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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A key Bacterial Reverse Mutation Assay according to OECD TG 471 was conducted in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli (WP2uvrA) in the presence and absence of a metabolic activation. The test item had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

A key in vitro Mammalian gene Mutation (Assay according to OECD TG 476 was conducted in CHO K1 Chinese hamster ovary cells at the Hprt locus in the presence and absence of a metabolic activation. The test item had no mutagenic activity on the growth of the mammalian cells under the test conditions used in this study.

An in vitro Micronucleus assay according to OECD TG 487 was conducted in human peripheral lymphocytes in the presence and absence of a metabolic activation. Preliminary results are provided; the study is not yet finalised and therefore supporting at this moment. In a key in vitro Micronucleus test with read-across substance Sodium bis (C11-14-isoalkyl, C13-rich) sulfosuccinate (CAS number 848588-96-5), the test item was negative for chromosome damage in human peripheral lymphocytes tested up to cytotoxic concentrations without and with metabolic activation.

Link to relevant study records

Referenceopen allclose all

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
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
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
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
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
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
Target gene:
histidine (Salmonella strains)
tryptophan (E.coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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.
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
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.
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
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
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
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
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
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 June 2020 to 6 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
adopted 29 July 2016
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008, B.17. "In vitro Mammalian Cell Gene Mutation Test”, (Official Journal L 142, 31/05/2008)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL: see confidential details

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: see confidential details

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was formulated in the selected vehicle (solvent) to provide a suitably concentrated stock solution as follows. The necessary amount of test item was weighed into a calibrated volumetric flask (no correction for purity of the test item was applied). Approximately 80% of the required volume of vehicle (solvent) was added and the formulation was stirred until homogeneity was reached (It was ultrasonicated 2x10 minutes), then the volume was adjusted to the required final level. From the stock solution, several dilutions were prepared using the selected vehicle (solvent) to prepare dosing solutions for lower doses. The vehicle (solvent) were filtered sterile using a 0.22 µm filter (Supplier: Millipore, Lot No.: MP183904G2, Expiry date: September 2021) before the preparation of the dosing formulations in each case. The stock solutions as well as all dilutions (dosing solutions) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood.
- Final dilution of a dissolved solid, stock liquid or gel: All dilutions (dosing solutions) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood.

OTHER SPECIFICS: see confidential datils
Target gene:
hprt locus at the X-chromosome
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO K1
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHO K1 cell purchased from American Type Culture Collection (ATCC)
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: Checking of mycoplasma infection was carried out for each batch of frozen stock; the cell line was tested negative.
- Number of passages if applicable:
- Methods for maintenance in cell culture: Cells were stored as frozen stocks in a liquid nitrogen tank. For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium (“culture medium”) and incubated at 37°C (± 0.5 °C) in a humidified atmosphere (5± 0.3% CO2 in air). When cells were growing well, subcultures were established in an appropriate number of flasks. Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture.
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: Prior to use in this test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium on 22 April 2016.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Four types of Ham's F12 medium were prepared:
*F12-1: Foetal bovine serum(FBS, heat inactivated) 1 % v/v, L-Glutamine 0.01 mL/mL, Antibiotic-Antimycotic solution 0.01 mL/mL*
*F12-5: FBS, heat inactivated) 5 % v/v, L-Glutamine 0.01 mL/mL, Antibiotic-Antimycotic solution 0.01 mL/mL*
*F12-10: FBS, heat inactivated) 10 % v/v, L-Glutamine 0.01 mL/mL, Antibiotic-Antimycotic solution 0.01 mL/mL*
*F12-SEL**: FBS, heat inactivated) 10 % v/v, L-Glutamine 0.01 mL/mL, Antibiotic-Antimycotic solution 0.01 mL/mL* (**Hypoxanthine-free Ham’s F-12 medium was used for preparation of the selection culture medium)
*Standard content of the antibiotic-antimycotic solution is 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B.

