Reaction products of Soya and Linseed Oil Fatty Acids with Bisphenol A diglycidylether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Soya/Linseed Oil Fatty Acid-BADGE reaction product was positive in in vitro assays but negative in in vivo assays (read-across from BADGE)

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:

14 August 2018 to 20 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD Test Guideline 471

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guideline 471, updated and adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: F288I1K351
- Expiration date of the lot/batch: 20 Jan 2020
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:Room temperature, protected from light
- Stability under test conditions: While the Certificate of Analysis indicates an expiry date (20 Jan 2020), it does not indicate the acceptable storage parameters for the test substance. Thus, the stability of the test
substance has not been determined to cover the period of shipment and storage at BioReliance
- Solubility and stability of the test substance in the solvent/vehicle: The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL with sonication at 31.7ºC for 5 minutes
in the solubility test conducted at BioReliance. Soya/Linseed Oil Fatty Acid-BADGE reaction product in DMSO, at concentrations of 106.4 and 0.320 mg/mL, was stable at room temperature for at least 3.6 hours
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: To achieve a solution, the most concentrated dilution was vortexed for 4 to 5 minutes in each assay.

Target gene:

The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria
require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. Thesestrains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision
repair system.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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 dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. The top dose was per the guideline Based upon preliminary toxicity assay results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate. In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO for the test substance and all positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in sterile water
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells

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:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION: - Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 3 in the mutagenicity assay
NUMBER OF CELLS EVALUATED: >/= 0.3 x 10^8 cells/plate
DETERMINATION OF CYTOTOXICITY
- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.

Evaluation criteria:

The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter). The mean and standard deviation of the number of revertants per plate were calculated
and reported.
For each replicate plating, the mean and standard deviation of the number of revertants per plate were ca lculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is an increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.

Statistics:

According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Mutagenicity Assay without S9 activation, Exposure Method: Plate incorporation assay

 

Strain	Substance	Dose level μg per plate	Mean revertants per plate	Standard Deviation	Ratio treated / solvent
TA98	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	15 13 16 11 9 11   12	2 5 3 2 2 5   4	1.3 1.1 1.3 0.9 0.8 0.9   ---
TA100	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	927 761 657 548 296 167   87	51 12 63 25 6 17   7	10.7 8.7 7.6 6.3 3.4 1.9
TA1535	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	46 29 34 26 19 16   12	1 5 7 4 4 3   5	3.8 2.4 2.8 2.2 1.6 1.3
TA1537	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	12 8 7 8 8 6   8	4 0 3 2 2 1   6	1.5 1.0 0.9 1.0 1.0 0.8
WP2uvrA	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	69 55 60 43 33 23   25	15 11 10 2 6 1   10	2.8 2.2 2.4 1.7 1.3 0.9
TA98	2NF	1.00	72	19	6.0
TA100	SA	1.00	765	49	8.8
TA1535	SA	1.00	721	59	60.1
TA1537	9AAD	75	546	133	68.3
WP2uvrA	MMS	1000	606	139	24.2

 

 

 

Mutagenicity Assay with S9 activation

Plate incorporation assay

 

Strain	Substance	Dose level μg per plate	Mean revertants per plate	Standard Deviation	Ratio treated / solvent
TA98	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	27 27 23 23 26 24   21	3 5 4 4 5 6   8	1.3 1.3 1.1 1.1 1.2 1.1
TA100	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	978 580 357 152 126 104   95	226 68 34 17 12 6   3	10.3 6.1 3.8 1.6 1.3 1.1
TA1535	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	416 458 222 50 16 18   16	77 30 11 5 4 5   5	26.0 28.6 13.9 3.1 1.0 1.1
TA1537	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	11 13 10 9 10 7   11	4 0 0 2 1 2   3	1.0 1.2 0.9 0.8 0.9 0.6
WP2uvrA	Soya/Linseed Oil Fatty Acid-BADGE reaction product         DMSO	5000 1500 500 150 50 15   ---	80 41 46 39 33 38   27	28 1 13 10 12 8   9	3.0 1.5 1.7 1.4 1.2 1.4
TA98	2AA	1	219	13	10.4
TA100	2AA	2	721	111	7.6
TA1535	2AA	1	88	4	5.5
TA1537	2AA	2	43	6	3.9
WP2uvrA	2AA	15	302	46	11.2

