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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Step 2 catalyst revealed no mutagenic activity in a bacterial mutagenicity test (according to guideline OECD testing guideline no. 471) in Salmonella typhimurium strains TA 1537, TA 1535, TA100, TA98 as well as Escherichia coli strain WP2 uvrA tested up to concentrations up to 5000 µg/plate with and without metabolic activation.

Step 2 Catalyst is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this study according to OECD testing guideline no. 487.

Step 2 Catalyst Mixture (Stripped) is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of this study performed according to OECD testing guideline no. 490.

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:

From 25 Apr 2018 to 08 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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

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)

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

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

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2 Mar 2018 to 22 Jul 2018

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:

as adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: peripheral human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: peripheral human lymphocytes
- Suitability of cells: according to guideline

For lymphocytes:
- Sex, age and number of blood donors: healthy, non smking volunteers, aged 23 to 35 years, one donor per assay
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: per assay, lymphocytes from one donor was used
- Mitogen used for lymphocytes: phytohaemagglutinin (Per culture 0.1 mL (9 mg/mL), received from Remel Europe Ltd., Dartford, United Kingdom)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 53 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 - 37.2°C).

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

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:
- at the 3 hours exposure: 0, 9.8, 19.5, 39.1, 78.1, 156.3 and 312.5 µg Step 2 Catalyst Mixture (Stripped)/mL culture medium with and without S9-mix
- at the 24 hours exposure: 0, 19.5, 39.1, 78.1, 156.3, 312.5 and 625 µg Step 2 Catalyst Mixture (Stripped)/mL culture medium without S9-mix
1st cytogenetic assay:
- 3 hours exposure: 0, 20, 160 and 320 µg Step 2 Catalyst Mixture (Stripped)/mL culture medium with and without S9-mix
2nd cytogenetic assay:
- 24 hours exposure: 0, 20, 80, 100, 120, 140 and 160 µg Step 2 Catalyst Mixture (Stripped)/mL culture medium without S9-mix

The highest tested concentration was determined by the solubility of Step 2 Catalyst Mixture (Stripped) in the culture medium.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: suitable for the test item and the test material

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: colchicine (migrated information)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium;

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Dose range finding:
- 3 hours exposure with and without S9-mix
- 24 hours exposure without S9-mix
1st cytogenetic assay: - 3 hours exposure with and without S9-mix
2nd cytogenetic assay: - 24 hours exposure without S9-mix

- Harvest time after the end of treatment (sampling/recovery times):
Dose range finding:
- 3 hours exposure: at the end of the exposure period, cells were washed twice and re-suspended in 5 mL culture medium with Cytochalasine B and incubated for another 24 hours (1.5 times normal cell cycle).
- 24 hours exposure: cytoB was added to the cells simultaneously with the test item at the 24 hours exposure time.
1st cytogenetic assay (i.e. 3 h expo): 24 hours after end of exposure
2nd cytogenetic assay (i.e. 24 h expo: cells are harvested at the end of the treatment period


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasine B obtained from Sigma; 5 µg/mL,
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
The following criteria for scoring of binucleated cells were used:
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)

Rationale for test conditions:

Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. Since Step 2 Catalyst Mixture (Stripped) was not cytotoxic, the highest concentration analyzed was determined by the solubility in the culture medium.

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: healthy volunteers

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
At a concentration of 312.5 µg/mL Step 2 Catalyst Mixture (Stripped) precipitated in the culture medium. In the dose-range finding study, at the 3 hours exposure time, blood cultures were treated with 9.8, 19.5, 39.1, 78.1, 156.3 and 312.5 µg Step 2 Catalyst Mixture (Stripped)/mL culture medium with and without S9-mix. At the 24 hours exposure time blood cultures were treated with 19.5, 39.1, 78.1, 156.3, 312.5 and 625 µg Step 2 Catalyst Mixture (Stripped)/mL culture medium without S9-mix.
After the 3 hour exposure time, 39% cytotoxicity was observed at the highest precipitating dose level of 312.5 µg/mL. At the 24 hours exposure time, 49% cytotoxicity was observed at the precipitating dose level of 156.3 µg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
1st cytogenicity test:
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei at a high cytotoxic dose level (89%). Although colchicine resulted in high cytotoxicity, a reproducible and detectable increase over the background was observed, which proves that the test conditions were adequate.
2nd cytogenicity test:
The positive control chemical mitomycin C produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei at a high cytotoxic dose level (98%). Although colchicine resulted in high cytotoxicity, a reproducible and detectable increase over the background was observed, which proves that the test conditions were adequate

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI - Step 2 Catalyst Mixture (Stripped) was not cytotoxic at the concentations tested
- other:
2nd cytogenicity test:
It appeared that a dose level of 140 µg/mL already precipitated in the culture medium. Therefore the cultures of 160 µg/mL were removed from the assay.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate
1st cytogenicity test:
Both in the absence and presence of S9-mix, Step 2 Catalyst Mixture (Stripped) did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.
2nd cytogenicity test:
Step 2 Catalyst Mixture (Stripped) did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei

Conclusions:

In conclusion, this test is valid and proved that Step 2 Catalyst Mixture (Stripped) is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this study according to OECD testing guideline no. 487.

Executive summary:

The objective of this study was to evaluate Step 2 Catalyst Mixture (Stripped) for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of Step 2 Catalyst Mixture (Stripped) was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD testing guideline no. 487.

Lot:6420 of Step 2 Catalyst Mixture (Stripped) was a red brown liquid. The vehicle of the test item was dimethyl sulfoxide.

In the first cytogenetic assay, Step 2 Catalyst Mixture (Stripped) was tested up to 320 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Step 2 Catalyst Mixture (Stripped) precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Step 2 Catalyst Mixture (Stripped) was tested up to 140 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. The test item precipitated at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system
(S9-mix) functioned properly.

Step 2 Catalyst Mixture (Stripped) did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, this test is valid and Step 2 Catalyst Mixture (Stripped) is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 03 Jul 2018 to 14 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

as adopted 29 July 2016

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:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells obtained from American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines

For cell lines:
- Absence of Mycoplasma contamination: check performed, only negatice cultures used
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
- Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
- Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Exposure medium
For 3 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
- Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/mL trifluorothymidine (TFT) (Sigma).
- Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
- Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 65 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.8 - 37.5°C).

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: concentration range of 20 to 625 µg/mL
1st mutagenicity test: 0, 2.4, 4.9, 9.8, 20, 39, 78, 156, 313 and 625 µg/mL
2nd mutagenicity test: 0, 2.4, 4.9, 9.8, 20, 39, 78, 156 and 313 µg/mL

Since the test item was not toxic up to the precipitating dose level and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. The highest test item concentrations showed a slight precipitate in the exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: suitable for the test item and the test model (i.e. cultured cells)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Dose range finder: 3 hours with and without metabolic activation and 24 hours without metabolic activation
1st mutagenicity test: 3 hours with and without metabolic activation
2nd mutagenicity test: 24 hours without metabolic activation
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days after treatement
- Selection time (if incubation with a selective agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): exposure time (either 3 or 24 hours) plus 2 days expression plus selection time (either 12 or 11 days) --> approximate total of 13 days
- Method used: microwell plates
- selective agent used: trifluorothymidine (TFT),5 µg TFT/mL selection medium indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Dose range finder: 8 x 10EXP6 cells (10EXP6 cells/mL for 3 hour treatment) or 6 x 10EXP6 cells (1.25 x 10EXP5 cells/mL for 24 hour treatment)
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose-range for the mutagenicity tests.

1st mutagenicity test:
8 x 10EXP6 cells (10EXP6 cells/mL for 3 hour treatment)
mutation frequency: cloning efficiency day 2 (scoring via the naked eye or the microscope) and mutation frequency (MTT staining)

2nd mutagenicity test:
6 x 10EXP6 cells (1.25 x 10EXP5 cells/mL for 24 hour treatment)
mutation frequency: cloning efficiency day 2 (scoring via the naked eye or the microscope) and mutation frequency (MTT staining)

- Criteria for small (slow growing) and large (fast growing) colonies:
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth (RTG); relative survival (RS)

Rationale for test conditions:

Eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods.
Since the test item was not toxic up to the precipitating dose level and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. The highest test item concentrations showed a slight precipitate in the exposure medium.

