Reaction products of disubstituted alkane, nickel, (Z/E)-substituted alkene, reaction mass of tetraalkyl-3,3’-di(propan-2-yl)-biphenyl-diyl tetrakis(dialkylphenyl) bis(phosphite) and tris(dialkylphenyl) phosphite and zinc halide

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:

From 25 Apr 2018 to 08 Aug 2018

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:

July 21, 1997

Deviations:

yes

Remarks:

No solubility test was performed. DMSO was selected as solvent based on the solubility findings in Charles River Laboratories Study No. 519589. This deviation is of no influence on the study results

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 31, 2008

Deviations:

yes

Remarks:

No solubility test was performed. DMSO was selected as solvent based on the solubility findings in Charles River Laboratories Study No. 519589. This deviation is of no influence on the study results

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Lot: 6420
Expiration date: 07/18/2019
Store under nitrogen

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: E. coli WP2 uvrA: The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate.
main study, direct plate assay: 52, 164, 512, 1600, 5000 µg/plate.
main study, pre-incubation assay: 17, 52, 164, 512, 1600, 5000 µg/plate.
maximum test concentration for soluble non-cytotoxic substances according to guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA1535), ICR-191(TA1537), 2-nitrofluorene (TA1537, TA98), Methylmethanesulfonate (TA100), 4-nitroquinoline N-oxide (WP2 uvrA)

Remarks:

without metabolic activtion

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar with and without preincubation

DURATION
- Preincubation period: none or 30 +/- 2 min at 37 °C
- Exposure duration: 48 +/- 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 +/- 4 h

NUMBER OF REPLICATIONS: triplicates for each dose level

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies are evaluated

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP 2 uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.


A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP 2 uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535,TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not performed

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Individual plate counts; (following pages)

LIST OF ABBREVIATIONS

n	Normal bacterial background lawn
a	Bacterial background lawn absent
NP	No precipitate
MP	Moderate precipitate

 

Dose range finding
Strain TA100

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	926		951		963		947	±	19
solvent control	93		128		99		107	±	19
1.7	122		82		118		107	±	22
5.4	103		102		101		102	±	1
17	125		95		113		111	±	15
52	110		93		105		103	±	9
164	127		127		128		127	±	1
512	98		91		103		97	±	6
1600	107		122		102		110	±	10
5000	173	n NP	166	n NP	155	n NP	165	±	9

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	1144		1277		1190		1204	±	68
solvent control	102		116		113		110	±	7
1.7	95		93		114		101	±	12
5.4	87		94		105		95	±	9
17	76		97		105		93	±	15
52	102		125		112		113	±	12
164	117		120		118		118	±	2
512	144		148		133		142	±	8
1600	125		146		150		140	±	13
5000	152	n NP	147	n NP	166	n NP	155	±	10

 

Dose range finding
Strain WP₂uvrA

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	1560		1284		1072		1305	±	245
solvent control	39		33		24		32	±	8
1.7	39		35		29		34	±	5
5.4	34		42		30		35	±	6
17	22		18		34		25	±	8
52	41		31		38		37	±	5
164	26		45		37		36	±	10
512	35		20		24		26	±	8
1600	30		24		31		28	±	4
5000	33	n NP	24	n NP	39	n NP	32	±	8

 

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	429		426		381		412	±	27
solvent control	45		27		31		34	±	9
1.7	44		27		23		31	±	11
5.4	41		35		29		35	±	6
17	44		39		42		42	±	3
52	41		41		38		40	±	2
164	38		38		38		38	±	0
512	46		58		44		49	±	8
1600	39		42		29		37	±	7
5000	50	n NP	45	n NP	54	n NP	50	±	5

 

 

Experiment 1

Strain TA1535

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	767		811		784		787	±	22
solvent control	7		8		11		9	±	2
52	12		10		7		10	±	3
164	10		3		5		6	±	4
512	14		11		15		13	±	2
1600	4		12		11		9	±	4
5000	12	n NP	14	n NP	19	n NP	15	±	4

 

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	267		291		294		284	±	15
solvent control	10		15		12		12	±	3
52	12		11		10		11	±	1
164	7		8		19		11	±	7
512	3		14		5		7	±	6
1600	16		25		14		18	±	6
5000	26	n NP	18	n NP	7	n NP	17	±	10

 

Experiment 1

Strain TA1537

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	854		883		909		882	±	28
solvent control	11		11		15		12	±	2
52	5		7		4		5	±	2
164	8		14		7		10	±	4
512	11		11		8		10	±	2
1600	12	n	3	n	3	n	6	±	5
5000^1	0	a NP	0	a NP	0	a NP	0	±	0

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	438		408		463		436	±	28
solvent control	18		12		8		13	±	5
52	4		7		5		5	±	2
164	10		3		4		6	±	4
512	18		10		5		11	±	7
1600	19	n	16	n	19	n	18	±	2
5000^1	0	a NP	0	a NP	0	a NP	0	±	0

1            No bacteria plated, not reported

 

Experiment 1A (repeat experiment, since no bacteria plated in the highest concentration of Experiment 1)

Strain TA1537

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	332		325		412		356	±	48
solvent control	0	n	3		0	n	1	±	2
5000	4	n MP	2	n MP	2	n MP	3	±	1

