4,4'-isopropylidenedi-2,6-xylol

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:

April - May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH S2B

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-isopropylidenedi-2,6-xylol

EC Number:

227-033-5

EC Name:

4,4'-isopropylidenedi-2,6-xylol

Cas Number:

5613-46-7

Molecular formula:

C19H24O2

IUPAC Name:

4,4'-isopropylidenedi-2,6-xylol

Test material form:

solid: particulate/powder

Details on test material:

- Purity: 99.74%
- Synonym: 4,4’-(1-methylethylidene)-bis(2,6-dimethylphenol)

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate with and w/out S9 activation (Preliminary toxicity and initial mutagenicity assay) – Plate incorporation method

2.0, 6.0, 20, 60, 200 and 600 micrograms per plate with all Salmonella tester strains and
60, 200, 600, 1800 and 5000 micrograms per plate with tester strain WP2 uvrA. (Independent repeat/confirmatory mutagenicity assay) – Plate incorporation method. Due to toxicity profiles that differed from that observed in the initial assay, tester strain TA100 in the absence of S9 activation and tester strain TA1537 in the presence of S9 activation were retested with an adjustment in dose levels: 0.15, 0.50, 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 micrograms per plate.

Vehicle / solvent:

DMSO (CAS No. 67-68-5); from EMD Chemicals Incorporated

Controls

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:

Preliminary Toxicity/Initial Mutagenicity Assay: The preliminary toxicity/initial mutagenicity assay was used to establish the dose range over which the test article would be assayed and to provide a preliminary mutagenicity evaluation. A vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor induced rat liver S9.

Confirmatory Mutagenicity Assay: The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Six to 10 dose levels of test article along with vehicle control and appropriate positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA in the presence and absence of Aroclor induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures: The test system was exposed to the test article via the plate incorporation method. On the day of its use, minimal top agar was melted and supplemented. Top agar, not used with S9 or Sham mix, was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was supplemented Vogel Bonner minimal medium E.

Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation.

Test article dilutions were prepared immediately before use. One half (0.5) milliliter of S9 or Sham mix, 100 microL of tester strain and 50 microL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45+/-2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 microL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37+/-2°C. Plates that were not counted immediately following the incubation period were stored at 2 8°C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation was scored relative to the vehicle control plate.

Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

Evaluation criteria:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and reported.
For the test article 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 article. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 150, 500 or 5000 microg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 150, 500 or 5000 microg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 60, 200, 500, 600, or 1500 microg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Generally observed beginning at 60, 200, 500, 600, or 1500 microg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the initial mutagenicity assay no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 or 1500 microg/plate. Toxicity was observed beginning at 150, 500 or at 5000 microg/plate with all test conditions except tester strain WP2 uvrA in the absence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 600 microg/plate with all Salmonella tester strains and 5000 microg/plate with tester strain WP2 uvrA.

For the Confirmatory Mutagenicity Assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 600 microg/plate. Toxicity was observed beginning at 60, 200 or at 600 microg/plate with all Salmonella tester strains except TA1537 in the presence of activation. Due to toxicity profiles that differed from that observed in the initial assay, tester strains TA100 and TA1537 in the absence or presence of S9 activation, respectively, were retested with adjustments to the dose levels. In experiment B3, no positive mutagenic responses were observed in either TA100 or TA1537 in the absence or presence of S9 activation, respectively. Precipitate was observed beginning at 500 microg/plate and toxicity was observed beginning at 500 or 1500 microg/plate.

Remarks on result:

other: Initial Mutagenicity Assay

Any other information on results incl. tables

Initial Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation: None

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DSMO)	13 ± 4	98 ± 6	6 ± 1	5 ± 1	22 ± 2
1.5	10 ± 2	93 ± 5	11 ± 1	7 ± 1	19 ± 5
5.0	11 ± 4	91 ± 13	7 ± 1	5 ± 1	21± 0
15	8 ± 1	95 ± 8	7 ± 3	6 ± 1	17 ± 6
50	9 ± 2	74 ±4	8 ± 4	4 ± 1	17 ± 3
150	0 ± 0	0 ± 0	6 ± 1	4 ± 1	15 ± 3
500	0 ± 0 P	0 ± 0 P	0 ± 0 P	4 ± 1 P	21 ± 12
1500	0 ± 0 P	0 ± 0 P	0 ± 0 P	3 ± 0 P	12 ± 0 P
5000	0 ± 0 P	0 ± 0 P	0 ± 0 P	2 ± 1 P	19 ± 1 P
Positive Control	199 ± 33	511 ± 6	456 ± 28	365 ± 58	385 ± 8

P = precipitate observed

 

Initial Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation: S9

 

Dose (microg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
Vehicle (DSMO)	13 ± 1	104 ± 8	9 ± 2	10 ± 1	38 ± 8
1.5	14 ± 9	99 ± 12	10 ± 2	8 ± 3	40 ± 1
5.0	12 ± 7	86 ± 9	8 ± 3	5 ± 1	33 ± 8
15	13 ± 2	72 ± 5	11 ± 2	5 ± 1	30 ± 6
50	15 ± 5	77 ± 16	7 ± 1	4 ± 1	28 ± 0
150	12 ± 1	91 ± 8	9 ± 0	4 ± 1	28 ± 1
500	0 ± 0 P	0 ± 0 P	0 ± 0 P	5 ± 3 P	27 ± 0 P
1500	0 ± 0 P	0 ± 0 P	0 ± 0 P	5 ± 4 P	26 ± 8 P
5000	0 ± 0 P	0 ± 0 P	0 ± 0 P	3 ± 0 P	11 ± 0 P
Positive Control	237 ± 18	590 ± 55	83 ± 14	47 ± 5	189 ± 13

P = precipitate observed

Confirmatory Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation: None

 

Dose (microg/plate)	TA98	TA100	TA1535	TA1537
Vehicle (DSMO)	23 ± 9	139 ± 11	11 ± 3	5 ± 5
2	20 ± 6	65 ± 8	13 ± 1	6 ± 3
6	24 ± 5	79 ± 7	10 ± 5	2 ± 2
20	24 ± 4	87 ± 15	11 ± 3	4 ± 1
60	18 ± 3	62 ± 13	11 ± 3	4 ± 3
200	10 ± 3	72 ± 9	11 ± 5	7 ± 3
600	0 ± 0 P	0 ± 0 P	0 ± 0 P	4 ± 1 P
Positive Control	305 ± 40	473 ± 34	629 ± 26	738 ± 53

P = precipitate observed

 

Dose (microg/plate)	WP2uvrA
Vehicle (DSMO)	14 ± 5
60	10 ± 3
200	11 ± 2
600	12 ± 1 P
1800	13 ± 3 P
5000	9 ± 2 P
Positive Control	295 ± 22

P = precipitate observed

 

Repeat Confirmatory Mutagenicity Assay for TA100

Mean Number of Revertants Per Plate

Activation: None

 

Dose (microg/plate)	TA100
Vehicle (DSMO)	83 ± 3
0.15	79 ± 1
0.50	86 ± 10
1.5	85 ± 2
5.0	86 ± 9
15	83 ± 1
50	82 ± 4
150	82 ± 5
500	55 ± 5 P
1500	51 ± 9 P
5000	55 ± 3 P
Positive Control	573 ± 9

P = precipitate observed

Confirmatory Mutagenicity Assay

Mean Number of Revertants Per Plate

Activation: S9

 

Dose (microg/plate)	TA98	TA100	TA1535	TA1537
Vehicle (DSMO)	32 ± 9	137 ± 14	10 ± 2	7 ± 2
2	26 ± 8	121 ± 14	9 ± 5	6 ± 3
6	28 ± 5	121 ± 18	11 ± 2	5 ± 1
20	30 ± 3	132 ± 12	11 ± 2	7 ± 4
60	25 ± 2	126 ± 4	10 ± 5	7 ± 3
200	25 ± 6	134 ± 16	12 ± 5	5 ± 3
600	0 ± 0 P	0 ± 0 P	12 ± 3 P	6 ± 2 P
Positive Control	577 ± 40	671 ± 159	94 ± 14	51 ± 14

P = precipitate observed

  

Dose (microg/plate)	WP2uvrA
Vehicle (DSMO)	20 ± 3
60	19 ± 2
200	19 ± 1
600	16 ± 4 P
1800	16 ± 5 P
5000	12 ± 1 P
Positive Control	188 ± 10

P = precipitate observed

 

Repeat Confirmatory Mutagenicity Assay for TA1537

Mean Number of Revertants Per Plate

Activation: S9

 

Dose (microg/plate)	TA1537
Vehicle (DSMO)	11 ± 1
0.15	9 ± 3
0.50	9 ± 3
1.5	8 ± 3
5.0	9 ± 4
15	9 ± 4
50	7 ± 2
150	8 ± 2
500	7 ± 4 P
1500	7 ± 3 P
5000	4 ± 0 P
Positive Control	76 ± 13

P = precipitate observed

Applicant's summary and conclusion

Conclusions:

negative with metabolic activation
negative without metabolic activation

All criteria for a valid study were met. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, TMBPA did not cause a positive response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.