 

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, Soya/Linseed Oil Fatty Acid-BADGE reaction
product did cause a positive mutagenic response with tester strains TA100, TA1535 and WP2 uvrA in the presence and absence of S9 activation. The study was concluded to be positive without conducting
a confirmatory (independent repeat) assay because the results were clearly positive; hence, no further testing was warranted

Executive summary:

The test substance, Soya/Linseed Oil Fatty Acid-BADGE reaction product, 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 preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Precipitate was observed beginning at 1000 μg per plate with all conditions. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation. Mutagenic responses were observed with tester strains TA100, 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 mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions. Positive mutagenic responses were observed (2.2- to 28.6-fold, maximum increases, outside the historical control limits) with tester strains TA100, TA1535 and WP2 uvrA in the presence and absence of S9 activation.

These results indicate Soya/Linseed Oil Fatty Acid-BADGE reaction product was positive for the ability to induce reverse mutations at selected loci of selected strains of Salmonella typhimurium (TA100 and

TA1535) 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 / micronucleus study

Remarks:

OECD 487

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2018 - 29 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 26 September 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Lot No.: F288I1K351
Purity: 100 %
Solubility Determination
DMSO was the vehicle of choice based on the information provided by the Sponsor, the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, upon sonication for five minutes at 31.7 ºC, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL.
Test Article Formulation Preparation
The formulations were prepared by weighing out the test article and adding DMSO to prepare the high concentration. From the high concentration serial dilutions were performed with DMSO to prepare the lower concentrations.

Species / strain / cell type:

primary culture, other:

Remarks:

Human Peripheral Blood Lymphocytes

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking individual (Males, 25 - 27 yrs of age). The donors had no recent history of radiotherapy, viral infection or the administration of drugs. This system has been demonstrated to be sensitive to the genotoxicity test for detection of micronuclei of a variety of chemicals (Clare et al., 2006).

Cytokinesis block (if used):

Cytochalasin B (cytoB) was dissolved in DMSO to a stock concentration of 2 mg/mL. It was used at 6 μg/mL concentration to block cytokinesis.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254-induced rat liver S9
- method of preparation of S9 mix ; The S9 mix was prepared on the day of use as indicated below:
Component Final Concentration in Culture Medium*
NADP (sodium salt) 1 mM
Glucose-6-phosphate 1 mM
Potassium chloride 6 mM
Magnesium chloride 2 mM
S9 homogenate 20 μL/mL
* RPMI 1640 Serum-free medium supplemented with 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

Based on the results of the preliminary toxicity test, the doses selected for testing in the micronucleus assay were as follows:
Treatment Condition Treatment Time Recovery Time Doses (μg/mL)
Non-activated 4 hr 20 hr 5, 15, 30, 40, 45, 50, 55, 60, 100
Non-activated 24 hr 0 hr 5, 20, 40, 45, 50, 55, 60, 70, 75, 100
S9-activated 4 hr 20 hr 50, 100, 200, 250, 300, 350, 400, 450, 500

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;

- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the information provided by the Sponsor, the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, upon sonication for five minutes at 31.7 ºC, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

vinblastine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : one

METHOD OF TREATMENT/ EXPOSURE:
Treatment was carried out by refeeding the cultures as follows:
Treatment Culture medium* (mL) Volume of S9 mix (mL) Volume of control / test substance dosing solution (μL)
Vehicle: Non-activated 5 - 50
Vehicle: S9-activated 4 1 50
Test Substance: Non-activated 5 - 50
Test Substance: S9-activated 4 1 50
Positive Control: Non-activated 5 - 50
Positive Control: S9-activated 4 1 50
* Complete medium for non-activated test system and serum-free medium for S9-activated test system

After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytoB at 6.0 μg/mL and returned to the incubator under standard conditions. For the 24 hour treatment in the non-activated study, cytoB (6.0 μg/mL) was added at the beginning of the treatment.


TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
4 hr non-activated
24 hr non-activated
4 hr activated
- Harvest time after the end of treatment (sampling/recovery times):
Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986). The cyto B exposure time for the 4 hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes).
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.


