SUMILIZER GP

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:

23 Apr - 24 Jun 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

2-Aminoanthracene was used as sole positive control for all strains in the presence with S9 mix; standard deviations were not calculated

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 2020

Deviations:

yes

Remarks:

2-Aminoanthracene was used as sole positive control for all strains in the presence with S9 mix; standard deviations were not calculated

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA/TSCA Guideline, 40 CFR Part 799, FR Vol.62, No.158 § 799.9510 TSCA bacterial reverse mutation test

Version / remarks:

adopted in 1997

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Testing Methods for New Chemical Substances , Kanpoan No.287, Eisei No.127, Heisei 09 • 10 • 31 Kikyoku No.2

Version / remarks:

adopted in 1997

Test guideline 6

Qualifier:

according to guideline

Guideline:

other: Ministry of Labour, Japan, Notification No.77 dated 1 September, 1988 and Notification No.67 dated 2 June, 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-450-4

EC Name:

-

Cas Number:

203255-81-6

Molecular formula:

C42H61O4P

IUPAC Name:

6-(3-(3-tert-Butyl-4-hydroxy-5-methylphenyl)propoxy)-2,4,8,1 0-tetra-tert-butyldibenz(d,f)(1,3,2)dioxaphosphepin

Method

Target gene:

his operon (S. typhimurium strains) and trp operon (E. coli strains)

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:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague Dawley rats treated with phenobarbital / benzoflavone
Source of S9 : Oriental Yeast Co., Ltd. (Tokyo, Japan)

Test concentrations with justification for top dose:

Dose finding experiment: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate with and without S9 mix
Main experiment: 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate without S9 mix and 19.5, 39.1, 78.1, 156 and 313 µg/plate with S9 mix
Dose levels for the main mutation experiment were selected based on the results of the dose-finding experiment, in which precipitation of the test substance in vehicle was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 0.01 µg/plate for TA 100 and WP2 uvrA, 0.1 µg/plate for TA 98 (-S9); 2-aminoanthracene, 0.5 µg/plate for TA 98, 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537 and 10 µg/plate for WP2 uvrA (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (dose-finding and main experiment)

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

NUMBER OF REPLICATIONS:
- Number of independent experiments: Following an initial dose-range finding study, a single experiment was performed in the main mutation study. The performance of the dose-range finding experiment was comparable to those of the main mutation study and can be considered as a valid independent experiment.
- Number of cultures per concentration: triplicates for each experiment (dose-finding and main experiment)

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 were observed

Evaluation criteria:

The test substance was judged to be positive (mutagenic potential) if the following criteria were met:
- the test chemical show a dose-dependent increase in the number of revertant colonies
- the increase is at least twice as many as that of the solvent control

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation was observed at 78.1 µg/plate without S9 mix and at 313 µg/plate with S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation of the test substance in vehicle was observed in the absence of S9 mix at concentrations ≥ 78.1 µg/plate and in the presence of S9 mix at concentrations ≥ 313 µg/plate. The observations were made in the dose finding experiment and in the main experiment.

RANGE-FINDING/SCREENING STUDIES:
Following an initial dose-range finding study, a single experiment was performed in the main mutation study. The performance of the dose-range finding experiment was comparable to those of the main mutation study, therefore the dose-finding experiment can be considered as a valid independent experiment.

HISTORICAL CONTROL DATA
- Positive historical control data: The positive control values were within the range of the historical control data (please refer to table 3 under "any other information on results incl. tables").
- Negative (solvent/vehicle) historical control data: The negative control values were within the range of the historical control data (please refer to table 3 under "any other information on results incl. tables").

Any other information on results incl. tables

Table 1: Results of the range-finding study

Dose-finding experiment: Pre-incubation test
Strain	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control DMSO
DMSO	95	109	8	14	21	29	21	36	7	12
Test item [µg/plate]
4.88 mean	95	112	7	10	26	30	19	38	7	11
19.5 mean	101	106	12	9	26	30	25	37	8	13
78.1 mean	88^#	106	7^#	10	24^#	31	28^#	40	6^#	11
313^# mean	88	102	9	9	24	30	20	40	6	14
1250^# mean	81	105	7	10	24	33	19	40	7	13
5000^#mean	90	97	8	7	23	26	12	31	4	9
Positive control
^§Mean	595	764	351	221	125	736	345	289	668	171
^§: Positive controls are: 9-aminoacridine (80 µg/plate for TA 1537 (-S9 mix)); sodium azide (0.5 µg/plate for TA 1535 (-S9 mix)); 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate for TA 100 and WP2 uvrA, 0.1 µg/plate for TA 98 (-S9)) and 2-aminoanthracene (0.5 µg/plate for TA 98, 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537 and 10 µg/plate for WP2 uvrA (+S9 mix)) #: Precipitation observed

Table 2: Results of the main mutagenicity experiment:

Main mutation experiment: Pre-incubation test
Strain	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
DMSO mean	121	84	7	7	29	28	18	32	7	15
Test item
4.88 mean	116	-	8	-	21	-	18	-	5	-
9.77 mean	128	-	11	-	28	-	28	-	5	-
19.5 mean	107	103	11	8	26	28	26	38	8	14
39.1 mean	110	93	9	8	26	32	23	31	9	13
78.1 mean	103^#	99	7^#	12	24^#	25	27^#	25	9^#	12
156 mean	-	99	-	9	-	25	-	30	-	12
313^# mean	-	96	-	5	-	27	-	32	-	15
Positive control
^§mean	703	704	348	220	184	497	321	261	855	136
^§: Positive controls are: 9-aminoacridine (80 µg/plate for TA 1537 (-S9 mix)); sodium azide (0.5 µg/plate for TA 1535 (-S9 mix)); 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate for TA 100 and WP2 uvrA, 0.1 µg/plate for TA 98 (-S9)) and 2-aminoanthracene (0.5 µg/plate for TA 98, 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537 and 10 µg/plate for WP2 uvrA (+S9 mix)) #: Precipitation observed

Table 3: Historical control data generated in the testing laboratory in Jul - Dec 1997

Strain	TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
Metabolic activation	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Vehicle control
Mean	95.8	103.8	7.8	9.2	19.0	24.0	21.7	35.6	7.3	12.5
SD	± 12.6	± 14.2	± 2.9	± 3.3	± 4.7	± 5.2	± 5.4	± 7.5	± 4.0	± 5.6
n	302	177	254	185	204	168	231	173	225	187
Positive control
Mean	565.2	627.2	315.6	194.8	160.1	556.9	410.5	320.5	713.3	159.6
SD	± 96.5	± 95.7	± 27.9	± 26.9	± 30.3	± 64.7	± 39.9	± 51.8	± 149.0	± 31.2
n	258	183	257	187	205	172	231	177	234	191
n: number of experiments performed

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative