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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2018 - August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial forward mutation assay

Test material

Constituent 1
Reference substance name:
Sulfuric acid, mono-C8-18-alkyl esters, magnesium salts, compds. with triethanolamine
EC Number:
287-840-3
EC Name:
Sulfuric acid, mono-C8-18-alkyl esters, magnesium salts, compds. with triethanolamine
Cas Number:
85586-38-5
IUPAC Name:
85586-38-5
Test material form:
solid
Remarks:
dried from of a reaction mixture, water and cyclohexanol where evaporated
Details on test material:
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: February 2020
Storage conditions: Room temperature
pH-value: Approx. 7 (moistened with water, determined by Bioassay Laboratories)
Specific details on test material used for the study:
see analytical report, study code 18L00152
Homogeneity: The homogeneity of the test substance was ensured by
mixing before preparation of the test substance solutions.
Storage stability: The stability of the test substance under storage conditions is
guaranteed until Feb 2020 as indicated by the sponsor, and
the sponsor holds this responsibility. The test facility is
organizationally independent from the BASF SE sponsor
division.

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
EXOGENOUS METABOLIC ACTIVATION
S9 fraction
The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALACapproved
laboratory in accordance with the German Animal Welfare Act and the effective
European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany
GmbH) received 80 mg/kg b.w. phenobarbital i.p. and ß-naphthoflavone orally (both supplied
by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with
a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The
day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum.
24 hours after the last administration, the rats were sacrificed, and the livers were prepared
using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and
washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three
volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at
+4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
S9 mix
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient
amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with
9 parts of S9 supplement (cofactors).
Test concentrations with justification for top dose:
1st and 2nd experiment
Doses: 0; 33; 100; 333; 1000; 2650 and 5300 μg/plate
Highest dose according to guideline (correction due to purity)
3rd experiment
Doses: 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate
Reason: Bacteriotoxicity was observed in the preincubation test. Therefore,
this experimental part were repeated with adjusted doses.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (= ultrapure water)

- Justification for choice of solvent/vehicle:Due to the good solubility of the test substance in water, water was used as vehicle.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

- Test substance added:
Standard plate test - Test tubes containing soft agar (overlay agar), which consists of
agar, NaCl and amino acid solution were kept in a water bath at about and the remaining components were added in the
following order: test solution, (vehicle or positive control), fresh bacterial culture, S9 mix (with metabolic activation),or
phosphate buffer (without metabolic activation), after mixing, the samples were poured onto Minimal glucose agar plates
Preincubation Test - test solution, vehicle or positive control, bacterial suspension and S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, soft agar was added and, after mixing, the samples were poured onto the agar plates.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
about 20 minutes
- Exposure duration/duration of treatment:
48 – 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Rationale for test conditions:
Selected concentration are max concentration according to guideline.
3rd Experiment was conducted at a top dose of 1000 µg/plate due to bacteriotoxicity in preincubation.
Evaluation criteria:
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain (see Appendix 5).
• The sterility controls revealed no indication of bacterial contamination (see Appendix 3).
• The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies compatible with the range of the historical positive control
data or above (see Appendix 5).
• Fresh bacterial culture containing approximately 109 cells per mL were used.

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least
doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli
WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and
TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix
or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative
control data under all experimental conditions in at least two experiments carried out
independently of each other.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: see other information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
other: see other information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: see other information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
other: see other information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
other: see other information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

A weak bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test only using tester strain TA 1535 without S9 mix at a concentration of 2650 µg/plate.

In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 100 µg/plate.

Decreased revertant numbers were observed at following concentrations (µg/plate):

Experiment

S9

TA 1535

TA 100

TA 1537

TA 98

E.coli

1st-SPT

Without

2650

-

-

-

-

With

 

 

-

 

 

2nd-PIT

Without

1000 – 5300

1000 – 5300

333 – 5300

333 – 5300

-

With

2650 – 5300

2650 – 5300

1000 – 5300

1000 – 5300

2650 – 5300

3rd-PIT

Without

100 – 1000

1000

333 – 1000

333 – 1000

n.t.

With

1000

1000

1000

1000

n.t.

- = no adverse effect observed

n.t. = not tested

Reduced background growth was observed at following concentrations (µg/plate):

Experiment

S9

TA 1535

TA 100

TA 1537

TA 98

E.coli

1st-SPT

Without

 

 

-

 

 

With

 

 

-

 

 

2nd-PIT

Without

2650 – 5300

2650 – 5300

333 – 5300

1000 – 5300

5300

With

2650 – 5300

2650 – 5300

2650 – 5300

2650 – 5300

-

3rd-PIT

Without

1000

-

-

1000

n.t.

With

1000

-

-

-

n.t.

- = no adverse effect observed

n.t. = not tested

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, it is concluded that Component in "confidential substance name" is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

The test substance Component in "confidential name" was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD 471 (BASF SE 2019).
STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
DOSE RANGE: 33 μg - 5300 μg/plate (SPT)
3.3 μg - 5300 μg/plate (PIT)
TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).
SOLUBILITY: No precipitation of the test substance was observed with and
without S9 mix.
TOXICITY: A bacteriotoxic effect was observed depending on the strain and
test conditions at and above 100 μg/plate 
MUTAGENICITY:
A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

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