- Cytochalasin B (cytoB) was dissolved in DMSO to a stock concentration of 2 mg/mL. It was used at 6 μg/mL concentration to block cytokinesis..
- Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986). The cyto B exposure time for the 4 hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes).
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange and identified by the BioReliance study number, treatment condition, dose level, test phase, harvest date, activation system, and replicate tube design.

- For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control.

- The slides from at least three test substance treatment groups were coded using random numbers by an individual not involved with the scoring process and scored for the presence of micronuclei based on cytotoxicity. A minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei. Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
• the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus



METHODS FOR MEASUREMENT OF CYTOTOXICITY
- For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control.
% Cytostasis (cytotoxicity) = 100 -100 {(CBPIt-1) /(CBPIc-1)}
t = test substance

Rationale for test conditions:

Standard valiated method.

Evaluation criteria:

Vehicle Controls
The frequency of cells with micronuclei should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are not extreme outliers (indicative of experimental or human error).
Positive Controls
The percentage of micronucleated cells must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).
Cell Proliferation
The CBPI of the vehicle control at harvest must be ≥ 1.4.
Test Conditions
The test substance must be tested using a 4-hour treatment with and without S9, as well as a 24-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.
Analyzable Concentrations
At least 2000 binucleated cells from at least three appropriate test substance concentrations.

Evaluation of Test Results
The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were 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 percentage of micronucleated 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:

lymphocytes: Human Peripheral Blood Lymphocytes

Remarks:

24hr treatment

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Statistically significant increase in micronuclei induction

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

39 - 60% at evaluated exposure conc

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: Human Peripheral Blood Lymphocytes

Remarks:

4 hr

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10 - 55% in evaluated exposure levels

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

1.3% Micronuclei inducaion

Species / strain:

lymphocytes: Human Peripheral Blood Lymphocytes

Remarks:

4 hr

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0 - 59% in examined exposure levels

Vehicle controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- The osmolality of the test substance doses in treatment medium was considered acceptable. The pH of the highest dose of test substance in treatment medium was 7.5.
-Visible precipitate was observed in treatment medium at the following doses: ≥ 500 μg/mL at the begining and conclusion of the treatment periods for all treatment times (4hr, 24 hr)
-Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 50 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 500 μg/mL in the S9-activated 4-hour exposure group.

Micronucleus Assay:

Precipitate:

Treatment Condition              Treatment Time                                                 Visible precipitate

At the beginning of Treatment period       At the conclusion of Treatment period

Non-activated                            4 hr                                   100 μg/mL                                          100 μg/mL

24 hr                                   ≥ 75 μg/mL                                        100 μg/mL

S9-activated                              4 hr                                   ≥ 100 μg/mL                                       ≥ 450 μg/mL

pH of the highest dose : 7.5

Cytotoxicity:

Treatment Condition              Treatment Time       Highest Evaluated Dose (μg/mL)       Cytotoxicity(%)

Non-activated                            4 hr                            40                                                 59

24 hr                            50                                                 60

S9 -activated                               4 hr                            200                                                 55

Definitive Assay: 4 hr Treatment w/o S9, 24hr harvest

Treatment (μg/mL)	Replicate Culture	Total # of cells counted	Percentage of Micronucleated Binucleated Cells per culture	Average Percent Micronucleated Binucleated Cells per Dose
DMSO	A	1000	0.20%	0.25%
	B	1000	0.30%
Soya/Linseed Oil Fatty Acid-BADGE reaction product
5	A	1000	0.20%	0.20%
	B	1000	0.20%
15	A	1000	0.50%	0.50%
	B	1000	0.50%
40	A	1000	0.50%	0.6%*
	B	1000	0.70%
*The Cochran-Armitage test was positive for a dose response (p ≤ 0.05) and the induction of micronuclei was slightly outside the historical 95% control limit of 0.06% to 0.59%

Definitive Assay: 4 hr Treatment + S9, 24hr harvest

Treatment (μg/mL)	Replicate Culture	Total # of cells counted	Percentage of Micronucleated Binucleated Cells per culture	Average Percent Micronucleated Binucleated Cells per Dose
DMSO	A	1000	0.10%	0.30%
	B	1000	0.50%
Soya/Linseed Oil Fatty Acid-BADGE reaction product
50	A	1000	0.30%	0.25%
	B	1000	0.20%
100	A	1000	0.40%	0.35%
	B	1000	0.30%
200	A	1000	0.30%	0.35%
	B	1000	0.40%
CP, 5	A	1000	1.40%	1.30%**
	B	1000	1.20%
** p ≤ 0.01, Fisher's exact test, relative to the solvent control.

Definitive Assay: 24 hr Treatment w/o S9, 24hr harvest

Treatment (μg/mL)	Replicate Culture	Total # of cells counted	Percentage of Micronucleated Binucleated Cells per culture	Average Percent Micronucleated Binucleated Cells per Dose
DMSO	A	1000	0.20%	0.25%
	B	1000	0.30%
Soya/Linseed Oil Fatty Acid-BADGE reaction product
5	A	1000	0.20%	0.20%
	B	1000	0.20%
40	A	1000	1.50%	1.6%**#
	B	1000	1.70%
40	A	1000	1.20%	1.45%**#
	B	1000	1.70%
VB, 7.5ng/ml	A	1000	0.70%	0.75%*
	B	1000	0.80%
* p ≤ 0.05; ** p ≤ 0.01, Fisher's exact test, relative to the solvent control. #The Cochran-Armitage test was positive for a dose response (p ≤ 0.05).

Conclusions:

Under the conditions of the assay described in this report, Soya/Linseed Oil Fatty Acid-BADGE reaction product was concluded to be equivocal for the induction of micronuclei in the non-activated 4-hour exposure group and positive for the induction of micronuclei in the non-activated 24-hour exposure group. Soya/Linseed Oil Fatty Acid-BADGE reaction product was concluded to be negative for the induction of micronuclei in the S9-activated test system.

Executive summary:

The test substance, Soya/Linseed Oil Fatty Acid-BADGE reaction product, was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 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 the test article being a UVCB. Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 50 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 500 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 500 μg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 100 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 50 to 500 μg/mL for the S9-activated 4-hour exposure group.

In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at doses ≥ 30 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 200 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 45 μg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at 100 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 450 μg/mL in the S9-activated 4-hour exposure group.

The doses selected for evaluation of micronuclei were 5, 15, and 40 μg/mL for the non-activated 4-hour exposure group; 50, 100, and 200 μg/mL for the S9-activated 4-hour exposure group; and 5, 40, and 50 μg/mL for the non-activated 24-hour exposure group.

In the non-activated 4-hour exposure group, no significant increase in micronuclei induction was observed at any dose (p ≥ 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was positive for a dose response (p ≤ 0.05). In addition, the induction of micronuclei at 40 μg/mL (0.60%) was slightly outside the historical 95% control limit of 0.06% to 0.59%.

In the S9-activated 4-hour exposure group, no significant or dose-dependent increases in micronuclei induction were observed at any dose (p ≥ 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the non-activated 24-hour exposure group, statistically significant and dose-dependent increases in micronuclei induction (1.60% and 1.45%) were observed at doses 40 and 50 μg/mL, respectively (p ≤ 0.01; Fisher’s Exact and Cochran-Armitage tests). In addition, the induction of micronuclei at 40 and 50 μg/mL was outside the historical 95% control limit of 0.06% to 0.60%.

These results indicate Soya/Linseed Oil Fatty Acid-BADGE reaction product was equivocal for the induction of micronuclei in the non-activated 4-hour exposure group, and positive for the induction of micronuclei in the non-activated 24-hour exposure group. Soya/Linseed Oil Fatty Acid-BADGE reaction product was negative for the induction of micronuclei in the presence of the exogenous metabolic activation system.

Reference 3

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
This read-across hypothesis corresponds to scenario 2 of the Read-Across Assessment Framework (RAAF), ECHA, March 2017 - different compounds have qualitatively similar properties - of the read-across assessment framework i.e. properties of the target substance are predicted to be quantitatively equal to those of the source substance. Namely, the source substance BADGE predicts the toxicological and ecotoxicological properties of the target substance Soya/Linseed Oil Fatty Acid-BADGE reaction product.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
for details see Justification for read-across attached to iuclid section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
for details see Justification for read-across attached to iuclid section 13

3. ANALOGUE APPROACH JUSTIFICATION
for details see Justification for read-across attached to iuclid section 13

4. DATA MATRIX
for details see Justification for read-across attached to iuclid section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

mouse lymphoma assay using L5178Y cell line as described by Clive et al., 1979.

Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.M. (1979). Validation and characterization of the L5178Y/TK+/- mouse lymphoma mutagen assay system. Mutat. Res 59:61-108

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase gene (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

The S-9 fraction was obtained from the livers of male Sprague-Dawley rats that were dosed with 500 mg/kg body weight of Aroclor 1254 and killed five days later.

Test concentrations with justification for top dose:

0.0018-0.013 mg/mL nonactivated; 0.032-2.4 mg/L activated

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Ethylmethane sulfonate without S9 and Benzo(a)pyrene with S9

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

Cells were treated with various test material concentrations, a negative control (vehicle) and a positive control (Ethylmethane sulfonate) concurrently for toxicity and mutagenicity.

Cells were counted on test day 2 for colony density. Cultures were selected for cloning for treated and both control groups, plated, and cultured for 10 days to determine viability. Mutant counts were recorded, and tutant frequency was calculated.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was noted in suspension growth at 0.18 mg/ml and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

toxicity was observed on suspension growth at 0.003 mg/ml and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Chemical analyses were performed on samples of EPON 828 used to dose the cells. The nominal concentration of both solutions was 100 mg/ml and the actual amount, as determined by analysis, was 104 +/- 1 mg/ml for Assay II (without S9) and 103 +/- mg/ml for Assay III (with S9). No anal;ysis was done on samples from Assay I as this was used as a preliminary assay to determine toxicity and establish dose levels for the mutagenicity assays (II and III).

In the absence of S-9 and EPON 828, there was a toxic effect on both suspension growth and soft agar growth and an increase in the mutant frequency at concentrations of 0.003 mg/ml and higher. The assay was repeated (Assay II) with 0.0075 mg/ml as the highest concentration and an additional five concentrations in decreasing 1/8 log10 dilutions. The results were similar to those obtained in the preliminary assay. There was evidence of a toxic effect on suspension growth and on soft agar growth commencing at 0.0024 mg/ml and a dose-related increase in mutants at all concentrations in the absense of S-9.

When EPON 828 was exposed to the cells in the presence of metabolic activation, no toxicity was noted on suspension growth or soft agar growth in Assay I at concentrations up to 0.1 mg/ml and there was no increase in the mutant frequency at any concentrations. Therefore in the repeat assay (III) a higher concentration, 0.24 mg/ml, was used and the other doses were set at 1/8 log 10 dilutions therefrom. Toxicity was apparent in suspension growth at 0.18 mg/ml and higher and in soft agar growth at 0.24 mg/ml. No increase in mutant frequency which exceeded 2-fold the background was noted at any concentration tested in the presence of S-9 metabolic activation.

Conclusions:

Interpretation of results: positive
BADGE was a direct acting mutagen in the mouse lymphoma gene mutation assay and such activity was eliminated by the addition of a metabolizing enzyme fraction derived from rat liver.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Soya/Linseed Oil Fatty Acid-BADGE reaction product was positive in in vitro assays but negative in in vivo assays (read-across from BADGE)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
This read-across hypothesis corresponds to scenario 2 of the Read-Across Assessment Framework (RAAF), ECHA, March 2017 - different compounds have qualitatively similar properties - of the read-across assessment framework i.e. properties of the target substance are predicted to be quantitatively equal to those of the source substance. Namely, the source substance BADGE predicts the toxicological and ecotoxicological properties of the target substance Soya/Linseed Oil Fatty Acid-BADGE reaction product.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
for details see Justification for read-across attached to iuclid section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
for details see Justification for read-across attached to iuclid section 13

3. ANALOGUE APPROACH JUSTIFICATION
for details see Justification for read-across attached to iuclid section 13

4. DATA MATRIX
for details see Justification for read-across attached to iuclid section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

Dominant lethal study in which male mice were orally gavaged with test material and subsequently mated with untreated female mice over a period of six weeks. The number of females pregnant and number of offspring in each litter was determined.

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

other: Tif: MAG f (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

Animals were 3-4 months of age at the time of test, were fed a standard rodent diet and water ad libitum, and were kept in environmentally-adequate housing facilities.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol (PEG 400)

Details on exposure:

The test material was administered orally in single doses to 20 male albino mice per group, which were then mated to untreated females from the same strain over a period of 6 weeks. At the end of each week, the 2 females per male were replaced by new ones, repeated for 6 weeks to cover the stages of the maturation of the germ cell from the A-spermatogonia to the mature spermatozoon. Doses of 3333 mg/kg and 10,000 mg/kg were given in polyethylene glycol (PEG 400). A control group was given only the vehicle.

