufactured by Agilent, Santa Clara, CA. PRM or Transpack were used to isolate the recoverable lambda shuttle DNA vectors from the genomic DNA and to package the lambda shuttle vector DNA using phage proteins and cofactors to create infectious lambda phage particles. Methods followed BioReliance SOP’s, based on Agilent instruction manual titled “λ Select-cII Mutation Detection System for Big Blue® Rodents” (Agilent, 2015) and Agilent instruction manual titled “Transpack Packaging Extract for Lambda Transgenic Shuttle Vector Recovery” (Agilent, 2009b).

METHOD OF ANALYSIS:
Isolated DNA was processed using Packaging Reaction Mix (PRM), which is used to isolate the recoverable lambda shuttle DNA vectors from the genomic DNA. Phage head, tail, and tail fibers from the packaging mix are then assembled around lambda shuttle vector DNA creating infectious lambda phage particles.
Packaged phage were incubated overnight at 37 ± 1.0°C, and then scored for plaque formation and titer determination; cII mutant selection plates were incubated for two days (nominally, 40-48 hours) at 24 ± 0.5°C, and then scored for mutant plaques. At least 125,000 phage were evaluated from at least 2 packagings for each dose and tissue.

The individual animal is considered the experimental unit. The mutant frequency (MF) was calculated (number of mutant phage / number of total phage screened) for each tissue analyzed from each animal. Since this ratio is extremely small and may not be normally distributed, a log10 transformation of the MF data was performed.
The statistical analysis of MF was conducted as follows: the positive control (Group 5) was independently compared to either of the vehicle controls (Group 1 and Group 6). In the second part of the analysis, test substance-treated groups were compared to their concurrent vehicle controls (i.e., Groups 2-3 vs. Group 1 and Groups 7-8 vs. Group 6). Lastly, the extended phase vehicle control (Group 6) was analyzed against the initial vehicle control (Group1), in order to assess the impact of potential differences in background mutation rates between phases.
In all instances, log10-transformed MF data from the vehicle control and treated groups were evaluated using a One-Way Analysis of Variance (ANOVA). The suitability of using the parametric ANOVA was confirmed by testing parameters of the log10-transformed MF data for normality and equal variance. If the data were normally distributed and exhibit equal variance, the parametric ANOVA analysis would be used; if either test failed, a
non-parametric method would be used.

Evaluation criteria:

Validity criteria:
Vehicle control values: The average mutant frequency of the vehicle controls should be within reasonable limits of the laboratory historical controls and literature values.
Positive control values: the positive control must induce a statistically significant increase in mutant frequency as compared with the concurrent vehicle control (P<0.05 will be considered significant) .

Criteria for a positive response: The test item will be considered to have produced a positive response if it induces a statistically significant increase in the frequency of cll mutants in any dose level outside the 95% control limits of the historical background mutant frequency range. Biological significance will be an important consideration ion the final determination of a positive response.

Criteria for a negative response: A test item will be considered to have produces a negative response if no significant increase in cll mutant frequency is observed.

Criteria for an equivocal response: equivocal responses will be evaluated by the study director on a case-by-case basis considering both statistical significance and biological relevance.

Statistics:

The incidence of all effects was analyzed separately by dose level. Dunnett’s test was conducted on body weight, body weight changes, and organ weight data. All statistics compared treated groups versus their concurrent control (i.e., Groups 2-5 vs. Group 1 and Groups 7-8 vs. Group 6), and were based on a significance value of p < 0.05.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Big Blue® Assay Results

Summary of Mutant Frequency Data

Dose Level (mg/kg/d)	Liver Mean [median] +- SD (x10 -6)	Duodenum  Mean [median] +- SD (x10 -6)	Glandular Stomach  Mean [median] +- SD (x10 -6)
0 (vehicle control, initial phase)	25.1 [26.5] +- 7.7	43.3 [40.0] +- 18.8	24.9 +- 4.5
50	24.7 +- 7.2	40.2 +- 20.1	19.1 +- 3.5
250	24.0 +- 6.3	29.3 +- 9.6	23.4 +- 6.9
20 mg/kg ENU (a)	239.1**###+- 65.2	840.9**[826.0##]+-85.8	512.7**###+-77.6
0 (vehicle control, Extended phase)	36.2 [37.6*]+- 5.2	34.6 [34.2] +-12.4	31.2 +- 9.3
500	25.0#+-3.8	26.4 +-8.9	19.7 +-0.9
1000	34.9 +-10.0	46.5 +- 12.0	28.5 +- 10.9

a = Days 1, 2, 3, 12, 19, and 26 only; SD = Standard Deviation.

* = Statistically significant (Kruskal-Wallis test, p < 0.05) compared to Group 1.

** = Statistically significant (One-way ANOVA, p < 0.001) compared to Group 1.

# = Statistically significantly lower (One-way ANOVA, p < 0.05) compared to Group 6.

## = Statistically significant (Kruskal-Wallis test, p < 0.01) compared to Group 6.

### = Statistically significant One-way ANOVA, p < 0.001) compared to Group 6.

 

 

Mutant frequencies (MF) for liver, duodenum, and glandular stomach in BADGE-treated animals were not statistically elevated over the correspondent controls at any dose level. MF in ENU-treated animals was statistically elevated over both vehicle controls for liver, duodenum, and glandular stomach, demonstrating the responsiveness of the test system to ENU, a directacting mutagen.

 

 

Formulation stability

BADGE formulated in 0.5% Methocel A4M methyl cellulose ethers with 0.1% Tween 80 in DI water, at concentrations of 4.66 and 121 mg/mL, was stable at 37°C for at least 3 days.

 

Mortality

Group 4 (1000 mg/kg/day) animals (initial phase) were terminated on Day 25, as directed by protocol amendment #2; all other animals survived until their scheduled terminal sacrifice on Day 31.

 

Clinical Signs

There were no remarkable clinical observations associated with BADGE treatment at doses up to and including 500 mg/kg/day in either the initial or extended phases.

Text Table 1 shows the comparison of post-dose cage-side observations between the 1000 mg/kg/day groups in the initial phase and the extended phase.

During the initial phase, severe signs of toxicity were noted post-dose (cage-side or unscheduled observations) at 1000 mg/kg/day (Group 4), beginning with Day 6, and these included: decreased motor activity, ruffled fur, hunched posture, squinty eyes, labored breathing, and diarrhea. After Day 9 however, most animals appeared normal. Diarrhea was also noted in two animals during detailed hands-on observations at 1000 mg/kg/day, on Day 8.

During the extended phase, signs of toxicity noted during cage-side or unscheduled observations (at 1000 mg/kg/day) included decreased motor activity, ruffled fur, hunched posture, and squinty eyes, starting with Day 9. All animals appeared normal after Day 12.

Thin appearance was also noted during hands-on observations at 1000 mg/kg/day (Group 8), on Day 8. No labored breathing or diarrhea were noted in the extended phase.

All other observations noted were unremarkable, and likely unrelated to the test substance administration as they also occurred in the control group. Red discharge from the left eye noted in a single animal at detailed hands-on observations on Days 22, 29, and 31, and as an unscheduled observation on Day 27, was considered an accidental injury and thus incidental to BADGE treatment. Overall, the occurrence of the signs of toxicity during cage-side or unscheduled observations seemed reduced in incidence and frequency in Group 8 compared to Group 4 with no diarrhea (evidence of severe toxicity) reported in Group 8 compared to Group 4.

 

Body Weights and Body Weight Gains

There were no statistically significant or otherwise remarkable differences in mean body weight between the concurrent control, and the BADGE-treated groups up to and including 500 mg/kg/day in both the initial and extended phase. In the extended phase, the mean body weight of the 1000 mg/kg/day animals (Group 8) was also not statistically significantly different from the concurrent control.

During the initial phase, toxicologically and statistically significant lower mean body weights were noted for the 1000 mg/kg/day dose group, starting with Day 8 (14% lower than control and a weight loss of 10.1% of the mean Day 1 body weight) and continuing through the last body weight on Day 22 (13% lower than the vehicle control). The severe, acute body weight losses coupled with the clinical observations occurring in the same time frame, were considered in the decision to terminate Group 4 and start the extended phase.

Although there were few statistically significant differences in comparisons of mean body weight of the BADGE-treated groups, there were notable, statistically significant differences in body weight gains that were related to BADGE treatment at doses of 250 mg/kg/day and higher.

In the initial phase, the mean body weight gain of the 250 mg/kg/day group was statistically significantly lower (25% of control) than the concurrent control in the interval from Day 22 to 29, but the overall weight gain (Day 1 to 31), was similar.

During the extended phase, the body weight changes at both 500 and 1000 mg/kg/day tended to be significantly lower with weight loss (2.4% and 5.7%, respectively) relative to the initial body weight, more notably during the first week of treatment. The overall (Day 1-31) mean body weight gains were 42% and 66% lower than control at 500 and 1000 mg/kg/day, respectively, and both of these were statistically significant differences.

A dose of 50 mg/kg/day was a clear no effect level for BADGE-related body weight changes.

For ENU-treated animals, the overall (Day 1-31) mean body weight gains were statistically significantly different, and 25% lower as compared to the vehicle control group.

 

Gross Necropsy Findings

A few gross observations were made in the livers of two control and two 1000 mg/kg/day animals in the extended phase; among these observations, a firm mass was noted in the median lobe of the liver for one 1000 mg/kg/day male. None of the gross necropsy findings were considered treatment-related.

 

Organ Weight Analysis

Organ weights were collected mainly for predicting the number of possible DNA extractions from a tissue, not for toxicity evaluation. Any statistically significant differences between BADGE-treated and control organ weights are considered incidental findings.

Conclusions:

There was no treatment-related mortality and no evidence of an increase in mutant frequency at the cII gene in liver, duodenum, or glandular stomach of F344 Big Blue® male rats after 28 days of once daily oral gavage treatment with BADGE, at doses ranging from 50 mg/kg/day up to the limit dose of 1000 mg/kg/day.

