Reaction mass of 3-phenyl-6-[(3-phenyl-3,4-dihydro-2H-1,3-benzoxazin-8-yl)methyl]-3,4-dihydro-2H-1,3-benzoxazine and 6,6'-methanediylbis(3-phenyl-3,4-dihydro-2H-1,3-benzoxazine)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: 19 april 2017 and Study Completion Date: 05 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Update and adopted 21 July 1997.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-methylenebis[3,4-dihydro-3-phenyl-2H-1,3-benzoxazine

EC Number:

604-043-1

Cas Number:

137836-80-7

Molecular formula:

C29 H26 N2 O2

IUPAC Name:

6,6'-methylenebis[3,4-dihydro-3-phenyl-2H-1,3-benzoxazine

Test material form:

solid: compact

Method

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. 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.

Species / strain

Species / strain / cell type:

other: TA98, TA100, TA1535, TA 1537 and Escherichia coli WP2 uvrA

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Not applicable

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:

- 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 par plate.
As no toxicity was observed, Precipitate was observed beginning at 333 µg per plate without S9 activation and beginning at 667 µg per plate with 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 50.0/ 150/ 500/ 1500 and 5000 µg per plate.

Vehicle / solvent:

DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 400 mg/mL with sonication at 33.7ºC for 5 minutes in the solubility test conducted at BioReliance.
CAS N°: 67-68-5
Lot: SHBB9319V
Purity: 99.95%

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Strain TA98 and TA1535.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in dmso

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Strain TA100 and TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Strain WP2 uvrA

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO

Positive control substance:

2-nitrofluorene

Remarks:

Strain TA98.

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in sterile water

Positive control substance:

sodium azide

Remarks:

Strain TA100 and TA1535.

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO

Positive control substance:

9-aminoacridine

Remarks:

Strain TA1537

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Dissolved in DMSO

Positive control substance:

methylmethanesulfonate

Remarks:

Strain WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

Number Cells Analyzed/Culture: 1.1 to 2.4 x10E8 cells per plate.

NUMBER OF REPLICATIONS:
- In the preliminary toxicity assay: Single plate per condition.
- In the mutagenicity assay: Triplicate

Preparation of Tester Strain
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.

Rationale for test conditions:

The following criteria must be met for the mutagenicity assay to be considered valid:

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.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.

To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.

The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.

A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).

Evaluation criteria:

Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated 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 a biologically relevant 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. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

not applicable.

Results and discussion

Test results

Key result

Species / strain:

other: Strain TA98, TA100, TA1535, TA1537 and E. Coli WP2 uvrA

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Precipitate was observed beginning at 500 µg per plate with all conditions

Vehicle controls validity:

valid

Untreated negative controls validity:

other: Solvent control valid

Positive controls validity:

valid

Additional information on results:

Please see"Any other information on results incl. tables.

Remarks on result:

other: No mutagenic

Any other information on results incl. tables

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Tester Strain Titer Results

	Tester Strain
	TA98	TA100	TA1535	TA1537	WP2 uvrA
Experiment	Titer Value (x 10^9cells per mL)
B1	1.8	1.1	1.5	2.0	2.4

Preliminary Toxicity Assay

The maximum dose of 5000 µg per plate was achieved using a concentration of 50.0 mg/mL and a 100 µL plating aliquot. No toxicity was observed. Precipitate was observed beginning at 333 µg per plate without S9 activation and beginning at 667 µg per plate with S9 activation.

Mutagenicity Assay

Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 50.0, 150, 500, 1500 and 5000 µg per plate. 

No toxicity was observed. Precipitate was observed beginning at 500 µg per plate with all conditions. 

