Sodium 1,4-diisotridecyl sulphonatosuccinate

Endpoint summary

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.

A key in vitro Micronucleus assay according to OECD TG 487 was conducted in human peripheral lymphocytes in the presence and absence of a metabolic activation. The test item is not clastogenic and aneugenic in human peripheral blood lymphocytes under the experimental conditions described in this study.

Link to relevant study records

Reference

Reference 1

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.

Reference 2

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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2020 to 21 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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

Species / strain / cell type:

lymphocytes: peripheral blood

Remarks:

Blood was collected from healthy adult, non-smoking, volunteers (aged 18 to 35 years).

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium or lithium heparin. Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells:
- Normal cell cycle time (negative control):

For lymphocytes:
- Sex, age and number of blood donors: Blood was collected from healthy adult, non-smoking, volunteers (aged 18 to 35 years).
- Whether whole blood or separated lymphocytes were used: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added.
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
*Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heatinactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/ streptomycin (50 U/mL and 50 μg/mL respectively) and heparin (30 U/mL).
*Lymphocytes cultures: Whole blood (0.4 mL) treated with heparin will be added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL phytohaemagglutinin (9 mg/mL) was added.

Cytokinesis block (if used):

Cytochalasin B (5 μg/mL), 24 hours incubation (1.5 times normal cell cycle)

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight). S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 m)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix. Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL phytohaemagglutinin (9 mg/mL)). The concentration of the S9-fraction in the exposure medium is 1.8% (v/v).

Test concentrations with justification for top dose:

Assay 1 (3 h exposure with and without S9): 0, 78, 156, 313 µg/mL culture medium. The test item precipitated in the culture medium at the highest dose level.
Assay 2 (24 h exposure without S9): 100, 150, 200 and 215 µg/mL culture medium. The highest dose level was chosen based on cytotoxicity (level of necrosis).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
The vehicle of the test item was dimethyl sulfoxide (DMSO).
Solvent for the positive controls: Hanks’ Balanced Salt Solution (HBSS) without calcium and magnesium.

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in dimethyl sulfoxide at concentrations of 250 mg/mL and below. The stock solution was treated with ultrasonic waves until the test item had completely dissolved. Test item concentrations were used within 1 hour after preparation.

- Justification for percentage of solvent in the final culture medium: The final concentration of the solvent in the culture medium was 1.0% (v/v).

Negative solvent / vehicle controls:

yes

Remarks:

The negative control used in the test system was the vehicle for the test item: DMSO.

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2 (The second assay was repeated)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Assay 1: 3 h exposure
Assay 2: 24 h exposure
- Harvest time after the end of treatment (sampling/recovery times):
Assay 1: 27 hours harvest time (3 h exposure + 24 h Cytochalasin B incubation)
Assay 2: 24 h harvest time (24 h exposure together with Cytochalasin B incubation)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
Cytochalasin B (5 µg/mL) 24h exposure
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol/ acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the cell pellet were fixated carefully with 3 changes of ethanol/acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 minutes with 6.7% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip with an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
At least 502 binucleated cells per culture were examined by light microscopy for micronuclei. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined binucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase was observed, this has no effect on the study integrity.
Assay 1: duplicate cultures: 1000 binucleated cells scored (except 1culture at 313 µg/mL test concentration with only 517 binucleated cells scored due to precipitation of the test item)
Assay 2: duplicate cultures: 1000 binucleated cells scored (selected concentrations of Cytogenetic Assay 2C; except 1 culture with only 971 binucleated cells scored due to heavy necrosis at 200 µg/mL concentration and another culture with only 502 binucleated cells scored due to heavy necrosis at 215 µg/mL concentration)
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
The following criteria for scoring of binucleated cells were used:
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cytokinesis-block proliferation index (CBPI)
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / Total number of cells

t = test item or control treatment culture
c = vehicle control culture
- Any supplementary information relevant to cytotoxicity: A minimum of 500 (with a maximum deviation of 5%) cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI). Three to four analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. At the 3 hours exposure time, the test item was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium. At the 24 hours exposure time, the highest dose level examined for micronuclei were the cultures that showed moderate to heavy necrosis whereas the level of necrosis of the lowest dose level was approximately the same as the level of necrosis of the solvent control. Also, cultures treated with an intermediate dose level were examined.
In the second cytogenetic assay the cytotoxicity was measured by the presence of lytic cells as evidence of necrosis. The level of necrosis as described in this study is as follows:
-: No necrosis obserced
+ Slight necrosis observed
++ Moderate necrosis observed
+++ Heavy necrosis observed.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
At least 502 binucleated cells per culture were examined by light microscopy for micronuclei. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined binucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase was observed, this has no effect on the study integrity.

Evaluation criteria:

An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

Graphpad Prism version 8.4.2 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
The Chi-square test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Assay 1 (3 h exposure)

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Assay 1 (3 h exposure)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Assay 2 (24 h exposure)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

highest dose of 215 µg/mL induced heavy necrosis (Assay 2, 24h exposure)

Vehicle controls validity:

valid

Positive controls validity:

valid

First Cytogenetic Assay

Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:

Without and with S9-mix: 78, 156 and 313 µg/mL culture medium

(3 hours exposure time, 27 hours harvest time).

Table 1 shows the cytokinesis-block proliferation index of cultures treated with various test item concentrations or with the positive or negative control items.

 

Table 1. Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in the First Cytogenetic Assay

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration (µg/mL)		Cytostasis (%)		Number of binucleated cells with micronuclei^1)
				1000		1000		2000
				A		B		A+B
0		0		3		2		5
78		-1		4		4		8
156		1		1		4		5
313^3)		22		5		2^2)		7
0.25-C		32		31		21		52^****
0.1 Colch		71		11		8		19^**

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration (µg/mL)		Cytostasis (%)		Number of binucleated cells with micronuclei^1)
				1000		1000		2000
				A		B		A+B
0		0		3		4		7
78		1		4		2		6
156		1		3		5		8
313^3)		3		3		3		6
15 CP		66		16		28		44^****

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or

       **** P < 0.0001.

¹⁾   1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

²⁾  517 binucleated cells were scored for the presence of micronuclei (see study plan deviation).

³⁾  The test item precipitated in the culture medium.

 

All dose levels were selected for scoring of micronuclei. Both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

Second Cytogenetic Assay

To obtain more information about the possible clastogenicity and aneugenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix: 10, 50, 75, 100, 125, 150, 200 and 250 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).

Table 2 shows the cytokinesis-block proliferation index and the level of necrosis (determined after cytogenetic assay 2C) of cultures treated with various test item concentrations or with the positive or negative control items.

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).

 

Table 2. Cytokinesis-Block Proliferation Index and Level of Necrosis of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in the Second Cytogenetic Assay
Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/mL		CBPI				Mean CBPI		% cytostasis		^1) Necrosis
0		1.69	-	1.74		1.71		0		-
10		1.69	-	1.71		1.70		1		-
50		1.65	-	1.68		1.66		7		-
75		1.66	-	1.70		1.68		5		-
100		1.71	-	1.73		1.72		-1		+
125		1.62	-	1.67		1.65		9		+
150		1.64	-	1.69		1.66		7		++
200		1.62	-	1.65		1.64		11		+++
250		^2)		^2)		^2)		^2)		^2)
0.15 MMC-C		1.34	-	1.39		1.37		49		ND
0.23 MMC-C		1.29	-	1.30		1.30		58		ND
0.05 Colch		1.02	-	1.02		1.02		97		ND

 

^1)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
^1)		Cell lysis
ND		Not Determined

Note: All calculations were performed without rounding off.

 

Based on these results (CBPI) the experiment was repeated in cytogenetic assay 2A with the following dose levels:

Without S9-mix: 10, 100, 150, 200, 210, 220, 230, 240 and 250 µg/mL culture medium

(24 hours exposure time, 24 hours harvest time).

Table 3 shows the cytokinesis-block proliferation index and the level of necrosis (determined after cytogenetic assay 2C) of cultures treated with various test item concentrations or with the positive or negative control items.

No appropriate dose levels could be selected for scoring of micronuclei since no dose level was available with a cytotoxicity of 55 ± 5%.

 

Table 3. Cytokinesis-Block Proliferation Index and Level of Necrosis of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in Cytogenetic Assay 2A

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/mL		CBPI				Mean CBPI		% cytostasis		^1) Necrosis
0		1.59	-	1.67		1.63		0		-
10		1.59	-	1.62		1.61		4		-
100		1.66	-	1.70		1.68		-9		+
150		1.54	-	1.55		1.55		13		+/++
200		1.56	-	^2)		1.56				++/^2)
210		^2)		^2)		^2)		^2)		^2)
220		^2)		^2)		^2)		^2)		^2)
230		^2)		^2)		^2)		^2)		^2)
240		^2)		^2)		^2)		^2)		^2)
250		^2)		^2)		^2)		^2)		^2)
0.15MMC-C		1.19	-	1.24		1.22		65		ND
0.23 MMC-C		1.22	-	1.24		1.23		63		ND
0.05 Colch		1.00	-	1.01		1.01		99		ND

 

^1)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
^2)		Cell lysis
ND		Not Determined

Note: All calculations were performed without rounding off.

 

Based on these results (CBPI) the experiment was repeated in cytogenetic assay 2B with the following dose levels:

Without S9-mix: 10, 100, 150, 170, 190, 210, 230 and 250 µg/mL culture medium

(24 hours exposure time, 24 hours harvest time).

Table 4 shows the cytokinesis-block proliferation index and the level of necrosis (determined after cytogenetic assay 2C) of cultures treated with various test item concentrations or with the positive or negative control items.

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).

 

Table 4. Cytokinesis-Block Proliferation Index and Level of Necrosis of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in Cytogenetic Assay 2B

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/mL		CBPI				Mean CBPI		% cytostasis		^1) Necrosis
0		1.93	-	1.94		1.93		0		-
10		1.87	-	1.90		1.89		5		-
100		1.90	-	1.95		1.92		1		+
150		1.88	-	1.89		1.88		5		+/++
170		1.88	-	1.90		1.89		5		+/++
190		1.85	-	1.86		1.86		8		++/+++
210		^2)		^2)		^2)		^2)		^2)
230		^2)		^2)		^2)		^2)		^2)
250		^2)		^2)		^2)		^2)		^2)
0.15 MMC-C		1.47	-	1.49		1.48		49		ND
0.23 MMC-C		1.42	-	1.46		1.44		53		ND
0.05 Colch		1.00	-	1.00		1.00		100		ND

 

^1)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
^2)		Cell lysis
ND		Not Determined

Note: All calculations were performed without rounding off.

 

Based on these results (CBPI), the experiment was repeated in cytogenetic assay 2C with the following dose levels:

Without S9-mix: 100, 150, 180, 185, 190, 195, 200, 205, 210 and 215 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).

Table 5 shows the cytokinesis-block proliferation index and the level of necrosis of cultures treated with various test item concentrations or with the positive or negative control items.

 

Table 5.Cytokinesis-Block Proliferation Index and Level of Necrosis of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in Cytogenetic Assay 2C
Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/mL		CBPI				Mean CBPI		% cytostasis		^1) Necrosis
0		1.81	-	1.86		1.83		0		-
100		1.85	-	1.89		1.87		-4		-
150		1.78	-	1.85		1.82		2		+
180		1.78	-	1.79		1.78		6		+++
185		1.78	-	1.82		1.80		4		+++
190		1.77	-	1.82		1.80		4		+++
195		1.71	-	1.76		1.74		12		+++
200		1.70	-	1.75		1.72		13		+++
205		1.72	-	1.75		1.74		12		+++
210		1.74	-	1.77		1.76		9		+++
215		1.65	-	1.70		1.67		19		+++
0.15 MMC-C		1.50	-	1.50		1.50		40		ND
0.23 MMC-C		1.40	-	1.43		1.41		51		ND
0.05 Colch		1.01	-	1.02		1.01		99		ND

 

^1)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
ND		Not Determined

Note: All calculations were performed without rounding off.

 

The results of the CBPI showed that all concentrations tested were not cytotoxic enough and could not be used for scoring of binucleated cells with micronuclei. However, a large number of lytic cells were observed at a concentration of 180 µg/mL and above which indicates necrosis and therefore cytotoxicity. In the second cytogenetic assay and cytogenetic assay 2A and 2B a concentration of 250 µg/mL, 200 µg/mL and 210 µg/mL showed cell lysis (no cells present on the slides) and these concentrations could not be scored for the presence of micronuclei. The next lower concentrations of 200 µg/mL, 150 µg/mL and 190 µg/mL showed a cytotoxicity below 14%. However, these concentrations showed moderate to heavy necrosis and low numbers of cells present on the slides. In this study the CBPI could not be used as a measure of cytotoxicity and therefore the level of necrosis was used to determine the dose levels that were selected for the scoring of micronuclei. The following concentrations were selected:

Without S9-mix: 100, 150, 200 and 215 µg/mL culture medium

(24 hours exposure time, 24 hours harvest time).

The test item induced a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. In addition, a dose related trend was observed (Table 6).

 

Table 6. Number Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with AEROSOL TR-70 E Lyophilized in Cytogenetic Assay 2C
Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration (µg/mL)		Cytostasis (%)		Necrosis ^3)		Number of binucleated cells with micronuclei^1)
						1000	1000	2000
						A	B	A+B
0		0		-		0	0	0
100		-4		-		1	0	1
150		2		+		2	1	3^*
200		13		+++		0	3^2)	3^*
215		19		+++		3	2^2)	5^**
0.15-C		40		ND		18	16	34^****
0.05 Colch		99		ND		20	11	31^****

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or
**** P < 0.0001.

¹⁾   1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

²⁾  971 and 502 binucleated cells were scored for the presence of micronuclei, respectively (see study plan deviation).

^3)-		No necrosis observed
+		Slight necrosis observed
++		Moderate necrosis observed
+++		Heavy necrosis observed
NA		Not applicable

 

Discussion

The ability of the test item to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on the solubility of the test item in the culture medium (3 hours exposure time) or on toxicity by means of the level of necrosis (24 hours exposure time).

The number of binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.

The positive control chemicals, mitomycin C, colchicine and cyclophosphamide produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the first cytogenetic assay, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

In the second cytogenetic assay at the 24 hours continuous exposure time, the test item induced a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. In addition, a dose related trend was observed (p = 0.012). However, the statistically significant increase was caused by the low number of cells with micronuclei in the solvent control cultures. The number of binucleated cells with micronuclei was well within the 95% control limits of the distribution of the historical negative control database at all concentrations tested and was therefore considered not biologically relevant. 

Conclusions:

In conclusion, this test is valid and that AEROSOL TR-70 E Lyophilized is not clastogenic and aneugenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate AEROSOL TR-70 E Lyophilized for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments. The study procedures described in this report are in compliance with the most recent OECD guideline.

A batch with purity of 99.42% was tested in the study; the vehicle of the test item was dimethyl sulfoxide.

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.

In the second cytogenetic assay, the test item was tested up to 215 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. The highest dose level was chosen based on cytotoxicity (level of necrosis).

The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C, colchicine and cyclophosphamide produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the first cytogenetic assay, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei.

In the second cytogenetic assay at the 24 hours continuous exposure time, the test item induced a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. In addition, a dose related trend was observed (p = 0.012). However, the statistically significant increase was caused by the low number of cells with micronuclei in the solvent control cultures. The number of binucleated cells with micronuclei was well within the 95% control limits of the distribution of the historical negative control database at all concentrations tested and was therefore considered not biologically relevant. 

In conclusion, this test is valid and AEROSOL TR-70 E Lyophilized is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

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:

A key 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:

A key 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.

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 second cytogenetic assay, the test item was tested up to 215 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. The highest dose level was chosen based on cytotoxicity (level of necrosis). The test item induced a dose dependent, statistically significant increase in the number of binucleated cells with micronuclei. In addition, a dose related trend was observed (p = 0.012). However, the statistically significant increase was caused by the low number of cells with micronuclei in the solvent control cultures. The number of binucleated cells with micronuclei was well within the 95% control limits of the distribution of the historical negative control database at all concentrations tested and was therefore considered not biologically relevant.

The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C, colchicine and cyclophosphamide produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test was concluded to be valid and the test substance was not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

 

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.