Sodium 1,4-diisotridecyl sulphonatosuccinate

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:

3 April 2020 - 17 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", 21 July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA 712-C-98-247, August 1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria", 30 May 2008

Test guideline 4

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The method described the above mentioned guidelines conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

sodium bis[C12-14 (branched) alkyl] sulfosuccinate

IUPAC Name:

sodium bis[C12-14 (branched) alkyl] sulfosuccinate

Test material form:

other: White viscous solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL : see confidential information

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL : see confidential information

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Distilled water was used as solvent to prepare the stock solution of the test material.
- Final dilution of a dissolved solid, stock liquid or gel: Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent.

OTHER SPECIFICS: see confidential information

Method

Target gene:

histidine (Salmonella strains)
tryptophan (E.coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : livers of phenobarbital/β-naphthoflavone-induced rats prepared by the Microbiological Laboratory of Charles River Laboratories Hungary Kft.

- method of preparation of S9 mix: according to Ames et al. [1] and Maron and Ames [2]
1. BRUCE N. AMES, JOYCE MCCANN and EDITH YAMASAKI: Methods for Detecting Carcinogens and Mutagens with the Salmonella /Mammalian-Microsome Mutagenicity Test. Mutation Research, 31: 347-364, 1975
2. DOROTHY M. MARON and BRUCE N. AMES: Revised Method for the Salmonella Mutagenicity Test. Mutation Research, 113: 173-215, 1983
Male Wistar rats (444-628 g, animals were 17-20 weeks old) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 2 September 2019.
On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The dates of preparation of S9 fractions for this study was 05 September 2019 (Charles River Laboratories Hungary code: E13142, Expiry date: 05 September 2021).

- concentration or volume of S9 mix and S9 in the final culture medium :
S9 Mix (containing 10 % (v/v) of S9)
Salt solution for S9 Mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration through a 0.22 μm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate buffer, pH 7.4: 500 mL
Rat liver homogenate (S9): 100 mL
Salt solution for S9 Mix (see above): 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.

In the standard plate incorparation Assay 1: The content of the tubes:
top agar: 2000 µL
vehicle or test item formulation (or reference controls): 50 µL
overnight culture of test strain: 100 µL
phosphate buffer (pH 7.4) or S9 mix: 500 µL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube.

In the standard pre-incubation procedure Assay 2:
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer were added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar was added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The sterility of the preparation was confirmed in each case. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Charles River Laboratories Hungary Kft. The mean protein concentration of the S9 fraction used was determined to be 24.5 g/L. The biological activity in the Salmonella assay of S9 was characterized in each case using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately.

Test concentrations with justification for top dose:

Concentrations for the main tests were selected on the basis of the Preliminary Compatibility Test and Preliminary Range Finding Test.
In the preliminary range finding experiment (plate incorporation, Salmonella typhimurium TA98 and TA100) following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 (plate incorporation) were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Assay 2 (pre-incubation) were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water was used as solvent to prepare the stock solution of the test material. In the study two vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals: distilled water or DMSO.

- Justification for choice of solvent/vehicle: Preliminary Compatibility Test was performed.
The solubility of the test item was examined using Distilled water, DMSO (Dimethyl sulfoxide) and N,N-Dimethylformamide (DMF). The test item was soluble at 100 mg test item/mL concentrations in Distilled water (after 15 minutes vortex). At 100 mg/mL concentration, good solubility was detected using DMSO and DMF. During formulation a piece of test item was detected in the solution and did not dissolve after approximately 25 minutes of vortex and 2 minutes of incubation in an ultrasonic water bath. As the piece still did not dissolve after 5 minutes of vortex again the formulation was diluted to 50 mg test item /mL. It dissolved after approximately 15 minutes of vortex to a homogeneous and dense solution. Due to the Sponsor’s request and better biocompatibility, Distilled water was selected as vehicle for the test item studies. Distilled water or DMSO were used as vehicle for the positive controls.. The obtained stock formulation (100 µL) with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension.

- Justification for percentage of solvent in the final culture medium:
Based on the solubility test, a 50 mg/mL stock solution was prepared in Distilled water.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

with and without S9

Negative solvent / vehicle controls:

yes

Remarks:

distilled water or DMSO: with and without S9

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD) in DMSO

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO 2 µg/plate all Salmonelaa strains with S9 and 50 µg/plate E. coli WP2uvra with S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentratio: triplicate
- Number of independent experiments : 2 Main assays (Assay 1:plate incorporation and Assay 2: preincubation; both with and without metabolic activation).

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation Assay 1); preincubation (Assay 2).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Assay 2 : incubated for 20 minutes at 37°C in a shaking incubator
- Exposure duration/duration of treatment: Assay 1: the plates were incubated at 37°C for 48(±1) hours; Assay 2: 20 minutes preincubation + 48(±1) hours plate incubation

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Assay 2: 20 minutes preincubation
- Selection time (if incubation with a selective agent): Assay 1 and 2: 48(±1) hours
- If a selective agent is used, indicate its identity, its concentration and, duration and period of cell exposure: not applicable
For the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 Histidine – Biotin solution (0.5 mM) was used as selective agent for 48(±1) hours exposure.
For Escherichia coli WP2 uvrA Tryptophan solution (2 mg/mL) was used as selective agent for 48(±1) hours exposure.
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: No inhibitory, cytotoxic effect of the test item was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation.

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Rationale for test conditions:

The purpose of this study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA strain in the presence and absence of activated rat liver S9 fraction.

Evaluation criteria:

The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests.
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to precipitating concentrations except in Assay 1 without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to precipitating concentrations except in Assay 2 without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to precipitating concentrations except in the Assays without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination:
Precipitate/slight precipitate was detected at the 5000 μg/plate concentration in the Assay 1 in Salmonella typhimurium TA 98 and TA100 strains with and without metabolic activation and in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA strains with metabolic activation.
Precipitate/slight precipitate was detected in the Assay 2 in all examined bacterial strains without metabolic activation on the plates at the 5000-50 μg/plate concentrations range; in Salmonella typhimurium TA98, TA1535 and Escherichia coli WP2 uvrA strains with metabolic activation on the plates at the 5000 and 1581 μg/plate concentrations and in Salmonella typhimurium TA 100 and TA1537 trains with metabolic activation on the plates at the 5000 μg/plate concentration.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
Precipitate/Slight precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation at 5000 µg/plate concentrations and with metabolic activation at 2500 µg/plate concentrations.
No inhibitory or toxic effects of the test item was observed in the preliminary experiment in both examined bacterial strains with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical method may be used as an aid in evaluating the test results.

Ames test:
In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
No inhibitory, cytotoxic effect of the test item was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation
Reduced colony number was observed at the 5000 μg/plate concentration in the Assay 1 in Salmonella typhimurium TA98 and TA100 strains without metabolic activation and in the Assay 2 in Salmonella typhimurium TA1537 strain also without metabolic activation .
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.40). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
In Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 50 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.42). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Untreated, negative (vehicle/solvent) and positive controls were run concurrently.
The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.
*Untreated control data
TA98 -S9 mix: mean: 22.0; SD: 5.3; range: 9-50 ; n= 2019
TA98 +S9 mix: mean: 28.0; SD: 6.7; range: 10-56 ; n= 2037
TA100 -S9 mix: mean: 100.8; SD: 18.9; range: 54-210 ; n= 2014
TA100 +S9 mix: mean: 108.3; SD: 17.9; range: 65-204 ; n= 2031
TA1535 -S9 mix: mean: 12.1; SD: 4.4; range: 1-46 ; n= 2025
TA1535 +S9 mix: mean: 11.7; SD: 3.5; range: 1-39 ; n=2036
TA1537 -S9 mix: mean: 7.9; SD: 3.4; range: 1-26 ; n= 2034
TA1537 +S9 mix: mean: 9.4; SD: 3.7; range: 1-29 ; n= 2043
E.coli-S9 mix: mean: 36.9; SD: 10.7; range: 11-82 ; n= 2025
E.coli +S9 mix: mean: 41.9; SD: 10.3; range: 16-89 ; n= 2022
*DMSO control data
TA98 -S9 mix: mean: 21.2; SD: 5.2; range: 6-55 ; n= 2172
TA98 +S9 mix: mean: 27.1; SD: 6.7; range: 11-67 ; n= 2189
TA100 -S9 mix: mean: 97.0; SD: 18.2; range: 40-217 ; n= 2163
TA100 +S9 mix: mean: 105.8; SD: 18.8; range: 53-229 ; n= 2181
TA1535 -S9 mix: mean: 12.2; SD: 4.4; range: 1-43 ; n= 2175
TA1535 +S9 mix: mean: 11.5; SD: 3.4; range: 2-33 ; n=2192
TA1537 -S9 mix: mean: 7.8; SD: 3.3; range: 1-27 ; n= 2190
TA1537 +S9 mix: mean: 9.1; SD: 3.6; range: 1-29 ; n= 2196
E.coli-S9 mix: mean: 36.0; SD: 10.7; range: 7-81 ; n= 2175
E.coli +S9 mix: mean: 41.0; SD: 10.2; range: 9-85 ; n= 2175
*Distilled water control data
TA98 -S9 mix: mean: 22.7; SD: 5.5; range: 11-45 ; n= 423
TA98 +S9mix: mean: 28.6; SD: 6.9; range: 10-53 ; n= 426
TA100 -S9 mix: mean: 100.4; SD: 19.7; range: 45-215 ; n= 2031
TA100 +S9 mix: mean: 108.2; SD: 19.9; range: 59-222 ; n= 2022
TA1535 -S9 mix: mean: 12.2; SD: 4.3; range: 2-47 ; n= 2037
TA1535 +S9 mix: mean: 11.5; SD: 3.3; range: 3-39 ; n=2040
TA1537 -S9 mix: mean: 8.6; SD: 3.5; range: 2-24 ; n= 429
TA1537 +S9 mix: mean: 9.9; SD: 3.7; range: 1-24 ; n= 426
E.coli-S9 mix: mean: 38.0; SD: 10.6; range: 12-84 ; n= 2055
E.coli +S9 mix: mean: 42.8; SD: 10.0; range: 13-91 ; n= 2040
*Positive reference control data
TA98 -S9 mix: mean: 372.6; SD: 97.9; range: 152-2336 ; n= 2019
TA98 +S9 mix: mean: 2411.0; SD: 267.8; range: 312-4918 ; n= 2037
TA100 -S9 mix: mean: 1203.6; SD: 179.2; range: 536-2120 ; n= 2013
TA100 +S9 mix: mean: 2424.7; SD: 246.9; range: 1116-5240 ; n= 2034
TA1535 -S9 mix: mean: 1167.1; SD: 172.9; range: 208-2440 ; n= 2025
TA1535 +S9 mix: mean: 228.3; SD: 112.5; range: 101-2216 ; n=2040
TA1537 -S9 mix: mean: 442.2; SD: 141.2; range: 149-2104 ; n= 2034
TA1537 +S9 mix: mean: 218.9; SD: 47.3; range: 117-838 ; n= 2043
E.coli-S9 mix: mean: 1038.2; SD: 136.1; range: 488-2496 ; n= 2028
E.coli +S9 mix: mean: 255.4; SD: 94.7; range: 125-2512 ; n= 2022

Applicant's summary and conclusion

Conclusions:

The test item AEROSOL TR-70 E Lyophilized (Batch Number: KB19J2101) had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/ β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 (Plate Incorporation Method) and an Assay 2 (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test item was dissolved in Distilled water. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

Precipitate/slight precipitate was detected at the 5000 μg/plate concentrationin the Assay 1 in Salmonella typhimurium TA 98 and TA100 strains with and without metabolic activation and in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA strains with metabolic activation.

Precipitate/slight precipitate was detected in the Assay 2 in all examined bacterial strains without metabolic activation on the plates at the 5000-50μg/plateconcentrations range; in Salmonella typhimurium TA98, TA1535 and Escherichia coli WP2uvrA strains with metabolic activation on the plates at the 5000 and 1581μg/plate concentrations and in Salmonella typhimurium TA 100 and TA1537 strains with metabolic activation on the plates at the 5000μg/plate concentration

No inhibitory, cytotoxic effect of the test item was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation.

Reduced colony number was observed in the Assay 1 in Salmonella typhimurium TA98 and TA100 strains without metabolic activation on the plates at the 5000 μg/plate concentration.

Reduced colony number was observed in the Assay 2 in Salmonella typhimurium TA1537 strain without metabolic activation on the plates at the 5000 μg/plate concentration.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. 

In conclusion, the test item AEROSOL TR-70 E Lyophilized (Batch Number:KB19J2101) had no mutagenic activity on the growth of the bacterial strainsunder the test conditions used in this study.