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	781		903		882		855	±	65
solvent control	3		4		1		3	±	2
5000	4	n MP	3	n MP	2	n MP	3	±	1

 

 

Experiment 1

Strain TA98

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	966		891		956		938	±	41
solvent control	30		23		22		25	±	4
52	15		16		22		18	±	4
164	31		14		10		18	±	11
512	14		14		10		13	±	2
1600	14		10		18		14	±	4
5000	18	n NP	16	n NP	12	n NP	15	±	3

 

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	997		914		899		937	±	53
solvent control	37		20		19		25	±	10
52	27		30		19		25	±	6
164	15		27		24		22	±	6
512	8		29		30		22	±	12
1600	15		8		20		14	±	6
5000	23	n NP	33	n NP	26	n NP	27	±	5

 

 

 

Experiment 2

Strain TA1535

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	1175		1303		1257		1245	±	65
solvent control	12		7		12		10	±	3
17	8		7		12		9	±	3
52	12		11		10		11	±	1
164	5		10		8		8	±	3
512	7		8		10		8	±	2
1600	10		4		7		7	±	3
5000	7	n NP	11	n NP	10	n NP	9	±	2

 

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	307		252		306		288	±	31
solvent control	5		15		8		9	±	5
17	8		7		10		8	±	2
52	20		7		14		14	±	7
164	5		15		14		11	±	6
512	12		10		12		11	±	1
1600	7		8		12		9	±	3
5000	14	n NP	15	n NP	24	n NP	18	±	6

 

Experiment 2

Strain TA1537

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	158		151		141		150	±	9
solvent control	7		4		3		5	±	2
17	3		1		5		3	±	2
52	16		10		5		10	±	6
164	1		3		5		3	±	2
512	10		8		4		7	±	3
1600	1		7		3		4	±	3
5000	3	n NP	4	n NP	5	n NP	4	±	1

 

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	241		246		245		244	±	3
solvent control	5		10		8		8	±	3
17	1		4		3		3	±	2
52	11		8		8		9	±	2
164	4		5		4		4	±	1
512	3		10		4		6	±	4
1600	8		8		4		7	±	2
5000	2	n NP	7	n NP	5	n NP	5	±	3

 

Experiment 2

Strain TA98

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	1598		1054		1445		1366	±	281
solvent control	18		10		12		13	±	4
17	20		7		10		12	±	7
52	14		14		14		14	±	0
164	10		23		12		15	±	7
512	10		14		12		12	±	2
1600	18		15		19		17	±	2
5000	18	n NP	10	n NP	18	n NP	15	±	5

 

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	577		623		726		642	±	76
solvent control	18		15		7		13	±	6
17	20		18		18		19	±	1
52	27		20		19		22	±	4
164	14		18		22		18	±	4
512	23		24		27		25	±	2
1600	30		15		18		21	±	8
5000	15	n NP	24	n NP	20	n NP	20	±	5

 

Experiment 2

Strain TA100

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	909		880		880		890	±	17
solvent control	103		106		110		106	±	4
17	99		107		121		109	±	11
52	122		107		102		110	±	10
164	97		114		102		104	±	9
512	117		146		102		122	±	22
1600	112		128		122		121	±	8
5000	132	n NP	122	n NP	128	n NP	127	±	5

 

 

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	1864		1963		2075		1967	±	106
solvent control	110		103		101		105	±	5
17	94		132		103		110	±	20
52	101		98		99		99	±	2
164	106		107		95		103	±	7
512	113		107		106		109	±	4
1600	105		140		139		128	±	20
5000	152	n NP	143	n NP	151	n NP	149	±	5

 

Experiment 2

Strain WP₂uvrA

 

WITHOUT S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	392		310		412		371	±	54
solvent control	29		30		24		28	±	3
17	37		26		34		32	±	6
52	26		48		24		33	±	13
164	37		41		39		39	±	2
512	27		29		33		30	±	3
1600	38		35		30		34	±	4
5000	31	n NP	34	n NP	45	n NP	37	±	7

WITH S9-MIX
plate	1		2		3		MEAN		SD
dose (µg/plate)
positive control	559		566		560		562	±	4
solvent control	37		38		34		36	±	2
17	49		58		30		46	±	14
52	39		41		45		42	±	3
164	46		39		45		43	±	4
512	31		52		29		37	±	13
1600	41		56		56		51	±	9
5000	49	n NP	39	n NP	37	n NP	42	±	6

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test material is not mutagenic in the bacterial reverse mutation assay with Salmonella typhimurium and Escherichia coli strains.

Executive summary:

A bacterial mutagenicity test (according to guideline OECD 471) in Salmonella typhimurium strains TA 1537, TA 1535, TA100, TA98 as well as Escherichia coli strain WP2 uvrA was performed with the test material in concentrations up to 5000 µg/plate with and without metabolic activation.

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. 

The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The negative (vehicle) control values were within the laboratory historical control data ranges, except the response for TA1537 in the absence of S9-mix, in the repeat of the first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (1 revertant colony) when compared against relevant historical control data (3 revertant colonies), the validity of the test was considered to be not affected.

In conclusion, the test item Step 2 Catalyst revealed no mutagenic activity under the conditions of this bacterial reverse mutation assay with Salmonella typhimurium and Escherichia coli strains.