4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with trimethylhexane-1,6-diamine

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:

22 August 2017 to 17 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with trimethylhexane-1,6-diamine

EC Number:

500-332-0

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with trimethylhexane-1,6-diamine

Cas Number:

153195-44-9

Molecular formula:

(C15 H16 O2 . C9 H22 N2 . C3 H5 Cl O)x

IUPAC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with trimethylhexane-1,6-diamine

Test material form:

liquid

Method

Target gene:

his

Species / strain

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:

rat liver S9

Test concentrations with justification for top dose:

plate incorporation assay: 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate (dose range based on preliminary assay to determine toxicity)

pre-incubation assay:
Salmonella strains TA100, TA1535 and TA1537 (without S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate.
All Salmonella strains (with S9) and Escherichia coli strain WP2uvrA and Salmonella strain TA98 (without S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Escherichia coli strain WP2uvrA (with S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

no

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1: in agar (plate incorporation); experiment 2: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times, or three times in the case of strains TA1535 and TA1537 which have relatively low spontaneous reversion rates, the concurrent solvent control for any tester strain, especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Experiment 1 – Without Metabolic Activation

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	88 98 96	(94) 5.3	25 20 18	(21) 3.6	22 21 20	(21) 1.0	14 23 22	(20) 4.9	10 6 11	(9) 2.6
	0.15 µg	85 77 95	(86) 9.0	18 23 19	(20) 2.6	N/T		N/T		11 12 13	(12) 1.0
	0.5 µg	88 75 90	(84) 8.1	26 16 17	(20) 5.5	N/T		N/T		9 13 10	(11) 2.1
	1.5 µg	89 70 89	(83) 11.0	18 18 13	(16) 2.9	20 18 31	(23) 7.0	17 24 27	(23) 5.1	10 13 13	(12) 1.7
	5 µg	70 77 78	(75) 4.4	15 22 23	(20) 4.4	24 21 19	(21) 2.5	19 16 21	(19) 2.5	11 14 6	(10) 4.0
	15 µg	64 65 48	(59) 9.5	14 25 24	(21) 6.1	21 22 23	(22) 1.0	20 14 21	(18) 3.8	6 9 8	(8) 1.5
	50 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	30 20 14	(21) 8.1	9 10 26	(15) 9.5	0 V 0 V 0 V	(0) 0.0
	150 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	500 µg	N/T		N/T		0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	N/T
	1500 µg	N/T		N/T		0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	N/T
	5000 µg	N/T		N/T		0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	N/T
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		391 456 437	(428) 33.4	451 459 499	(470) 25.7	802 900 968	(890) 83.5	187 208 210	(202) 12.7	186 241 260	(229) 38.4

N/T         Not tested at this dose level

T         Toxic, no bacterial background lawn

V         Very weak bacterial background lawn

Experiment 1 – With Metabolic Activation

S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100 †		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	65 65 86	(72) 12.1	19 29 29	(26) 5.8	26 36 35	(32) 5.5	17 19 27	(21) 5.3	17 9 9	(12) 4.6
	0.15 µg	73 78 71	(74) 3.6	N/T		N/T		N/T		N/T
	0.5 µg	66 59 74	(66) 7.5	N/T		N/T		N/T		N/T
	1.5 µg	66 68 67	(67) 1.0	25 28 16	(23) 6.2	27 22 36	(28) 7.1	26 16 22	(21) 5.0	9 6 14	(10) 4.0
	5 µg	69 66 62	(66) 3.5	23 27 25	(25) 2.0	23 36 30	(30) 6.5	26 22 23	(24) 2.1	10 21 8	(13) 7.0
	15 µg	59 66 62	(62) 3.5	22 27 31	(27) 4.5	32 32 35	(33) 1.7	21 16 29	(22) 6.6	8 16 7	(10) 4.9
	50 µg	33 43 38	(38) 5.0	17 11 11	(13) 3.5	24 29 20	(24) 4.5	28 30 21	(26) 4.7	9 6 18	(11) 6.2
	150 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	13 20 14	(16) 3.8	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	500 µg	N/T		0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	1500 µg	N/T		0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	5000 µg	N/T		0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1109 1439 1247	(1265) 165.7	271 272 280	(274) 4.9	198 239 249	(229) 27.0	180 195 184	(186) 7.8	429 433 558	(473) 73.4

