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Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of a bacterial reverse gene mutation assay conducted according to OECD TG 471, the test item is considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-01-10 to 2020-04-15

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:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name: N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide
- Lot/batch no.: 2019001
- Purity: 98.78%
- Appearance/physical state: white crystalline powder
- Expiry Date: 2020-10-28
- Storage: at room temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Target gene:

S. typhimurium: histidine (his)
E. coli: tryptophan (trp)

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:

Type and composition of metabolic activation system:
- source of S9 : The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Gießen, Germany. Male Sprague Dawley rats were induced with phenobarbital/β-naphthoflavone.

- method of preparation of S9 mix:
100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP.
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts, liver preparation 0.5 parts.
During the experiment the S9 mix is stored on ice.

- concentration or volume of S9 mix and S9 in the final culture medium:
Plate incorporation method: 500 µl S9 mix in 2700 µL.
Pre-incubation method: 100 μL of the test item-preparation is pre-incubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL) for 60 min at 37 °C prior to adding the overlay agar (2000 μL) and pouring onto the surface of a minimal agar plate.

- quality controls of S9: The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

Pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Main experiment I: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Main experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Top dose corresponds to maximum recommended dose according to OECD TG 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
- Justification for percentage of solvent in the final culture medium: see above

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Samples of each tester strain were grown by culturing for 12 h at 37 °C in S. typhimurium medium (Nutrient Broth) and E. coli medium (Luria Bertani), respectively, to the late exponential or early stationary phase of growth (approx. 10E9 cells/mL).
- Test substance added in agar (plate incorporation) in Experiment I and via preincubation in Experiment II

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 min at 37 °C
- Exposure duration/duration of treatment: After solidification, the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).

Rationale for test conditions:

The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). Test conditions in the main experiment were based on the results of the pre-experiment.

Evaluation criteria:

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM 101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test item was observed in all tester strains used in experiment I at concentrations of 2500 μg/plate and higher (with and without metabolic activation) and in experiment II at concentrations of 1000 μg/plate and higher (with and without metabolic activation). The observed precipitation did not interfere with the scoring, thus it did not impact the results.

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
Ames test:
- Signs of toxicity : No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I. The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA1537 at a concentration of 316 μg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.
In experiment II toxic effects of the test item were observed in tester strains TA98 and TA1535 at concentrations of 100 μg/plate and higher (without metabolic activation). In tester strain TA100 toxic effects of the test item were noted at concentrations of 1000 μg/plate and higher (without metabolic activation). In tester strain TA1537 toxic effects of the test item were observed at concentrations of 316 μg/plate and higher (without metabolic activation).

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: Please refer to section "Any other information on results incl. tables" for more details.
- Negative (solvent/vehicle) historical control data: Please refer to section "Any other information on results incl. tables" for more details.

Table 1: Historical Laboratory Control Data of the Negative Control without S9

	TA98	TA100	TA1535	TA1537	WP2 uvrA (pKM 101)
Mean	28.4	95.4	15.6	15.6	165.9
SD	8	16.3	5.9	6.1	38
Min	14	44	5	3	99
Max	61	143	35	35	242
RSD [%]	28.2	17	37.8	39.2	22.9
n	976	1023	926	927	40

Table 2: Historical Laboratory Control Data of the Positive Control without S9

	TA98	TA100	TA1535	TA1537	WP2 uvrA (pKM 101)
Substance Conc./plate	4-NOPD 10 μg	NaN3 10 μg	NaN3 10 μg	4-NOPD 40 μg	MMS 1 μL
Mean	443.2	614.2	826.2	123.3	1918.9
SD	168.7	208.2	362.2	49.4	975.2
Min	89	166	28	33	1045
Max	2013	2493	1863	570	4144
RSD [%]	38.1	33.9	43.8	40	50.8
n	985	1034	935	934	36

Table 3: Historical Laboratory Control Data of the Negative Control with S9

	TA98	TA100	TA1535	TA1537	WP2 uvrA (pKM 101)
Mean	29.9	92.7	14.1	16.2	212.5
SD	7.3	14	5.3	6.3	56.7
Min	15	60	4	5	105
Max	60	154	37	41	359
RSD [%]	24.5	15.1	37.5	38.8	26.7
n	974	1020	923	924	51

Table 4: Historical Laboratory Control Data of the Positive Control with S9

	TA98	TA100	TA1535	TA1537	WP2 uvrA (pKM 101)
Substance Conc./plate	2-AA 2.5 μg	2-AA 2.5 μg	2-AA 2.5 μg	2-AA 2.5 μg	2-AA 10 μg
Mean	1305.9	1054.5	185.7	187.3	717.8
SD	792.7	611	130.1	104	207.9
Min	70	119	19	23	304
Max	3609	2920	1856	1476	1065
RSD [%]	60.7	57.9	70.1	55.5	29
n	976	1026	929	929	48

Conclusions:

In a bacterial reverse mutation assay conducted according to OECD TG 471, the test item did not increase the number of revertants either with or without metabolic activation under the experimental conditions described. The test item is therefore considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria performed conducted according to OECD TG 471, S. typhimurium strains TA98, TA100, TA1535, TA 537 and E. coli strain WP2 uvrA (pKM 101) were exposed to N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide (98.78% purity) at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in experiment I (plate incorporation method) and at concentrations of 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in experiment II (pre-incubation test) in the presence and absence of mammalian metabolic activation (S9-mix).

The test item was tested up to the recommended limit concentration and precipitation was observed in all tester strains used in experiment I at concentrations of 2500 μg/plate and higher (with and without metabolic activation) and in experiment II at concentrations of 1000 μg/plate and higher (with and without metabolic activation).

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test item is therefore considered to be non-mutagenic in this bacterial reverse mutation assay.

The study is classified as acceptable as it satisfies the requirement for Test Guideline  OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay in bacteria performed according to OECD TG 471, S. typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strain WP2 uvrA (pKM 101) were exposed to N-(4-((4-(3-phenylureido)phenyl)sulfonyl)phenyl)benzenesulfonamide (98.78% purity) at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in experiment I (plate incorporation method) and at concentrations of 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in experiment II (pre-incubation test) in the presence and absence of mammalian metabolic activation (S9-mix).

The test item was tested up to the recommended limit concentration and precipitation was observed in all tester strains used in experiment I at concentrations of 2500 μg/plate and higher (with and without metabolic activation) and in experiment II at concentrations of 1000 μg/plate and higher (with and without metabolic activation).

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background. Thus, the test item is therefore considered to be non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Based on the information provided in this section, classification for mutagenicity according to the CLP criteria set out in Annex I of Regulation (EC) No. 1272/2008 is not warranted.