Duration of treatment / exposure:

One dose. Test material was dissolved in polyethylene glycol 400.

Frequency of treatment:

once

Post exposure period:

Each male mouse was allowed to mate with two untreated females beginning six hours after receiving a single oral dose of test material. Each group of two untreated females remained with the treated male mouse for a week. After one week the females were removed and replace by another group of two untreated females.

Remarks:

Doses / Concentrations:
Doses of 3333 mg/kg and 10,000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

20 male mice/group

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Females were necropsied on the 14 day of gestation. The number of live embryos and embryonic deaths were listed. In addition, the uteri were placed in a solution of ammonium sulphide in order to detect sites of early embryonic resorptions.

Statistics:

To compare the total number of implantations indicating pre-implantation loss, the t-test or Mann-Whitney's U-test was used. The total numbers of mated and pregnant dams or embryonic deaths were compared with the aid of the X2-test or Fisher's exact test.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

In-life observations included diarrhea in males of the high dose group two days after treatment.

Vehicle controls validity:

valid

Additional information on results:

In-life observations included diarrhea in males of the high dose group two days after treatment. There were no adverse effects on females associated with any of the groups.

The data on mating ratio, numbers of implantations, and embryonic deaths are comparable for all groups.

The data on mating ratio, on the numbers of implantations and embryonic deaths were comparable for all groups.

The diarrhea observed two days after dosing male mice was most likely the result of the vehicle rather than the test material.

Conclusions:

Interpretation of results: negative The test material was negative in the dominant lethal assay.

Reference 2

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
This read-across hypothesis corresponds to scenario 2 of the Read-Across Assessment Framework (RAAF), ECHA, March 2017 - different compounds have qualitatively similar properties - of the read-across assessment framework i.e. properties of the target substance are predicted to be quantitatively equal to those of the source substance. Namely, the source substance BADGE predicts the toxicological and ecotoxicological properties of the target substance Soya/Linseed Oil Fatty Acid-BADGE reaction product.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
for details see Justification for read-across attached to iuclid section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
for details see Justification for read-across attached to iuclid section 13

3. ANALOGUE APPROACH JUSTIFICATION
for details see Justification for read-across attached to iuclid section 13

4. DATA MATRIX
for details see Justification for read-across attached to iuclid section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

Type of assay:

transgenic rodent mutagenicity assay

Species:

rat

Strain:

other: F344 Big Blue

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. Homogeneity evaluations were performed by analyzing two samples each from the top, middle, and bottom levels of the dose formulation. Thus, there were 6 samples in total, 2 from each location (top, middle, bottom). The Relative Standard Deviation was calculated for each level as well as for the average of all 3 levels.

The range, as indicated in the Text Table 1 (below), represents the highest and lowest average value seen among the top, middle, and bottom levels. There was a wider than anticipated degree of variability between sampling levels for the high-dose group (1000 mg/kg) in Phase 1. Homogeneity at this level improved in Phase 2 as demonstrated by the results of the homogeneity analysis in that phase. The High-dose group from Phase 1 was not used in the assessment of mutagenicity.


Stability testing was conducted on the first and last dose formulations prepared in “Phase 1” and then once (first dose formulation) during Phase 2. Unfortunately, the first stability test in Phase 1 was inadvertently performed at room temperature and thus not relevant to stability of the dose formulations which were maintained at 37C. The second stability test was conducted at 37C however the results were not reported in the study report because the T=0 homogeneity criteria were not met. We have added the T=3-day results from the second stability analysis to Text Table 1 (below) for completeness. The stability test performed in Phase 2 was also done at 37C and demonstrated stability over the dose range.

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.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A bacterial reverse mutation assay as well as an in vitro micronucleus test are available for the target substance Soya/Linseed Oil Fatty Acid-BADGE reaction product. Supporting data are available for the source substances BADGE and docosanoic acid. A mouse lymphoma cell mutagenicity assay is not available for the target substance, but for the source substance BADGE. Since Soya/Linseed Oil Fatty Acid-BADGE reaction product and BADGE were positive for mutagenicity in vitro, additional in vivo data on the source substance BADGE are presented. A justification for read-across is attached to iuclid section 13.

 

In vitro tests

Bacterial Reverse Mutation Assay

The test substance, Soya/Linseed Oil Fatty Acid-BADGE reaction product, 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 preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. Precipitate was observed beginning at 1000 μg per plate with all conditions. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation. Mutagenic responses were observed with tester strains TA100, 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 mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions. Positive mutagenic responses were observed (2.2- to 28.6-fold, maximum increases, outside the historical control limits) with tester strains TA100, TA1535 and WP2 uvrA in the presence and absence of S9 activation.

These results indicate Soya/Linseed Oil Fatty Acid-BADGE reaction product was positive for the ability to induce reverse mutations at selected loci of selected strains of Salmonella typhimurium (TA100 and TA1535) and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

 

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay indicate that the source substance BADGE did not cause a positive increase in the mean number of revenants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9). Hence, BADGE-2HCL was considered to be non mutagenic in this assay.

Other bacterial reverse mutation assays indicate that the compound does not induce frameshift mutations, but has the ability to induce base pair substitutions in tester strains TA100 and TA1535 in the presence and absence of metabolic activation.

 

Docosanoic acid did not induce gene mutations in the S. typhimurium and E. coli strains (S. typhimurium TA100, TA1535, TA98, TA1537, E. coli WP2 uvrA). No toxicity was observed up to a concentration of 5000 μg/plate, with or without metabolic activation.

 

in vitro cytogenicity / micronucleus test

The test substance, Soya/Linseed Oil Fatty Acid-BADGE reaction product, was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 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 the test article being a UVCB. Cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses ≥ 50 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 500 μg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 500 μg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 100 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 50 to 500 μg/mL for the S9-activated 4-hour exposure group.

In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was observed at doses ≥ 30 μg/mL in the non-activated 4-hour exposure group; at doses ≥ 200 μg/mL in the S9-activated 4-hour exposure group; and at doses ≥ 45 μg/mL in the non-activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at 100 μg/mL in the non-activated 4 and 24-hour exposure groups, and at doses ≥ 450 μg/mL in the S9-activated 4-hour exposure group.

The doses selected for evaluation of micronuclei were 5, 15, and 40 μg/mL for the non-activated 4-hour exposure group; 50, 100, and 200 μg/mL for the S9-activated 4-hour exposure group; and 5, 40, and 50 μg/mL for the non-activated 24-hour exposure group.

In the non-activated 4-hour exposure group, no significant increase in micronuclei induction was observed at any dose (p ≥ 0.05; Fisher’s Exact test). However, the Cochran-Armitage test was positive for a dose response (p ≤ 0.05). In addition, the induction of micronuclei at 40 μg/mL (0.60%) was slightly outside the historical 95% control limit of 0.06% to 0.59%.

In the S9-activated 4-hour exposure group, no significant or dose-dependent increases in micronuclei induction were observed at any dose (p ≥ 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the non-activated 24-hour exposure group, statistically significant and dose-dependent increases in micronuclei induction (1.60% and 1.45%) were observed at doses 40 and 50 μg/mL, respectively (p ≤ 0.01; Fisher’s Exact and Cochran-Armitage tests). In addition, the induction of micronuclei at 40 and 50 μg/mL was outside the historical 95% control limit of 0.06% to 0.60%.

These results indicate Soya/Linseed Oil Fatty Acid-BADGE reaction product was equivocal for the induction of micronuclei in the non-activated 4-hour exposure group, and positive for the induction of micronuclei in the non-activated 24-hour exposure group.

 

BADGE induced chromosome damage in vitro: BADGE is reported to be very stable in the absence of metabolizing enzymes (S9 mix) but is rapidly hydrolyzed to a diglycol (BADGE*2H2O) by S9 mix. BADGE but not BADGE*2H2O, induced micronuclei and gene mutations at the HPRT locus in cultured Chinese hamster V79 cells. The induced micronuclei consisted of acentric chromosomal fragments and did not contain whole chromosomes/chromatids, as was shown by staining with CREST antikinetochore antibodies. BADGE exhibits clastogenic and mutagenic potential, which is lost after hydrolysis of the epoxide rings and converted to aneuploidogenic potential after cleavage to bisphenol A.