Experiment 2 – Without Metabolic Activation

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	107 86 78	(90) 15.0	19 19 20	(19) 0.6	19 16 26	(20) 5.1	26 22 36	(28) 7.2	13 14 16	(14) 1.5
	0.015 µg	102 78 99	(93) 13.1	24 21 19	(21) 2.5	N/T		N/T		21 15 12	(16) 4.6
	0.05 µg	101 83 91	(92) 9.0	11 25 18	(18) 7.0	17 17 16	(17) 0.6	25 29 28	(27) 2.1	12 17 15	(15) 2.5
	0.15 µg	96 92 101	(96) 4.5	11 23 14	(16) 6.2	19 23 22	(21) 2.1	30 39 34	(34) 4.5	15 16 15	(15) 0.6
	0.5 µg	71 72 97	(80) 14.7	12 17 21	(17) 4.5	19 17 32	(23) 8.1	26 29 29	(28) 1.7	14 11 13	(13) 1.5
	1.5 µg	84 81 77	(81) 3.5	16 13 21	(17) 4.0	23 17 18	(19) 3.2	27 32 20	(26) 6.0	13 18 17	(16) 2.6
	5 µg	33 S 48 S 28 S	(36) 10.4	11 S 5 S 6 S	(7) 3.2	34 17 14	(22) 10.8	16 22 16	(18) 3.5	13 S 16 S 15 S	(15) 1.5
	15 µg	52 S 49 S 51 S	(51) 1.5	0 S 2 S 1 S	(1) 1.0	18 S 15 S 8 S	(14) 5.1	21 S 11 S 11 S	(14) 5.8	14 S 12 S 12 S	(13) 1.2
	50 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	12 S 10 S 11 S	(11) 1.0	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0
	150 µg	N/T		N/T		0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	N/T
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		1559 1632 1454	(1548) 89.5	1684 1514 1647	(1615) 89.4	842 871 837	(850) 18.4	295 303 318	(305) 11.7	643 478 683	(601) 108.7

 

Experiment 2 – With Metabolic Activation

S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	80 79 87	(82) 4.4	28 33 21	(27) 6.0	36 38 23	(32) 8.1	35 28 36	(33) 4.4	18 19 13	(17) 3.2
	0.05 µg	98 102 98	(99) 2.3	26 21 23	(23) 2.5	N/T		26 27 33	(29) 3.8	14 15 15	(15) 0.6
	0.15 µg	98 105 77	(93) 14.6	29 26 23	(26) 3.0	22 30 34	(29) 6.1	34 26 26	(29) 4.6	19 8 14	(14) 5.5
	0.5 µg	97 88 86	(90) 5.9	15 36 23	(25) 10.6	31 27 28	(29) 2.1	32 27 30	(30) 2.5	13 14 8	(12) 3.2
	1.5 µg	93 103 85	(94) 9.0	22 31 24	(26) 4.7	27 42 24	(31) 9.6	30 30 34	(31) 2.3	14 15 14	(14) 0.6
	5 µg	98 104 97	(100) 3.8	28 26 21	(25) 3.6	28 28 34	(30) 3.5	32 29 29	(30) 1.7	9 10 12	(10) 1.5
	15 µg	96 86 100	(94) 7.2	19 17 14	(17) 2.5	25 17 21	(21) 4.0	27 28 27	(27) 0.6	13 18 9	(13) 4.5
	50 µg	48 S 17 S 20 S	(28) 17.1	7 S 9 S 10 S	(9) 1.5	22 29 27	(26) 3.6	14 S 18 S 19 S	(17) 2.6	8 S 5 S 7 S	(7) 1.5
	150 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	14 S 14 S 6 S	(11) 4.6	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	500 µg	N/T		N/T		0 V 0 V 0 V	(0) 0.0	N/T		N/T
Positive controls S9-Mix (+)	Name Dose Level No. of Reverants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		2371 2194 2233	(2266) 93.0	290 293 312	(298) 11.9	265 401 425	(364) 86.3	249 245 216	(237) 18.0	490 469 438	(466) 26.2

 

Applicant's summary and conclusion

Conclusions:

Badge-TMD adduct was considered to be non mutagenic under the conditions of this test.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2 uvrA were treated with Badge-TMD adduct using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was initially 1.5 to 5000 mg/plate. However, the test item induced excessive toxicity to three of the Salmonella strains (TA100 (with and without S9) and TA1535 and TA1537 (without S9)) and consequently part of the experiment was repeated using an amended dose range of 0.15 to 150 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of Experiment 1 (and repeat), and ranged between 0.015 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. Eight test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the toxic limit of the test item following the change in test methodology.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate or the toxic limit,depending on bacterial strain type and presence or absence of S9-mix. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 µg/plate in the absence of S9-mix and 150 µg/plate in the presence of S9-mix. The toxic limit of the test item was employed as the maximum dose in Experiment 2. The test item again induced a toxic response in the second mutation test after employing the pre-incubation method with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 µg/plate and from 50 µg/plate in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. 

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). 

Badge-TMD adduct was considered to be non mutagenic under the conditions of this test.