Executive summary:

This study investigated the effect of BADGE (Bisphenol A Diglycidyl Ether; CAS 1675-54-3) on mutant frequency at the cII gene in liver, glandular stomach, and duodenum from male transgenic Fischer 344 (F344) Big Blue® rats. The Big Blue® Assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (OECD, 2013).

The study consisted of 47 transgenic F344 Big Blue® male rats assigned to eight groups, as detailed below. The initial study design used 5 groups of 6 male rats each: one vehicle control (Group 1), 3 BADGE-treated groups (2, 3 and 4) and one positive control (Group 5) (initial phase). Due to signs of excessive toxicity coupled with concerns regarding formulation homogeneity, the high dose (Group 4) animals were terminated early, and a second cohort (extended phase) of 17 male rats was added, drawn from the same breeding group to include: one vehicle control (Group 6, 5 animals),

and two BADGE-treated groups (Groups 7 and 8, 6 animals each) (extended phase).

Groups 1 and 6 received the vehicle (0.5% Methocel A4M methyl cellulose ethers with 0.1% Tween 80 in deionized water). Test substance-treated animals received BADGE, formulated in the vehicle, at the dose levels presented in the table below. Animals in Groups 1-3 and 6-8 were dosed once daily via oral gavage for 28 consecutive days. Animals in Group 4 were dosed once daily by oral gavage for 25 consecutive days, except on Day 8 (as directed by the Study Director). Positive control animals (Group 5) receivedN-ethyl-N-nitrosourea (ENU) in buffer solution, pH 6.00 by oral gavage at 20 mg/kg/day, on Days 1, 2, 3, 12, 19, and 26. All doses were administered based upon body weight at a volume of 10 mL/kg body weight (bw).

The Group 4 animals were terminated by carbon dioxide (CO2) overdose on Day 25, and discarded without necropsy. The remaining animals were terminated by CO2 overdose on Day 31. A partial necropsy was performed for animals in Groups 1-3 and 5-8; the liver, duodenum, glandular stomach, testes, and cauda were collected, weighed, flash frozen, and stored at or below -60°C.

Liver, duodenum, and glandular stomach from the first five surviving animals/group were processed for DNA isolation and analysis of cII mutants, following BioReliance SOPs. Samples of liver, duodenum, and glandular stomach from the extended study were retained in 10% neutral buffered formalin (10% NBF) for possible histopathology.

All animals survived to their scheduled termination. There were no remarkable, BADGE-related clinical observations at dose levels up to and including 500 mg/kg/day. At 1000 mg/kg/day, BADGE-related clinical observations included transient observation of decreased motor activity, ruffled fur, hunched posture, and squinty eyes that began on Day 6 or Day 9 for the initial and extended dosing phases, respectively. Slight to moderate diarrhea and labored breathing were noted on Days 6, 7, and 8 (and once on Day 19) only at 1000 mg/kg/day during the initial phase, but these effects were not observed during the extended phase.

There were no statistically significant or otherwise remarkable differences in mean body weight between the concurrent control, and the BADGE-treated groups up to and including 500 mg/kg/day in both the initial and extended phases. In the extended phase, the mean body weight of the 1000 mg/kg/day animals was also not statistically significantly different from the concurrent control. Although there were several statistically significant differences of mean body weights and/or body weight gains (bwg) for the BADGE-treated group that was terminated early on Day 25 (Group 4, 1000 mg/kg/day), there were also a few intervals of statistically significantly lower absolute bwg compared to the concurrent control, that were related to BADGE treatment, among the other groups: a single interval (Days 22-29) at 250 mg/kg/day in the initial phase, and several intervals, including Days 1-31 for both 500 and 1000 mg/kg/day, in the extended phase. The bwg data also included single intervals for each dose level with significantly higher bwg values compared to controls.

Repeated treatment with BADGE, up to a limit dose of 1000 mg/kg/day did not result in elevated mutant frequencies (MF) at thecIIgene in liver, duodenum, or glandular stomach of F344 Big Blue® male rats. The lack of mutation induction in these portals of entry and systemic tissues obviated the need to analyze the testes or cauda for mutations. The treatment with ENU produced statistically significant increases in MF for all tissues evaluated, demonstrating the utility of the test system to detect and quantify induced mutants following exposure to a known direct-acting mutagen. The study design and results obtained met protocol-specified assay acceptance criteria and were consistent with the study requirements of OECD TG 488 for transgenic rodent mutation assays.

In conclusion, there was no treatment-related mortality and no evidence of an increase in mutant frequency at the cII gene in liver, duodenum, or glandular stomach of F344 Big Blue® male rats after 28 days of once daily oral gavage treatment with BADGE, at doses ranging from 50 mg/kg/day up to the limit dose of 1000 mg/kg/day.

 

Justification for classification or non-classification

No classification is neccessary based on negative results in Transgenic Rodent testing on an similar substance.