 

Docosanoic acid did not induce structural chromosomal aberrations in the absence or presence of an exogenous metabolic activation system.

 

in vitro gene mutation study in mammalian cells

BADGE was tested in the mouse lymphoma cell mutagenicity assay for its ability to induce gene mutation at the thymidine kinase locus in the absence and presence of activation by a rat liver microsome (S-9) fraction. Cells were exposed to concentrations of the test item up to and including those which resulted in toxicity to the cells.

In the absence of activation, the test item in DMSO produced a dose-related cytotoxic effect at concentrations of 0.0024 mg/ml and higher and a dose-related increase in the mutant frequency at concentrations from 0.0018 to 0.0056 mg/ml. The positive control (EMS, 0.8 mg/ml) produced a mutant frequency approximately 20 times that of the untreated control. In the presence of activation EPON 828 produced cytotoxic effects at concentrations of 0.18 mg/ml and higher; however, there was no increase in mutant frequency at any concentration tested. The positive control (P[a]P, 0.005 mg/ml) produced mutant frequency values greater than twofold that of the untreated control.

It can be concluded that BADGE is a direct acting mutagen in the mouse lymphoma gene mutation assay and that this activity is removed by the addition of a rat liver metabolizing enzyme fraction.

 

In vivo tests

Transgenic rodent mutagenicity assay

This study investigated the effect of BADGE on mutant frequency at the cII gene in liver, glandular stomach, and duodenum from male transgenic Fischer 344 (F344) Big Blue® rats in accordance with OECD Test Guideline 488.

The initial study design used 5 groups of 6 male rats each: vehicle control, 50, 250, and 1000 mg/kg bw/d, positive control (20 mg/kg bw/d ENU). Due to signs of excessive toxicity coupled with concerns regarding formulation homogeneity, the high dose 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 (5 animals), and two BADGE-treated groups (500, and 1000 mg/kg bw/d, respectively) (extended phase).

Animals were dosed once daily via oral gavage for 28 consecutive days, positive control animals (Group 5) received N-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. Liver, duodenum, and glandular stomach from the first five surviving animals/group were processed for DNA isolation and analysis of cII mutants.

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 the cII gene 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.

 

In vivo micronucleus assay

The objective of this in vivo assay was to evaluate the ability of BADGE to induce micronuclei in bone marrow polychromatic erythrocytes of ICR mice. The test article was suspended in corn oil and dosed by oral gavage at 500, 2500 and 5000 mg/kg as specified by the sponsor. The animals were dosed with the test article and were killed 24, 48 and 72 hours after dosing for extraction of the bone marrow. Ten animals (five males and five females) were randomly assigned to each dose/kill time group. Negative and positive control groups killed 24 hours after dosing were included in the assay. BADGE did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.

 

Chinese hamsters were orally gavaged on two consecutive days with 0, 825, 1650 and 3300 BADGE/kg in 20 ml/kg polyethylene glycol 400. Cyclophosphamide, 128 mg/kg in 20 ml/kg PEG400, was used as the positive control. 24 hours after the last gavage, the animals were sacrificed and bone marrow was harvested from the shafts of both femurs. Cells from the bone marrow were fixed on slides and 1000 bone marrow cells each were scored per animal and the following anomalies were registered: a) single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronuclei in leucopoietic cells, e) polyploid cells.

The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo. Mutagenic effects present themselves in interphase cells in form of nucleus anomalies of bone marrow cells. These anomalies occur in interphase cells as a consequence of damage during the mitotic process. The increase in anomalies shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations.

The bone marrow smears from animals treated with various doses of the test item showed no significant differences from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group.

By contrast, a positive control experiment with cyclophosphamide (128 mg/kg) yielded 7.73% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (PEG 400) alone.

It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with BADGE.

 

Conclusion

Overall, BADGE was positive in a number of in vitro assays but negative in in vivo assays. Based on read-across, the negative outcome of the in vivo studies is also applicable to the target substance Soya/Linseed Oil Fatty Acid-BADGE reaction product.

Justification for classification or non-classification

The available data demonstrate, that although Soya/Linseed Oil Fatty Acid-BADGE reaction product appears to be genotoxic in in vitro test systems, in vivo it is not clastogenic or genotoxic. As such, there is no requirement for classification, nor is there a need to perform further studies to assess potential Germ cell mutagenicity.