N,N'-bis{N-[N-(4-chlorophenyl)carbamimidoyl]carbamimidoyl}hexane-1,6-bis(aminium) diundec-10-enoate

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:

February 17- March 28, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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 reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis{N-[N-(4-chlorophenyl)carbamimidoyl]carbamimidoyl}hexane-1,6-bis(aminium) diundec-10-enoate

EC Number:

813-944-0

Cas Number:

1884575-91-0

Molecular formula:

C44H70Cl2N10O4

IUPAC Name:

N,N'-bis{N-[N-(4-chlorophenyl)carbamimidoyl]carbamimidoyl}hexane-1,6-bis(aminium) diundec-10-enoate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 001/CHUA/01 16
- Expiration date of the lot/batch: December, 2017
- Purity test date: January, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability under test conditions: Two years

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

To establish a dose response effect, 5 dose levels with adequately spaced intervals were tested. The maximum dose level was 0.158 mg/plate both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

Vehicle / solvent:

The solubility of test item in RO water was insoluble.. The test item was found soluble in Dimethyl sulfoxide (DMSO) at 50 mg/mL to give final treatment concentration of 5 mg/plate (recommended maximum test concentration for soluble in-cytotoxic substances). Therefore, DMSO was chosen as solvent for the study.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 1.)* 2-Aminoanthracene, 2-AA, 2.)* 4-Nitro-o-phenylenediamine, 4-NOPD

Details on test system and experimental conditions:

The histidine dependent strains are derived from Salmonella typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa) mutation they possess a faulty lipopolysaccharide envelope, which enables substances to penetrate the cell wall more easily. A further mutation (deletion of the uvrB gene) causes an inactivation of the excision repair system. The latter alteration also includes a deletion in the nitrate reductase and biotin genes. In the strains TA 98,
TA 100, and TA 102 the R-factor plasmid pKMlOl carries umu DC analogous genes that a-re involved in error-prone repair mechanism and the ampicillin resistance marker. The strain TA 102 does not contain the uvrb-mutation. Additionally TA 102 contains the multicopy plasmid pAQ I, which carries the hisG428 mutation and a tetracycline resistance gene. TA 102 contains the ochre mutation in the hisG gene. When summarised, the mutations of the bacterial strains used in this study can be described as follows: Salmonella typhimurium

Strains Genotype Type of mutation indicated
TA 1537 his C 3076; rfa; uvrB Frame shift mutations
TA 98 his D 3052; rfa; uvrB; R-factor Frame shift mutations
TA 1535 his G 46; rfa; uvrB Base-pair substitutions
TA 100 his G 46; rfa; uvrB; R-factor Base-pair substitutions
TA 102 his G 428; rfa; uvrB; R-factor Transitions/Transversions


Regular checking (once in three months) of the properties of the Salmonella typhimurium strains regarding the membrane permeability and ampicillin and tetracycline resistance as well as histidine dependence is performed in RCC Laboratories India Private Limited. In this way it is ensured that the experimental conditions set down by Ames are fulfilled. The bacterial strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were obtained from Xenometrix AG, Gewerbestrasse 25, CH-4123 AUschwil (Switzerland).

Rationale for test conditions:

To select an appropriate solvent and dose concentration range of the test item to be tested , solubility and precipitation test were performed.

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice [strains TA 98, TA I 00 and TA I 02) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of solvent control such an increase is not considered biologically relevant.

Statistics:

The colonies were counted manually. The mean values of the plates for each concentration together with standard deviation were compared to the spontaneous reversion rates. Microsoft Office Excel based calculation were used for descriptive statistical analysis.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In TA 98 cyto-toxicity was observed in the treated concentrations 1.582 and 5 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In TA 100 cyto-toxicity was observed in the treated concentrations 1.582 and 5 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The pre-experiment was performed with TA 100 and TA 98 strain of Salmonella typhimurium and with eight different concentrations of the test item prepared with (-YI 0) half log intervals and three plates were used for each dose level and for the controls. 5 mg/plate was selected as the highest dose in the pre-experiment based on the solubility and precipitation test. Following doses were selected for the pre-experiment i.e., 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate. The results were evaluated based on the reduction of revertant colony count and bacterial background lawn.

In TA 98 and TA 100, cyto-toxicity was observed in the treated concentrations 1.582 and 5 mg/plate (T7 to T8), moderate inhibition was observed in the treated concentrations 0.501 mg/plate (T6) and there was no reduction in colony count as well as background lawn in any of the following concentrations tested; 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate both in absence and in the presence of metabolic activation, when compared to that of the vehicle control group. Based on the pre-experiment (Cytotoxicity) results, 0.158 mg/plate was selected as the highest dose for the main study (Trial-I and Trial-II) both in the presence and in absence of the metabolic activation.

Trial-I was performed with five concentrations of test item along with the negative, vehicle and concurrent positive controls with the remaining three strains i.e. TA 1537, TA 1535 and TA 102 by the plate incorporation method. For the TA 98 and TA 100 revertant colony counts were directly incorporated in the Trial-I from the pre-experiment up to the required five concentrations 0.002 mg/plate (Tl) to 0.158 mg/plate (TS). Following concentrations of test item were prepared with the (-Y 10) half log intervals:
0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate Both in the absence (-S9) as well as in the presence of metabolic activation (+S9). The plates were treated and incubated at 37 ± 2°C for 48 hours. There was no distinct increase in the revertant colony count in any of the five strains observed at any of the test concentrations. Positive controls resulted in an unequivocal response in all the five tester strains, compared to respective controls used.

Trial-II was performed independently with all the five tester strains along with the negative, vehicle and positive controls by pre-incubation method for the confirmation of the Trial-I results. Following concentrations of the test item with ('110) half log intervals were prepared: 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate, both in the presence (+S9) as well as in the absence (-S9) of metabolic activation. The concentration of positive controls used was same as used in the plate incorporation assay. The test
item, negative, vehicle and positive controls were pre-incubated along with 500 ~LL of metabolic activation mix (+S9)/Buffer (-S9) and 100 μL of bacterial culture for 60 minutes at 37 ± 2 °C in an incubator. After pre-incubation, 2 mL of top agar was mixed with the pre-incubation mixture and poured on minimal glucose agar plates. The treated plates were incubated for 48 hours in an incubator. No significant increase in the revertant counts was observed in any of the five tester strains preincubated with the test item.
The positive controls showed an unequivocal increase in revertant counts with all the five tester strains and the respective controls used, hence confirming the non-mutagenic activity of the test item.

Any other information on results incl. tables

See study report pages 21 for info on induvial experiments

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item CHUA did not induce gene mutations either by base pair substitution or by frame shifts in the genome of the strains used under the conditions of the assay, with and without metabolic activation.

Executive summary:

This study was performed to assess the mutagenic potential of CHUA to induce gene mutations in comparison to vehicle (solvent) control according to the plate incorporation method (Trial-I) and the pre-incubation method (Trial-II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations: 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). (The test item concentration values has been incorporated up to three decimal places in the report). No significant increase in revertant colony numbers in any of the tester strains were observed following treatment with CHUA at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The spontaneous reversion rates in the negative, vehicle and positive controls were within the range of our historical data.

The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.