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

not performed

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 20 to 625 µg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.
After the 3 hour treatement and after 24 and 48 hours of subculture the relative suspension growth was 2 and 25% at the test item concentration of 625 µg/mL compared to the relative suspension growth of the solvent control in the absence and presence of S9-mix, respectively. Based on this results concentrations of 2.4 up to 625 µg/mL were chosen for the first mutagenicity experiment.
After the 24 hour treatement and after additional 24 hours of subculture the relative suspension growth was 25% at the test item concentration of 313 µg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 625 µg/mL. Due to high cytotoxicity the concentration of 625 µg/mL was not further examined in the mutagenicity test with prolonged treatment period (2nd mutagenicity test).

STUDY RESULTS
- Concurrent vehicle negative and positive control data yielded the expected results.

Gene mutation tests in mammalian cells:
1st mutagenicity test:
Based on the results of the dose-range finding test and solubility test, the following
dose-range was selected for the first mutagenicity test in the absence and presence of
S9-mix with a 3 hour treatment period: 2.4, 4.9, 9.8, 20, 39, 78, 156, 313 and 625 µg/mL exposure medium.
- Evaluation of toxicity
To minimize the selection of too many dose levels with precipitation in the exposure medium, the dose level of 625 µg/mL was not selected for mutation frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus in the absence and presence of S9-mix: 2.4, 4.9, 9.8, 20, 39, 78, 156 and 313 µg/mL exposure medium.
No toxicity was observed up to and including the highest tested dose level.
- Evaluation of the mutagenicity
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with Step 2 Catalyst Mixture (Stripped) either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

2nd mutagenicity Test
To obtain more information about the possible mutagenicity of Step 2 Catalyst Mixture (Stripped), a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 2.4, 4.9, 9.8, 20, 39, 78, 156 and 313 µg/mL exposure medium.
- Evaluation of toxicity
No toxicity was observed up to and including the highest tested dose level and all dose levels were selected to measure mutation frequencies at the TK-locus.
- Evaluation of mutagenicity
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

Mutation frequency per 106 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 808 684 1669
SD 239 206 843
n 136 124 146
Upper control limit
(95% control limits) 1465 1222 4196
Lower control limit
(95% control limits) 152 146 -859
SD = Standard deviation
n = Number of observations (single culture of positive controls)
Distribution historical positive control data from experiments performed between November 2014 and November 2017

- Negative (solvent/vehicle) historical control data:
Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

Mutation frequency per 10EXP6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 96 92 96
SD 29 30 29
n 268 248 285
Upper control limit
(95% control limits) 160 152 160
Lower control limit
(95% control limits) 32 31 32
SD = Standard deviation
n = Number of observations (single culture of solvent controls)
Distribution historical negative control data from experiments performed between November 2014 and November 2017 (different solvents used).

Conclusions:

Step 2 Catalyst Mixture (Stripped) was not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of this study performed according to OECD testing guideline no. 490.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of Step 2 Catalyst Mixture (Stripped) by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  

The study procedures described in this report were based on the most recent OECD testing guideline no. 490. 

Batch Lot 6420 of Step 2 Catalyst Mixture (Stripped) was a red brown liquid. The vehicle of the test item was dimethyl sulfoxide.

In the first experiment, Step 2 Catalyst Mixture (Stripped) was tested up to concentrations of 313 µg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, Step 2 Catalyst Mixture (Stripped) was again tested up to concentrations of 313 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test item precipitated in the culture medium at this dose level. This is the highest concentration recommended in the guidelines.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. 

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Step 2 Catalyst Mixture (Stripped) did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, Step 2 Catalyst Mixture (Stripped) did not induce a biologically relevant increase in the mutation frequency.

In conclusion, Step 2 Catalyst Mixture (Stripped) is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Due to the consistent negative findings in all reliable in vitro assays according to current testing guidelines no classification for mutagenicity for Step 2 catalyst is recommended according to the criteria of Regulation (EC) No 1272/2008.