[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 Jun 2020

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Product of 1,1,1-Trimethylol propane triacrylate with aziridine and 1-cyclooctyl-1,4-diazocane and 4-hydroxy-4-methylpentan-2-one

Molecular formula:

C6H12N2 - C47H77O16N4

IUPAC Name:

Reaction Product of 1,1,1-Trimethylol propane triacrylate with aziridine and 1-cyclooctyl-1,4-diazocane and 4-hydroxy-4-methylpentan-2-one

Test material form:

liquid

Details on test material:

Name of test item: Emuldur 3643
Test item No.: 19/0071-2
Batch identification: 27771924U0
Purity: 100 % (UVCB*, for details see analytical characterization No.20L00150) *Substances of unknown or variable composition, complex reaction products.
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: July 15, 2021
Storage conditions: Ambient (RT)
Physical state / color: Liquid / colorless to yellowish, clear

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction
The S9 fraction was prepared according to Ames et al. (1, 2) at BASF SE in an AAALACapproved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive (experimental conduct with records and documentation in general
accordance with the GLP principles).
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 -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.
The preparation date and protein content of the S9 fraction were given in the Appendix (part historical data).

S9 mix
The S9 mix was prepared freshly prior to each experiment (1, 2). 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). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer (6) is prepared by mixing a Na2HPO4 solution with a 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.

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (top dose as requested by the guideline)

Vehicle / solvent:

dimethyl sulfoxide (DMSO)
Due to the insolubility of the test substance in 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.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene (2-AA) with S9, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 4-nitroquinoline-N-oxide (4-NQO) without S9

Details on test system and experimental conditions:

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) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 109 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.
The use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were
obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.

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 strains by 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 were checked for the following characteristics at regular intervals:
deep rough character (rfa); UV sensitivity ( uvrB); ampicillin resistance (R factor plasmid).
E. coli WP2 uvrA was checked for UV sensitivity.
Histidine and tryptophan auxotrophy were checked in each experiment via the spontaneous rate.

EXOGENOUS METABOLIC ACTIVATION
S9 fraction
The S9 fraction was prepared according to Ames et al. (1, 2) at BASF SE in an AAALACapproved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive (experimental conduct with records and documentation in general
accordance with the GLP principles).
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 -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.
The preparation date and protein content of the S9 fraction were given in the Appendix (part historical data).

S9 mix
The S9 mix was prepared freshly prior to each experiment (1, 2). 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). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer (6) is prepared by mixing a Na2HPO4 solution with a 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 (see Appendix - part historical data).

DOSES
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was 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.

TEST SUBSTANCE PREPARATIONS
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
The test substance was dissolved in dimethyl sulfoxide (DMSO).
To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
The further concentrations were diluted according to the planned doses.
All test substance formulations were prepared immediately before use.

ANALYSIS OF TEST SUBSTANCE PREPARATION
The stability of the test substance in the vehicle DMSO was not determined analytically,
because the test substance was administered immediately after preparation and is usually
stable.

EXPERIMENTAL PROCEDURE
Choice of the vehicle
Due to the insolubility of the test substance in 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.

Mutagenicity tests
Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was 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) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive 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 were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

• 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) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive 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 were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK).
Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Controls
Negative controls / Vehicle controls
Each experiment included negative controls in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).
• Sterility control
Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strain.
• 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. Vehicle controls were used for several BASF projects done in parallel. Details were described in the raw data.

Positive controls
The following positive controls were used to check the mutability of the bacteria and the activity
of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC) (Sigma-Aldrich; 98%)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA
The stability of the selected positive controls was well-defined under the selected culture conditions, since they were well-established reference mutagens. Positive controls were used for several BASF projects done in parallel. Details were described in the raw data.

Scope of tests and test conditions
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 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 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 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: An increase of revertants was observed in the standard plate test
using tester strains TA 1535, TA 100 and E. coli both with and without
S9 mix. To confirm the results, a repeat experiment was performed.

Rationale for test conditions:

as specified by the guideline

Evaluation criteria:

see "any other information on materials and methods incl. tables"

Statistics:

N/A

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions at and above 2500 μg/plate.
No test substance precipitation was observed with and without S9 mix.
The additional treated plates for sterility control showed no contamination in all performed experiments.

Any other information on results incl. tables

Discussion

According to the results of the present study, the test substance led to adistince, relevant and dose-dependent increase in the number of his+ and trp+ revertants using tester strain TA 1535, TA100 and E. coli WP2 uvrA both with and without S9 mix. The increase of his+ and trp+

revertants was reproducible in two experiments carried out independently of each other. Based on the assessment criteria described (Paragraph 3.8.5.) the test substance has to be considered positive.

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. The number of revertant colonies in the negative controls, with and without S9 mix, were within the range of the historical negative control data for each tester strain (see Appendix – part

historical data).

In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies compatible with the range of the historical positive control data (see see Appendix – part historical data).

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test substance is mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.