Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Test Item: Sulfonium compounds, C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts)
- BASF Test Item No.: 01/0686-2
- Batch Number: 12000229U0
- Purity: 100% (UVCB, for details see analytical report No. 12L00196)
- Expiration Date: February 21, 2013
- Physical state, appearance: Liquid, yellowish
- Storage conditions: Room temperature
- Stability in Solvent: Not indicated by the Sponsor

Method

Target gene:
- Salmonella typhimurium: histidine
- Escherichia coli: tryptophan
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:
phenobarbital/ß-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd Experiment: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate
3rd Experiment: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 mix for all strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix for strains TA 1535 and TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
Without S9 mix for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix for strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix for strain E. coli WP2 uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) are thawed at room temperature, and 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g/L Difconutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. As a rule, a germ density of ≥ 108 bacteria/mL is reached. These cultures
grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strains mentioned is in accordance with the current scientific recommendations for the conduct of this assay. The Salmonella strains TA 98, TA 100 and TA 1537 were obtained from (former) KNOLL Aktiengesellschaft, Ludwigshafen, Germany, on 30 Oct 1989. The Salmonella strain TA 1535
and the Escherichia coli strain were obtained from Merck KGaA, Darmstadt, Germany (22 Apr 2010 and 09 Sep 1991, respectively).

Salmonella typhimurium
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined (2, 3, 4). The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are
induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances. The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strainsby the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537
and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98. The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions (5). The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Checking the tester strains
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid). E. coli WP2 uvrA is checked for UV sensitivity.Histidine and tryptophan auxotrophy is checked in each experiment via the spontaneous rate.

EXOGENOUS METABOLIC ACTIVATION
S9 fraction
The S9 fraction is prepared according to Ames et al. (1, 2) at BASF SE in an AAALAC approved 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 β-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals are housed in Makrolon cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a relative humidity of 30 - 70%. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours). Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats are sacrificed, and the livers are prepared using sterile solvents and glassware at a temperature of +4°C. The livers are weighed and washed in a weight-equivalent volume of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9 000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (so-called S9 fraction) are stored at -70°C to -80°C.

S9 mix
The S9 mix is prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient amount of S9 fraction is thawed at room temperature and 1 volume of S9 fraction is mixed with 9 volumes of S9 supplement (cofactors). This preparation, the so-called S9 mix, is kept on ice until used. The concentrations of the cofactors in the S9 mix are: MgCl2 8 mM, KCl 33 mM, glucose-6-phosphate 5 mM, NADP 4 mM and phosphate buffer (pH 7.4) 15 mM. The phosphate buffer (6) is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4:1. To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

DOSES
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al. (1, 2).
• Salmonella typhimurium Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept
in a water bath at about 42 - 45°C, and the remaining components are added in the following order: 0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture, 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation). After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. Composition of the minimal glucose agar: 980 mL ultrapure water, 20 mL Vogel-Bonner E medium, 15 g Difco bacto agar, 20 g D-glucose, monohydrate. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.
Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order: 0.1 mL test solution or vehicle (negative control), 0.1 mL fresh bacterial culture, 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation),
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. (5), with the exception of the amino acid solution, which has previously been added to the soft agar: 300 mL solution B (agar), 100 mL solution A (saline solution), 8 mL solution C (glucose solution), 10 mL solution D (casein solution) After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.
Preincubation Test
The experimental procedure is based on the method described by Yahagi et al. (7) and Matsushima et al. (8). 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at
37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar isadded and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Titer determination
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments. In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. Test tubes containing 2-mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C,
and the remaining components are added in the following order: 0.1 mL vehicle (without and with test substance), 0.1 mL fresh bacterial culture (dilution: 10-6), 0.5 mL S9 mix In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Negative controls / Vehicle controls:
Each experiment includes negative controls in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).
• Sterility control: Additional plates are treated with soft agar, S9 mix, buffer, vehicle or the test substance but without the addition of tester strains
• Vehicle control: The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.
Scope of tests and test conditions
1st Experiment: Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA. Doses: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate Type of test: Standard plate test with and without S9 mix Number of plates: 3 test plates per dose or per control
2nd Experiment: Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA. Doses: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate Type of test: Standard plate test with and without S9 mix Number of plates: 3 test plates per dose or per control. Reason: Bacteriotoxicity was observed in the standard plate test
3rd Experiment: Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA. Doses: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate. Type of test: Preincubation test with and without S9 mix. Number of plates: 3 test plates per dose or per control. Reason: No mutagenicity was observed in the standard plate test
Evaluation criteria:
Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
depending on the strain and test conditions from 100 μg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance Sulfonium compounds,C11-14-alkylbis(hydroxyethyl), 2-hydroxyethyl sulfates (salts) was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia
coli WP2 uvrA.
MUTAGENICITY
Standard plate test,Tests without S9 mix: TA 1535, TA 100,TA 1537, TA 98, E. coli WP2 uvrA:No relevant increase in the number of his+ or trp+ revertants.
Standard plate test,Tests with S9 mix: TA 1535, TA 100,TA 1537, TA 98, E. coli WP2 uvrA: No relevant increase in the number of his+ or trp+ revertants.
Preincubation test Tests without S9 mix: TA 1535, TA 100,TA 1537, TA 98, E. coli WP2 uvrA: No relevant increase in the number of his+ or trp+ revertants.
Preincubation test Tests with S9 mix: TA 1535, TA 100,TA 1537, TA 98, E. coli WP2 uvrA: No relevant increase in the number of his+ or trp+ revertants.
TOXICITY:
Strong bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate and in the preincubation test depending on the strain and test conditions from 100 μg/plate onward.
DISCUSSION:
According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard
plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control
data or above (see Appendix 6)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion