Sodium 1,4-dicyclohexyl sulphonatosuccinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered substance (solid fraction) was tested in following in vitro genotoxicity assay: Bacterial Reverse Mutation Assay using Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli (WP2 uvrA) was tested negative up to concentrations of 5000 μg solid fraction of test item /plate in the presence and absence of metabolic activation (Plate incorporation and Pre incubation).

Read across was performed for the genotoxicity endpoints with category members Docusate sodium (CAS 577-11-7), and Sodium 1,4-diisodecyl sulphonatosuccinate (CAS 29857-13-4) and Sodium 1,4 – diisotridecyl sulphonatosuccinate (CAS 55184-72-0) based on the read across justification for the Diester Sulfosuccinate Category group.

Based on the experimental data, there was no evidence for bacterial and mammalian gene mutation potential, nor was there any indication of chromosome aberration potential. In conclusion, based on the registered substance and on the read across with experimental data, there is no genotoxic potential for the test substance.

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:

9 April 2020 to 11 June 2021

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine (Salmonella); 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:

- source of S9
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Charles River Laboratories Hungary Kft. according to Ames et al. [1] and Maron and Ames [2]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
The composition of solution refers to 1000 mL.
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
*Induction of Liver Enzymes
Male Wistar rats (444-628 g, animals were 17-20 weeks old and 433-642 g, animals were 13-17 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 were 02 September 2019 and 13 January 2020.
* Preparation of Rat Liver Homogenate S9 Fraction
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 were 05 September 2019 and 16 January 2020 (Charles River Laboratories Hungary code: E13142 and E13222, Expiry date: 05 September 2021 and 16 January 2022).
- method of preparation of S9 mix
The 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.
- concentration or volume of S9 mix and S9 in the final culture medium :
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
(S9 Mix (containing 10 % (v/v) of S9)
- 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 were determined to be 24.5 g/L and 28.0 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:

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 solid fraction of test item /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 solid fraction of test item/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg solid fraction of test item /plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled Water (vehicle for the test item)
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. The following chemicals were used for vehicle (solvent) control groups: Dimethyl sulfoxide (DMSO) and Distilled Water
- Justification for choice of solvent/vehicle: 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 solid fraction of test item /mL concentration using Distilled water, DMSO and DMF. Due to the better biocompatibility Distilled water was selected as vehicle for the study. The obtained stock formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: Preliminary Range Finding Test, Assay 1 (plate incorporation), Assay 2 (preincubation)

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

Assay 1: TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No
- Exposure duration/duration of treatment: 48(±1) hours at 37°C
Assay 2: TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Yes (20 minutes at 37°C in a shaking incubator)
- Exposure duration/duration of treatment: 20 minutes + 48(±1) hours at 37°C

FOR GENE MUTATION:

Evaluation criteria:

(1)Criteria for Validity:
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.
(2) Criteria for a Positive Response:
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.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
(3) Criteria for a Negative Response:
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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Assay 2 at 5000 µg solid fraction/plate 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:

cytotoxicity

Remarks:

In Assay 2 at 1581 µg solid fraction/ plate without S9. Reduced colony number in Assy 2 at 5000 µg/solid fraction/plate without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Assay 2 at 1581 µg solid fraction/plate without S9.

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:

cytotoxicity

Remarks:

In Assay 2 at 1581 µg solid fraction/plate 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 nor precipitates, but tested up to recommended limit concentrations

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: No precipitate was detected on the plates in the main tests in the all examined Bacterial strains with and without metabolic activation.

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 solid fraction of test item/plate.
No precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation.
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: See Table 2 and 3 under Any other information on results incl. tables

Ames test:
- Signs of toxicity: 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. In Assay 1 no inhibitory, cytotoxic effect of the test item was observed in all Salmonella typhimurium bacterial strains and Escherichia coli WP2 uvrA strain with and without metabolic activation.
In Assay 2 inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development) was observed in all Salmonella typhimurium strains without metabolic activation at 5000 μg solid fraction of test item/plate concentration and in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation on the plates at the 1581 μg solid fraction of test item/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 solid fraction of test item/plate concentration.
-Revertants:
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 strain at 15.81 μg solid fraction of test item/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.24). However, there was no dose-response relationship, the observed mutation factor values were above 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 and 5 μg solid fraction of test item/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.33). 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)
- Positive historical control data: See under Any other information on results incl. tables
- Negative (solvent/vehicle) historical control data: See under Any other information on results incl. tables