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

HISTORICAL CONTROL DATA:

Historical Negative and Positive Control Values 2015 Revertants per plate
		Activation
		None					Rat Liver
Strain	Control	Mean	SD	Min	Max	95% CL	Mean	SD	Min	Max	95% CL
TA98 (2015)	Neg	16	5	6	43	6 -26	23	7	5	53	9 -37
TA98 (2015)	Pos	190	191	42	2468		329	176	51	1786
TA100 (2015)	Neg	90	12	62	233	66 -114	98	15	63	157	68 -128
TA100 (2015)	Pos	697	172	239	1767		671	284	138	2692
TA1535 (2015)	Neg	13	5	2	35	3 -23	13	5	3	33	3 -23
TA1535 (2015)	Pos	624	196	50	2509		137	110	24	1060
TA1537 (2015)	Neg	7	3	1	20	1 -13	9	3	2	23	3 -15
TA1537 (2015)	Pos	392	292	24	2887		73	53	19	574
WP2 uvrA (2015)	Neg	25 8	7	73	9 -41	28	28	8	10	96	12 -44
WP2 uvrA (2015)	Pos	336	112	89	1026		352	117	78	1409
SD=standard deviation; Min=minimum value; Max=maximum value; 95% CL = Mean ±2 SD (but not less than zero); Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control  Metabolic activation  Test substance  Dose Level (μg/plate) Revertant Colony Counts (Mean +/- SD)               Without metabolic Activation      TA98 TA100  TA1535  TA1537  WP2 uvrA     DMSO  100.0  12 +/- 3 79 +/- 9  10 +/- 4  7 +/- 2  26 +/- 5      Test item 50  12 +/- 3 80 +/-4  8 +/- 3  8 +/- 2  24 +/- 1      Test item  150  12 +/- 1 89+/-9 11 +/- 2  6 +/-1   20 +/- 1     Test item  500p  11 +/-2 75 +/-12  12 +/-3  7 +/-2  20 +/-2      Test item  1500p  9 +/- 1 71 +/-6  9 +/- 2 7 +/-3  17 +/-2      Test item  5000 p 9 +/-4  65 +/-8  8 +/-1 5 +/-2  14 +/-5    2NF  1.00  105 +/-31            SA  1.00   723+/-57 698 +/-26         9AAD  75.0        913 +/-126      MMS  1000          323 +/-22 Key to positive controls: SA: sodium azide 2AA: 2-aminoanthracene 9AAD: 9-Aminoacridine 2NF: 2-nitrofluorene MMS: methyl methanesulfonate p: Precipitate

Metabolic activation  Test substance  Dose Level (μg/plate) Revertant Colony Counts (Mean +/- SD)               Without metabolic Activation      TA98 TA100  TA1535  TA1537  WP2 uvrA     DMSO  100.0  19 +/-2 98 +/-7 10 +/- 2  7 +/- 1  27 +/- 2      Test item 50  22 +/-4 87 +/-12  12 +/-4  8 +/- 1  22 +/- 4      Test item  150  22 +/- 1 85 +/-12 9 +/-1  9 +/-2   27 +/- 1     Test item  500p  19 +/-1 115 +/-2  10 +/-1  9 +/-1 27 +/-5     Test item  1500p  16 +/-2 90 +/-5  9 +/- 2 8 +/-3  25 +/-1      Test item  5000 p 16 +/-1  91 +/-11 8 +/-1 7 +/-1  21 +/-4    2AA  1.00  264 +/-13            2AA  2.00   894 +/-51 74 +/-16      2AA  15.0          437 +/-47 Key to positive controls: SA: sodium azide 2AA: 2-aminoanthracene 9AAD: 9-Aminoacridine 2NF: 2-nitrofluorene MMS: methyl methanesulfonate p: Precipitate

Applicant's summary and conclusion

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, Formaldehyde, reaction products with benzenamine and 4,4'-methylenebis[phenol] did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Executive summary:

The test substance, Formaldehyde, reaction products with benzenamine and 4,4'-methylenebis[phenol], was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimuriumand at the tryptophan locus ofEscherichia colistrain 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. No toxicity was observed. Precipitate was observed beginning at 333 µg per plate without S9 activation and beginning at 667 µg per plate with 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 50.0, 150, 500, 1500 and 5000 µg per plate. No toxicity was observed. Precipitate was observed beginning at 500 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate Formaldehyde, reaction products with benzenamine and 4,4'-methylenebis[phenol] was negative for the ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.