For the 5-hour treatments, at least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x10E7 cells were placed in flasks and incubated for about approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 5-hour treatment contained 1% (v/v) serum (F12-1, for treatment without metabolic activation) or 5% (v/v) serum (F12-5, for treatment with metabolic activation). A suitable volume (100 µL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). In case of experiment with metabolic activation, 1.0 mL of S9-mix was added to the cultures (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 5-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), the cultures were washed thoroughly with F12-10 medium (culture medium). Then, dishes were covered with appropriate amount of fresh F12-10 medium (10-60 mL) and incubated for 19 hours at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x105 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.

For the 24-hour treatment, at least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x10E7 cells were placed in flasks and incubated for approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 24-hour treatment contained 5% serum (F12-5). A suitable volume (100 µL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 24 hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
Duplicate cultures were used for each treatment. Solubility of the test item in the cultures was visually examined at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment.


Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used
- source of S9 : Male Wistar rats (331-421 g animals were 7-9 weeks old at the initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Initiation date of the induction of liver enzymes used for preparation S9 used in this study was 13 May 2019.
- method of preparation of S9 mix: On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) 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 minutes 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 date of preparation of S9 fraction for this study was 16 May 2019 (Test Facility internal code: E13090, Expiry date: 16 May 2021).
The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of the test Facility. The protein concentration of the S9 fraction used in the study was determined to be 27.3 g/L. The sterility of the preparation was confirmed.
Prior to addition to the culture medium the S9-mix was kept in an ice bath.
- concentration or volume of S9 mix and S9 in the final culture medium: For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to 9 mL of cell culture medium to give a total of 10 mL (the same ratio was applied in those cases when higher treatment volume was used). The final concentration of the liver homogenate in the test system was 3%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The biological activity in the Salmonella assay of S9 was characterized using the two mutagens (2-Aminoanthracene and Benzo(a)pyrene), that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.

The S9-mix was prepared as follows:
HEPES* : 20 mM Concentration of the stock solution; 0.2 mL/mL Concentration in the mix
KCl: 330 mM Concentration of the stock solution; 0.1 mL/mL Concentration in the mix
MgCl2: 50 mM Concentration of the stock solution; 0.1 mL/mL Concentration in the mix
NADP**: 40 mM Concentration of the stock solution; 0.1 mL/mL Concentration in the mix
D-Glucose 6 phosphate (Monosodium salt): 50 mM Concentration of the stock solution; 0.1 mL/mL Concentration in the mix
F12-10: - Concentration of the stock solution; 0.1 mL/mL Concentration in the mix
S9 fraction: - Concentration of the stock solution; 0.3 mL/mL Concentration in the mix
*HEPES = N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**NADP= β-Nicotinamide-adenine dinucleotide-phosphate
Test concentrations with justification for top dose:
In the preliminary experiment, a 5-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed with a range of test concentrations to determine toxicity immediately after the treatments.
Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test according to the OECD No. 476 guideline instructions (up to the cytotoxicity limit), as follows:
Assay 1 and Assay 1 repeated
5-hour treatment in the presence of S9-mix:
120, 100, 90, 80, 70, 30, 10 and 3.33 µg/mL
5-hour treatment in the absence of S9-mix:
15, 14, 13, 12, 11, 10, 3.33, 1.11, 0.37 and 0.12 µg/mL.
Assay 2 and Assay 2 repeated
5-hour treatment in the presence of S9-mix:
120, 100, 90, 80, 70, 30, 10 and 3.33 µg/mL
24-hour treatment in the absence of S9-mix:
45, 40, 35, 30, 10, 3.33, 1.11 and 0.37 µg/mL.
The tested concentration range in the study was considered to be adequate as concentrations up to the highest practical concentration (based on the cytotoxicity) were examined in the study. The examined concentration range covered the range from producing cytotoxicity to no cytotoxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Propylene glycol (PG) was used for vehicle of the test item. Dimethyl sulfoxide (DMSO) was used as vehicle of the positive control materials in the study.

- Justification for choice of solvent/vehicle: Based on the available information (method development study was performed at the Test Facility, study code: 20/027-316ANE), propylene glycol (abbreviated as PG in the raw data and study documents) was selected as vehicle for this study in agreement with the Sponsor. The test item was soluble at 200 mg/mL concentration in PG. This vehicle (solvent) is compatible with the survival of the cells and the S9 activity.
The test item was formulated in the selected vehicle (solvent) to provide a suitably concentrated stock solution as follows. The necessary amount of test item was weighed into a calibrated volumetric flask (no correction for purity of the test item was applied). Approximately 80% of the required volume of vehicle (solvent) was added and the formulation was stirred until homogeneity was reached (It was ultrasonicated 2x10 minutes), then the volume was adjusted to the required final level. From the stock solution, several dilutions were prepared using the selected vehicle (solvent) to prepare dosing solutions for lower doses. The vehicle (solvent) were filtered sterile using a 0.22 µm filter (Supplier: Millipore, Lot No.: MP183904G2, Expiry date: September 2021) before the preparation of the dosing formulations in each case. The stock solutions as well as all dilutions (dosing solutions) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood.

Untreated negative controls:
yes
Remarks:
to demonstrate that the selected vehicle (solvent) had no mutagenic effects
Negative solvent / vehicle controls:
yes
Remarks:
Propylene glycol
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration:
Preliminary Toxicity Test: single (positive controls not included)
Main Mutation assays: treatment in duplicate; plating for survival in triplicate; plating for viability in triplicate; plating for selection of the mutant phenotype: 5 parallels per sample
- Number of independent experiments: 6 (4 were planned, 2 repeat assays had to be done: see notes below)
Assay 1, a 5-hour treatment with metabolic activation
Assay 1 repeated, a 5-hour treatment without metabolic activation
Assay 2, 5-hour treatment with metabolic activation
Assay 2 repeated, 24-hour treatment without metabolic activation
Note 1: In Assay 1, in the absence of S9-mix (5-hour treatment), excessive cytotoxicity of the test item was observed. The selected concentration intervals seemed to be not sufficiently refined to evaluate at least four test concentrations to meet the acceptability criteria (appropriate cytotoxicity). Therefore, an additional experiment (Assay 1 repeated) was performed to use more closely spaced concentrations with modified concentrations to give further information about the cytotoxic effects and to meet the acceptability criteria.
Note 2: In Assay 2, in the absence of S9-mix (24-hour treatment), excessive cytotoxicity of the test item was observed. The selected concentration intervals seemed to be not sufficiently refined to evaluate at least four test concentrations to meet the acceptability criteria (appropriate cytotoxicity). Therefore, an additional experiment (Assay 2 repeated) was performed to use more closely spaced concentrations with modified concentrations to give further information about the cytotoxic effects and to meet the acceptability criteria

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): at least 2x10E6 cells (test); at least 2x10E7 cells (positive control)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: /
- Exposure duration/duration of treatment: 5 h treatment (Assay 1, Assay 1 repeated, Assay 2), 24 h treatment (Assay 2 repeated)
- Harvest time after the end of treatment (sampling/recovery times):
19 hours (Assay 1, Assay 1 repeated and Assay 2).
0 hours (Assay 2 repeated): harvest immediate after treatment

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days during which time the HPRT-mutation was expressed
- Selection time (if incubation with a selective agent): 7 days with 6-thioguanine
- Fixation time (start of exposure up to fixation or harvest of cells): After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.
- If a selective agent is used, indicate its identity, its concentration and, duration and period of cell exposure: 6-thioguanine (6-TG) final volume: 10 mL; final 6-TG concentration: 10 µg/mL; 7 days incubation
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: at least 2x106 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x107 cells were placed in flasks and incubated for about approximately 24 hours before treatment
- Criteria for small (slow growing) and large (fast growing) colonies: not applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative survival (RS)
- Any supplementary information relevant to cytotoxicity: /

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The test item iss considered to be mutagenic if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
5. The historical control range is considered when deciding if the result is positive.
Rationale for test conditions:
To evaluate the potential of the test item to induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase (Hprt) enzyme locus in CHO K1 Chinese hamster ovary cells in the absence and presence of a rat liver metabolising system.
Evaluation criteria:
The assay is considered valid if all of the following criteria are met (based on the relevant guidelines):
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the general historical control data.
2. The positive control chemicals induced a clear increase in mutant frequency.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8.
4. At least four test item concentrations in duplicate cultures were presented.

The test item is considered to be mutagenic in this assay if the following criteria are met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary).
According to the relevant OECD 476 guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response.
Statistics:
The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency).
In the statistical analysis, negative trends were not considered significant.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
K1
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
5 h exposure (Assay 1)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
marked cytotoxicity of the test item was observed (90 µg/mL concentration showed a relative survival of 20%).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
K1
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
5 h exposure (Assay 1 repeated)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
marked cytotoxicity of the test item was observed (15 µg/mL concentration showed a relative survival of 13%).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
5 h exposure (Assay 2)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
marked cytotoxicity of the test item was observed (100 µg/mL showed a relative survival of 12%).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
K1
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
24 h exposure (Assay 2 repeated)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
marked cytotoxicity of the test item was observed (30 µg/mL concentration showed a relative survival of 19%).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There were no relevant changes in pH after treatment in any cases.
- Data on osmolality: There were no relevant changes in osmolality after treatment in any cases.
- Possibility of evaporation from medium: vapour pressure ≤ 4.8*10-3Pa at 20 °C
- Water solubility: >170 g/L
- Precipitation and time of the determination: no
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 µg/mL (the recommended maximum concentration).
No insolubility was detected in the preliminary experiment. The concentrations selected for the main experiments were based on results of the performed Preliminary Toxicity Test according to the OECD No. 476 guideline instructions (up to the cytotoxicity limit). Eight concentrations were selected for the main experiments.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays, and the observed values were in the expected range (5-20 x 10E-6) as shown in the OECD No. 476 guideline.
The cloning efficiencies for the negative (vehicle) controls on Days 1 and 8 were within the target range of 60-140% and 70-130% in all assays.
The positive controls (DMBA in the presence of metabolic activation and EMS in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays


For all test methods and criteria for data analysis and interpretation:
Assay 1 (5 h exposure with S9): An evaluation was made using data of six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).
Assay 1 repeated (5 h exposure without S9): An evaluation was made using data of all ten concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).
Assay 2 (5 h exposure with S9: An evaluation was made using data of seven concentrations. Statistically significant increase in mutant frequency (at p<0.05 level) was observed in this experiment at the lowest tested concentration (3.33 µg/mL) although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (10.2 x 10E-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10E-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment).Therefore, it was concluded as biologically not relevant increase. No dose response to the treatment was observed (a trend analysis showed no effect of treatment). In overall, this experiment was concluded as negative.
Assay 2 repeated (24 h exposure without S9): An evaluation was made using data of five concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).



Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
For the 5-hour treatments, at least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x10E7 cells were placed in flasks and incubated for about approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 5-hour treatment contained 1% (v/v) serum (F12-1, for treatment without metabolic activation) or 5% (v/v) serum (F12-5, for treatment with metabolic activation). A suitable volume (100 µL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). In case of experiment with metabolic activation, 1.0 mL of S9-mix was added to the cultures (higher volume using the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 5-hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), the cultures were washed thoroughly with F12-10 medium (culture medium). Then, dishes were covered with appropriate amount of fresh F12-10 medium (10-60 mL) and incubated for 19 hours at 37°C (±0.5°C) in a humidified atmosphere (5±0.3% CO2 in air).
After the 19-hour incubation period, cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.

For the 24-hour treatment, at least 2x10E6 cells were placed in each of a series of sterile dishes (diameter approx. 100 mm) and in case of the positive control at least 2x107 cells were placed in flasks and incubated for approximately 24 hours before treatment at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). On the treatment day, plating medium was removed and appropriate amount of fresh medium was added to the cells. Treatment medium for the 24-hour treatment contained 5% serum (F12-5). A suitable volume (100 µL) of vehicle (solvent), test item solution or positive control solution was added to the 10 mL final volume (the same ratio was applied in those cases when higher than 10 mL final volume was used). After the 24 hour incubation period at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air), cells were washed twice with phosphate buffered saline (PBS), detached with trypsin-EDTA solution and counted using a haemocytometer. In samples where sufficient cells survived, cell number was adjusted to 2x10E5 cells/mL. Cells (10 mL cell suspension) were transferred to dishes for growth through the expression period or diluted to be plated for survival.
Duplicate cultures were used for each treatment. Solubility of the test item in the cultures was visually examined at the beginning and end of the treatments. Measurement of pH and osmolality was also performed after the treatment.

Plating for survival: Following adjustment of the cultures to 2x10E5 cells/mL, samples from these cultures were diluted to 40 cells/mL using F12-10 medium.Five mL suspension (200 cells/dish) per each culture were plated into 3 parallel dishes (diameter was approx. 60 mm). The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing

o Number of cells plated in selective and non-selective medium
Non-selective (plating for viability): At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10E5 cells/mL, then further diluted to 40 cells/mL using F12-10 medium. Five mL of cell suspension (200 cells/dish) per each culture were plated in F12-10 medium in 3 parallel dishes (diameter was approx. 60 mm) for a viability test. The dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 5 days for colony growing.
Selective (plating for selection of the mutant phenotype): At the end of the expression period (Day 8), cell number in the samples was adjusted to 4x10E5 cells/mL. 1 mL of the adjusted cell suspension and 4 mL of F12-SEL medium were added into Petri dishes (diameter approx. 100 mm, 5 parallels per sample) for each sample. An additional 5 mL of F12-SEL medium containing 20 µg/mL
6-thioguanine (6-TG) was added to the dishes (final volume: 10 mL, final 6-TG concentration: 10 µg/mL) to determine mutation frequency. Dishes were incubated at 37°C (± 0.5°C) in a humidified atmosphere (5±0.3% CO2 in air) for 7 days for colony growing.

o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: mutation frequency ((Number of 6-TG resistant mutants per 10E6 clonable cells):
*Positive control DMBA:
5-hour, S9+: Mean: 905.2; SD: 562.7; min 141.2; max 2119.4; n=27
*Positive control EMS:
5-hour, S9-: Mean: 445.6; SD: 118.6; min 239.6; max 636.6; n=13
24-hour, S9-: Mean: 1176.6; SD: 610.9; min 363.1; max 2449.8; n=14
DMBA = 7,12-Dimethylbenz[a]anthracene
EMS = Ethyl methanesulfonate
S9+ = in the presence of S9-mix
S9- = in the absence of S9-mix

- Negative (solvent/vehicle) historical control data: mutation frequency ((Number of 6-TG resistant mutants per 10E6 clonable cells):
*Untreated control
5-hour, S9+: Mean: 18.3; SD: 15.1; min 5.1; max 64.1; n=27
5-hour, S9-: Mean: 20.7; SD: 16.4; min 5.5; max 55.5; n=13
24-hour, S9-: Mean: 19.0; SD: 17.2; min 3.3; max 58.0; n=14
*DMSO control
5-hour, S9+: Mean: 21.8; SD: 15.9; min 5.4; max 57.3; n=29
5-hour, S9-: Mean: 18.9; SD: 11.6; min 6.5; max 47.4; n=13
24-hour, S9-: Mean: 18.4; SD: 14.4; min 6.8; max 48.5; n=14
DMSO = Dimethyl sulfoxide
S9+ = in the presence of S9-mix
S9- = in the absence of S9-mix
Conclusions:
No mutagenic effect of AEROSOL TR-70 E Lyophilized was observed either in the presence or absence of a metabolic activation system and the assay was valid based on negative and positive control under the conditions of this HPRT assay.

Executive summary:

An in vitro mammalian cell assay [1-2] was performed in CHO K1 Chinese hamster ovary cells at the Hprt locus to evaluate the potential of AEROSOL TR-70 E Lyophilized to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The design of this study was based on the Commission Regulation (EC) No. 440/2008 and OECD No. 476 guideline, and the study was performed in compliance with Charles River Laboratories Hungary Kft. standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Propylene glycol was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:

Assay 1 and Assay 1 repeated

5-hour treatment in the presence of S9-mix:

120, 100, 90, 80, 70, 30, 10 and 3.33 µg/mL

5-hour treatment in the absence of S9-mix:

15, 14, 13, 12, 11, 10, 3.33, 1.11, 0.37 and 0.12 µg/mL.

Assay 2 and Assay 2 repeated

5-hour treatment in the presence of S9-mix:

120, 100, 90, 80, 70, 30, 10 and 3.33 µg/mL

24-hour treatment in the absence of S9-mix:

45, 40, 35, 30, 10, 3.33, 1.11 and 0.37 µg/mL.

Note: In Assay 1, in the absence of S9-mix (5-hour treatment) and in Assay 2, in the absence of S9-mix (24-hour treatment), excessive cytotoxicity of the test item was observed. The selected concentration intervals seemed to be not sufficiently refined to evaluate at least four test concentrations to meet the acceptability criteria (appropriate cytotoxicity). Therefore, additional experiments (Assay 1 repeated and Assay 2 repeated) were performed to use more closely spaced concentrations with modified concentrations to give further information about the cytotoxic effects and to meet the acceptability criteria.

 

In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.

 

In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no relevant changes in pH and osmolality after treatment in any cases.

 

In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (90 µg/mL concentration showed a relative survival of 20%). An evaluation was made using data of six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

In Assay 1 repeated, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (15 µg/mL concentration showed a relative survival of 13%). An evaluation was made using data of all ten concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

 

In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (100 µg/mL showed a relative survival of 12%). An evaluation was made using data of seven concentrations. Statistically significant increase in the mutation frequency (at p<0.05 level) was observed in this experiment at the lowest tested concentration (3.33 µg/mL), although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (10.2 x 10-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. In overall, this experiment was concluded as negative.

In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (30 µg/mL concentration showed a relative survival of 19%). An evaluation was made using data of five concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

 

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least five evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the maximum recommended concentrations or cytotoxic range in each test). The overall study was considered to be valid.

 

In conclusion, no mutagenic effect of AEROSOL TR-70 E Lyophilized was observed either in the presence or absence of a metabolic activation system and the assay was valid based on negative and positive control under the conditions of this HPRT assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached Read-across information
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality:
The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
Osmolality: with a semi-micro osmometer.
No relevant changes in pH or osmolality of the formulations were noted.
- Water solubility: The test item was completely dissolved in aqua ad iniectabilia.

RANGE-FINDING/SCREENING STUDIES:
In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg test item/mL medium were employed. Cytotoxicity was noted starting at the concentration of 250 µg test item/mL. Hence, 250 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
Data from vehicle and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the concurrent vehicle controls or positive controls for an experiment.
The micronucleus frequencies of the vehicle controls without and with metabolic activation for the last 8 or 7 studies (most recent background data, not audited by the QAU-department) are given as follows:
Micronucleus frequency per 1000 cells
Without metabolic activation (4-h or 20-h exposure)
Untreated control (n = 8)
Mean: 4.9
SD: 2.0
range: 1 - 9
Vehicle control (n = 8)
Mean: 7.2
SD: 4.6
range: 1 - 18
Mitomycin C Positive control (n = 7)
Mean: 95.8
SD: 66.1
range: 24 - 286
Colchicine Positive control (n = 7)
Mean: 25.4
SD: 10.2
range: 7 - 43
With metabolic activation (4-h exposure)
Vehicle control (n = 8)
Mean: 10.8
SD: 6.2
range: 2 - 25
Cyclophosphamide Positive control (n = 7)
Mean: 60.3
SD: 37.8
range: 20 – 147
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study cytotoxicity was noted at the top concentration of 250 µg/mL in the experiments without and with metabolic activation.
Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the read-across test item tested up to cytotoxic concentrations of 250 µg test item/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively
Executive summary:

Test sample of read-across substance Sodium bis(C11-14-isoalkyl, C13-rich) sulfosuccinate was assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.