Table 1. Historical Control Data  (Period of 2015-2020)

Untreated control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E.coli	TA98	TA100	TA1535	TA1537	E.coli
Mean	20.9	98.3	13.2	8.8	40.6	25.6	104.6	12.3	9.9	44.1
St.dev.	4.4	12.7	3.5	3.2	9.4	5.8	13.2	3.2	3.6	9.4
Range	11-50	67-152	1-33	2-26	14-77	13-54	67-152	3-39	1-29	16-89
n	1296	1297	1302	1314	1302	1311	1314	1314	1323	1299
DMSO control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.5	95.4	13.0	8.4	39.6	24.9	101.0	11.8	9.4	43.0
St.dev.	4.2	12.1	3.5	3.1	9.7	5.4	13.5	3.1	3.5	9.5
Range	7-41	60-145	3-34	1-27	12-75	11-50	53-165	2-33	1-29	9-76
n	1428	1419	1425	1449	1425	1443	1443	1449	1455	1431
Distilled water control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.5	97.5	13.2	9.3	41.5	25.9	103.1	12.1	10.4	44.6
St.dev.	4.4	12.5	3.3	3.4	9.1	5.6	13.6	3.1	3.7	9.0
Range	13-37	58-150	2-32	3-20	17-72	15-45	59-164	3-34	3-24	13-76
n	288	1332	1332	303	1341	291	1314	1326	300	1320
DMF control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	19.6	93.5	12.9	9.2	42.0	23.8	96.2	11.9	10.5	43.3
St.dev.	3.9	13.3	3.1	3.4	10.8	5.5	13.2	3.0	3.6	10.9
Range	11-33	57-121	6-24	2-18	16-70	11-37	68-143	3-21	3-19	18-72
n	114	114	114	117	111	114	114	114	114	111
Acetone control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.9	97.1	12.9	8.2	40.1	25.5	102.2	11.5	9.1	44.1
St.dev.	4.3	10.0	3.6	2.8	8.5	5.6	11.3	3.0	3.2	8.7
Range	11-35	63-126	6-32	2-17	21-63	16-44	66-132	4-19	1-19	20-70
n	219	222	222	225	219	219	222	225	225	219
Positive reference control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	395.9	1130.9	1138.8	413.3	1020.6	2391.6	2408.0	219.0	214.0	239.5
St.dev.	99.4	82.5	102.0	34.0	114.4	146.2	115.7	32.1	22.3	35.6
Range	182-2336	536-1480	568-2004	208-629	488-2496	312-2736	1116-3104	101-418	147-424	127-384
n	1296	1296	1302	1314	1305	1311	1317	1317	1323	1299

TA98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella typhimurium TA1537, E. coli: Escherichia coli WP2 uvrA; n: number of cases

98: Salmonella typhimurium TA98, TA100: Salmonella typhimurium TA100, TA1535: Salmonella typhimurium TA1535, TA1537: Salmonella

Table 2: Summary Table of the Assay 1

 

Concentrations (μg solid fraction of test item /plate	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	21.3	22.7	86.3	111.7	16.7	17.0	12.0	13.3	40.0	43.0
	MF	0.90	0.94	0.95	1.06	1.09	1.02	1.09	1.03	1.00	1.00
DMSO control	Mean	22.0	23.0	--	116.7	--	14.7	130.	12.0	--	42.3
	MF	0.93	0.96	--	1.10	--	0.88	1.18	0.92	--	0.98
Distilled water control	Mean	23.7	24.0	90.7	105.7	15.3	16.7	11.0	13.0	40.0	43.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	16.7	23.7	100.0	111.3	14.0	14.0	10.3	11.0	38.7	41.7
	MF	0.70	0.99	1.10	1.05	0.91	0.84	0.94	0.85	0.97	0.97
1581	Mean	17.7	23.0	98.7	111.3	14.0	12.0	9.7	13.0	38.0	40.3
	MF	0.75	0.96	1.09	1.05	0.91	0.72	0.88	1.00	0.95	0.94
500	Mean	19.0	21.7	108.3	110.3	14.7	14.7	11.0	11.7	39.7	42.7
	MF	0.80	0.90	1.19	1.04	0.96	0.88	1.00	0.90	0.99	0.99
158.1	Mean	17.7	22.0	100.0	109.0	13.0	15.7	10.3	12.3	41.3	42.7
	MF	0.75	0.92	1.10	1.03	0.85	0.94	0.94	0.95	1.03	0.99
50	Mean	17.0	24.0	99.0	120.7	17.7	12.0	10.7	11.7	37.0	42.3
	MF	0.72	1.00	1.09	1.14	1.15	0.72	0.97	0.90	0.93	0.98
15.81	Mean	17.0	20.3	95.7	116.7	16.3	14.0	13.7	9.3	40.0	42.7
	MF	0.72	0.85	1.06	1.10	1.07	0.84	1.24	0.72	1.00	0.99
NDP (4µg)	Mean	409.3	--	--	--	--	--	--	--	--	--
	MF	18.61	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2469.3	--	2472.0	--	212.7	--	210.7	--	--
	MF	--	107.36	--	21.19	--	14.50	--	17.56	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	246.7
	MF	--	--	--	--	--	--	--	--	--	5.83
SAZ (2µg)	Mean	--	--	1128.0	--	1081.3	--	--	--	--	--
	MF	--	--	12.44	--	70.52	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	405.3	--	--	--
	MF	--	--	--	--	--	--	31.18	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1098.7	--
	MF	--	--	--	--	--	--	--	--	24.47	--

 

Table 3: Summary Table of the Assay 2

 

Concentrations (μg solid fraction of test item /plate	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	17.3	21.0	82.3	98.3	17.0	17.7	9.3	11.0	42.3	44.0
	MF	0.93	0.84	0.98	0.95	1.02	1.00	1.17	0.89	1.03	1.01
DMSO control	Mean	18.3	23.3	--	94.0	--	17.0	11.0	11.7	--	42.7
	MF	0.98	0.93	--	0.91	--	0.96	1.38	0.95	--	0.98
Distilled water control	Mean	18.7	25.0	84.0	103.3	16.7	17.7	8.0	12.3	41.0	43.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	11.3	21.3	51.7	93.3	8.3	17.0	0.0	6.3	23.0	43.7
	MF	0.61	0.85	0.62	0.90	0.50	0.96	0.00	0.51	0.56	1.00
1581	Mean	16.7	22.0	56.0	102.7	12.0	17.7	8.0	12.0	37.3	47.0
	MF	0.89	0.88	0.67	0.99	0.72	1.00	1.00	0.97	0.91	1.08
500	Mean	16.7	22.3	92.0	103.7	16.7	16.0	10.3	11.3	42.3	47.0
	MF	0.89	0.89	1.10	1.00	1.00	0.91	1.29	0.92	1.03	1.08
158.1	Mean	17.3	23.0	92.7	104.7	17.7	15.3	9.0	11.7	43.0	46.0
	MF	0.93	0.92	1.10	1.01	1.06	0.87	1.13	0.95	1.05	1.05
50	Mean	16.7	20.7	91.3	96.7	15.7	16.7	10.7	9.7	43.7	43.7
	MF	0.89	0.83	1.09	0.94	0.94	0.94	1.33	0.78	1.07	1.00
15.81	Mean	17.0	26.7	94.7	108.0	14.7	17.3	10.0	11.7	41.7	46.7
	MF	0.91	1.07	1.13	1.05	0.88	0.98	1.25	0.95	1.02	1.07
5	Mean	18.0	25.3	82.3	95.0	15.7	16.7	10.7	12.0	40.7	46.0
	MF	0.96	1.01	0.98	0.92	0.94	0.94	1.33	0.97	0.99	1.05
NDP (4µg)	Mean	421.3	--	--	--	--	--	--	--	--	--
	MF	22.98	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2468.0	--	2461.3	--	219.0	--	207.7	--	--
	MF	--	105.77	--	26.18	--	12.88	--	17.80	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	246.7
	MF	--	--	--	--	--	--	--	--	--	5.78
SAZ (2µg)	Mean	--	--	1082.7	--	1217.3	--	--	--	--	--
	MF	--	--	12.89	--	73.04	--	--	---	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	414.7	--	--	--
	MF	--	--	--	--	--	--	37.70	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1056.0	--
	MF	--	--	--	--	--	--	--	--	25.76	--

 

um TA1537, E. coli: Escherichia coli WP2 uvrA; n: number of cases

Conclusions:

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 registered substance 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 WP2 uvrA) 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 solid fraction of test item /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 solid fraction of test item/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg solid fraction of test item /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.

No precipitate was detected on the plates in the main tests in all examined Bacterial strains with and without metabolic activation.

No inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test.

No inhibitory, cytotoxic effect of the test item was observed in Assay 1.

In Assay 2 inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development) was observed in all Salmonella typhimurium strains without metabolic activation at 5000 μg solid fraction of test item/plate concentration and in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation on the plates at the 1581 μg solid fraction of test item/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 solid fraction of test item /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 registered substance had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached Read-across justification

Reason / purpose for cross-reference:

read-across source

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 read-across test item 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 read-across test item 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 read-aross test item 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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached Read-across justification

Reason / purpose for cross-reference:

read-across source

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 read-across test item 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 read-across test item 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 read-across test item 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)

Additional information

Bacterial mutagenicity

A key Bacterial Reverse Mutation Assay according to OECD Test Guidance No. 471 was performed for the registered substance using Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli (WP2 uvrA) in the presence and absence of metabolic activation (Tóth-Gönczöl, 2021). 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 solid fraction of test item /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 solid fraction of test item/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg solid fraction of test item/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. No precipitate was detected on the plates in the main tests in all examined Bacterial strains with and without metabolic activation. No inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test. No inhibitory, cytotoxic effect of the test item was observed in Assay 1. In Assay 2 inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development) was observed in all Salmonella typhimurium strains without metabolic activation at 5000 μg solid fraction of test item/plate concentration and in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation on the plates at the 1581 μg solid fraction of test item/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 solid fraction of test item /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 registered test substance CAS 23386-52-9 had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study (Tóth-Gönczöl, 2021).

 

Read-across

- A supporting study for bacterial mutagenicity was available for read-across substance Docusate sodium (CAS 577-11-7) with 5 Salmonella thyphimurium strains (Clare, 1993); the study was conducted according to OECD 471 and GLP guidelines, and was considered to be reliable, adequate and relevant. After a range-finder experiment showing cytotoxicity at the highest concentration of 5000 µg/plate, maximum test concentrations of 1000 and 2500 µg/plate were chosen for the main experiment 1. In experiment 1, concentrations were close to the limit of toxicity, therefore for experiment 2, concentrations for all strains were maximally 2000 µg/plate without S9 and 2500 µg/plate with S9. In both experiments, Docusate sodium did not result in statistically significant increases in revertant number of colonies, both with and without S9.

- In a supporting Ames test, read-across substance Sodium 1,4-diisodecyl sulphonatosuccinate (CAS 29857-13-4) containing 63-67% active ingredient was tested for mutagenicity with the strains TA100, TA1535, TA1537, TA1538, TA98 of Salmonella typhimurium and Escherichia coli WP2uvrA (Müller, 1988). The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test compound proved not to be toxic to the bacterial strains. 5000 µg/plate was chosen as top dose level for the mutagenicity study. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Netzer SB 10 did not result in relevant increases in the number of revertant colonies. It can be stated that the test substance is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

- A supporting Bacterial Reverse Mutation Assay was conducted with read-across substance Sodium 1,4 – diisotridecyl sulphonatosuccinate (CAS 55184-72-0) 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 TA98 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 TA100 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

Read-across

- A key in vitro mammalian cell assay was performed with read-across substance Sodium 1,4 – diisotridecyl sulphonatosuccinate (CAS 55184-72-0) 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.

 

Chromosomal aberration

Read-across

- A key in vitro micronucleus test in human peripheral lymphocytes was conducted with read-across substance Sodium 1,4 – diisotridecyl sulphonatosuccinate (CAS 55184-72-0) 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.

 

Conclusion

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial mutagenicity (registered and read-across substances) and mammalian mutagenicity and chromosomal aberration (read-across substances). Based on the available results, there were no indications of mutagenicity or genotoxicity, and no further testing is needed. The substance can be considered to have no mutagenic or genotoxic potential.

Further information supporting the safety of the test substance is provided in the read-across justification (read-across substances: Docusate sodium (CAS No. 577-11-7), Sodium 1,4-diisodecyl sulphonatosuccinate (CAS 29857-13-4) and Sodium 1,4 – diisotridecyl sulphonatosuccinate (CAS 55184-72-0)) for the Diester category, showing that the group was negative in vitro for bacterial/mammalian mutagenicity and chromosomal aberration and in vivo for micronuclei formation  (justification with data matrix separately attached in Section 13).

 

Justification for classification or non-classification

Based on the negative findings with the registered substance and read across substances, the test item does not need to be classified and has no obligatory labelling requirement for genotoxicity according to the CLP regulation (No. 1272/2008 of 16 December 2008).