The read-across test item was completely dissolved in aqua ad iniectabilia. Aqua ad iniectabilia served as the vehicle control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg/mL medium were employed. Cytotoxicity was noted starting at the concentration of 250 µg test item/mL. Hence, 250 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

In the main study cytotoxicity was noted at the top concentration of 250 µg/mL in the experiments without and with metabolic activation.

Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation.

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated with the read-across test item at concentrations of 31.3, 62.5 or 125 µg test item/mL medium (4 h or 20-h exposure) in the absence of metabolic activation ranged from 5.0 to 8.5 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentrations. The dose level of 250 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation.

Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. In this test the following frequencies were observed: vehicle control: 8.0 or 5.0 micronuclei per 1000 binucleated cells and untreated controls: 9.5 or 8.0 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively). Vehicle and untreated control values fell within acceptation ranges.

 Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the read-across test item at concentrations of 31.3, 62.5 or 125 µg test item/mL medium (4-h exposure) in the presence of metabolic activation ranged from 2.5 to 10.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. The dose level of 250 µg test item/mL medium led to cytotoxicity, not enough cells of sufficient quality were available for evaluation.

Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. In this test the following frequencies were obsereved: vehicle control: 8.5 or 8.0 micronuclei per 1000 binucleated cells and untreated controls: 10.0 or 8.5 micronuclei per 1000 binucleated cells. Vehicle and untreated control values fell within acceptation ranges.

Under the present test conditions, the read-across test item tested up to cytotoxic concentrations of 250 µg test item/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data were available for the registered substance.

Bacterial mutagenicity:

A key Bacterial Reverse Mutation Assay was conducted in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli (WP2uvrA) in the presence and absence of a metabolic activation (Tóth-Gönczöl, 2020). Based on the results of a preliminary experiment, the examined test concentrations in the Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate (plate incorporation) and 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate in Assay 2 (preincubation). Precipitate/slight precipitate was detected at the 5000 μg/plate concentration in the Assay 1 in S. typhimurium TA 98 and TA100 strains with and without metabolic activation and in S.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 S. typhimurium TA98, TA1535 and E. 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 strains with metabolic activation on the plates at the 5000 μg/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 analysable 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 strains under the test conditions used in this study.

Mammalian mutagenicity:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the Hprt locus to evaluate the potential of AEROSOL TR-70 E Lyophilized to cause gene mutation (Kovács, 2020). Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). Propylene glycol was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows: Assay 1 and Assay 1 repeated (5-hour treatment in the presence of S9-mix: 120, 100, 90, 80, 70, 30, 10 and 3.33 µg/mL; 5-hour treatment in the absence of S9-mix: 15, 14, 13, 12, 11, 10, 3.33, 1.11, 0.37 and 0.12 µg/mL) - Assay 2 and Assay 2 repeated (5-hour treatment in the presence of S9-mix: 120, 100, 90, 80, 70, 30, 10 and 3.33 µg/mL; 24-hour treatment in the absence of S9-mix: 45, 40, 35, 30, 10, 3.33, 1.11 and 0.37 µg/mL).

Note: In Assay 1, in the absence of S9-mix (5-hour treatment) and in Assay 2, in the absence of S9-mix (24-hour treatment), excessive cytotoxicity of the test item was observed. The selected concentration intervals seemed to be not sufficiently refined to evaluate at least four test concentrations to meet the acceptability criteria (appropriate cytotoxicity). Therefore, additional experiments (Assay 1 repeated and Assay 2 repeated) were performed to use more closely spaced concentrations with modified concentrations to give further information about the cytotoxic effects and to meet the acceptability criteria.

In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no relevant changes in pH and osmolality after treatment in any cases. In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (90 µg/mL concentration showed a relative survival of 20%). An evaluation was made using data of six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). In Assay 1 repeated, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (15 µg/mL concentration showed a relative survival of 13%). An evaluation was made using data of all ten concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (100 µg/mL showed a relative survival of 12%). An evaluation was made using data of seven concentrations. Statistically significant increase in the mutation frequency (at p<0.05 level) was observed in this experiment at the lowest tested concentration (3.33 µg/mL), although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (10.2 x 10E-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10E-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. In overall, this experiment was concluded as negative. In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (30 µg/mL concentration showed a relative survival of 19%). An evaluation was made using data of five concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least five evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the maximum recommended concentrations or cytotoxic range in each test). The overall study was considered to be valid.

In conclusion, no mutagenic effect of AEROSOL TR-70 E Lyophilized was observed either in the presence or absence of a metabolic activation system and the assay was valid based on negative and positive control under the conditions of this HPRT assay.

Cytogenicity:

An in vitro micronucleus test in human peripheral lymphocytes was conducted with the registered substance using human peripheral lymphocytes both in the presence and absence of metabolic activation (Buskens, 2020). The test is employing 2 exposure times without S9 mix (3 and 27 hours) and one exposure time with S9 mix (3 hours). The harvesting time was 27 hours after the end of 3h exposure and 24 h after 24 h exposure. Each treatment was conducted in duplicate. Preliminary results are provided; the study is not yet finalised and therefore supporting at this moment.

In the first cytogenetic assay, the test item was tested up to 313 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. The test item precipitated in the culture medium at this dose level. The test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix in the first cytogenetic assay.

In the second cytogenetic assay , human lymphocytes were exposed for 24 hours in the absence of S9-mix at 10, 50, 75, 100, 125, 150, 200 and 250 µg/mL culture medium: no appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 200 µg/mL not enough cytotoxicity was observed (11%), whereas the next higher concentration of 250 µg/mL was too toxic for scoring (cell lysis). The experiment was repeated in cytogenetic assay 2A at 10, 100, 150, 200, 210, 220, 230, 240 and 250 µg/mL culture medium: no appropriate dose levels could be selected for scoring of micronuclei since no dose level was available with a cytotoxicity of 55 ± 5%. The experiment was repeated in cytogenetic assay 2B at  10, 100, 150, 170, 190, 210, 230 and 250 µg/mL culture medium: no appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 190 µg/mL not enough cytotoxicity was observed (8%), whereas the next higher concentration of 210 µg/mL was too toxic for scoring (cell lysis). The experiment will be repeated at 100, 150, 180, 185, 190, 195, 200, 205, 210 and 215 µg/mL culture medium.

A key in vitro micronucleus study was still available for a structurally similar test substance Sodium bis (C11-14-isoalkyl, C13-rich) sulfosuccinate (CAS number 848588-96-5). Justification for this read across is separately attached in Section 13. This sudy was conducted using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals (Flügge, 2013c). The test was carried out employing 2 exposure times without S9 mix: (4 hours and 20 hours) and one exposure time with S9 mix (repeated). The harvesting time was 24 hours after the end of exposure. Each treatment was conducted in duplicate. The test item was completely dissolved in aqua ad iniectabilia, which also served as the vehicle control. Based on a preliminary experiment, cytotoxicity was noted starting at a concentration of 250 µg test item/mL in the experiment without and with metabolic activation. Hence, 250µg/mL were employed as the top concentration for the main test without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). There was no increase in micronuclei up to the cytotoxic concentration when compared to control both with and without metabolic activation. Under the present test conditions, the test item tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of any chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant damage.

 

Justification for classification or non-classification

Based on these results and according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for genetic toxicity.