Sodium 1,2-diisobutoxycarbonylethanesulphonate

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 key in vitro genotoxicity assays:

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

-In vitro mammalian cell gene mutation in CHO K1 Chinese hamster ovary (CHO) cells at the Hprt locus was tested negative up to 2000 µg/mL for 5 hours with and without metabolic activation and up to 2000 µg/mL for 24 hours without metabolic activation .

-In vitro Micronucleus in cultured human lymphocytes was tested negative up to 2000 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction (first assay) and in the second assay for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix (second assay).

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:

27 April 2020 to 16 April 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 (Eschericha coli)

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: 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 composition of solution refers to 1000 mL.
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.
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 ± 10°C. The dates of preparation of S9 fractions for this study were 05 September 2019 and 16 January 2020 (Charles River Laboratories Hungary code: E13142 (it was used in the Preliminary Range Finding Test) and E13222 (it was used in main tests), 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.
*0.2 M Sodium Phosphate Buffer, pH 7.4
-Solution A (880 mL)
Na2HPO4 x 12 H2O 71.63 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
-Solution B (120 mL)
NaH2PO4 x H2O 27.6 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
0.2 M Sodium phosphate buffer pH 7.4:
Solution A 880 mL
Solution B 120 mL
- concentration or volume of S9 mix and S9 in the final culture medium:
*Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates were properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
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. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.
*Assay 2 followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Assay 1. Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled.
Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37°C 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. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.
- 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:

Preliminary Range Finding Test: Based on the solubility test, a 100 mg solid fraction of test item /mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and 4 times approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg solid fraction of test item/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used.
Test Item Concentrations in the Mutagenicity Tests (Assay 1 and Assay 2): Based on the results of the preliminary test, a 100 mg solid fraction of test item/mL stock solution was prepared in Distilled water, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg solid fraction of test item/plate.
Examined concentrations in Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg solid fraction of test item/plate.
Examined concentrations in Assay 2 were 5000, 1581, 500, 158.1, 50, 15.8 and 5 μg solid fraction of test item/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; Distilled water

- Justification for choice of solvent/vehicle: 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 solubility of the test item was examined using Distilled water. The test item was soluble at 100 mg test item/mL concentrations in Distilled water. Therefore, 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 in a Preliminary Compatibility Test.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-,nitro-1,2-phenylene-diamine (NPD); in DMSO; 4 µg/plate

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; in DMSO; 2 µg/plate; in all Salmonella strains with S9 and in DMSO 50 µg/plate in 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: two main tests: In Assay 1, the plate incorporation method was used. In Assay 2, the pre-incubation method was used.

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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C in a shaking incubator (Assay 2)
- Exposure duration/duration of treatment: 48(±1) hours at 37°C (Assay1) and 48(±1) hours plus 20 minutes at 37°C (Assay 2)

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 observed in Assay 1. In Assay 2 inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Salmonella typhimurium TA100, TA1537 strains and Escherichia coli strain without metabolic activation at 5000 μg solid fraction of test item/plate concentration.

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 Excel software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Evaluation criteria:

*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.
*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.
*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.

Statistics:

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.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg solid fraction test item/plate concentration in Assay 2 (no cytotoxicity in Assay 1)

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg solid fraction test material/plate concentration in Assay 2 (no cytotoxicity in Assay 1)

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg solid fraction test item/plate concentration in Assay 2 (no cytotoxicity in Assay 1)

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):
- Preliminary Compatibility Test:
The solubility of the test item was examined using Distilled water. The test item was soluble at 100 mg test item/mL concentrations in distilled water (clear solution). Therefore, 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. The test item was soluble in the top solution (clear solution).
-Preliminary Range Finding Test:
Based on the solubility test, a 100 mg solid fraction of test item /mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and 4 times approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg solid fraction of test item/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used. Each sample (including the untreated, negative (solvent) and positive controls) was tested in triplicate. 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: The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA100 strain at 15.81 μg solid fraction of test item/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.21). 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.
In Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 500 μg solid fraction of test item/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.44). 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.

Ames test:
- Signs of toxicity: In the Assay 1 no inhibitory, cytotoxic effect of the test item was observed in all bacterial strains with and without metabolic activation.
In Assay 2 inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Salmonella typhimurium TA100, TA1537 strains and Escherichia coli strain without metabolic activation at 5000 μg solid fraction of test item/plate concentration.

Table 1: 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	19.7	21.0	82.3	93.3	14.7	14.7	14.7	11.7	44.3	45.7
	MF	1.00	0.90	1.02	1.03	1.00	0.98	1.26	0.97	1.02	0.99
DMSO control	Mean	19.3	22.3	--	92.0	--	13.3	11.3	11.0	--	45.3
	MF	0.98	0.96	--	1.01	--	0.89	0.97	0.92	--	0.99
Distilled water control	Mean	19.7	23.3	80.7	90.7	14.7	15.0	11.7	12.0	43.3	46.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	17.0	19.0	78.7	92.7	15.7	14.0	11.0	12.3	42.7	44.7
	MF	0.86	0.81	0.98	1.02	1.07	0.93	0.94	1.03	0.98	0.97
1581	Mean	16.7	20.0	74.7	91.7	14.3	14.7	10.7	11.0	43.0	45.0
	MF	0.85	0.86	0.93	1.01	0.98	0.98	0.91	0.92	0.99	0.98
500	Mean	16.0	17.0	74.3	97.7	13.7	15.7	9.0	12.0	43.0	43.3
	MF	0.81	0.73	0.92	1.08	0.93	1.04	0.77	1.00	0.99	0.94
158.1	Mean	16.3	20.3	84.0	97.7	15.0	15.3	9.0	11.7	42.7	46.0
	MF	0.83	0.87	1.04	1.08	1.02	1.02	0.77	0.97	0.98	1.00
50	Mean	16.0	18.0	86.7	99.0	13.3	14.3	9.7	11.7	43.0	45.3
	MF	0.81	0.77	1.07	1.09	0.91	0.96	0.83	0.97	0.99	0.99
15.81	Mean	16.3	18.0	97.3	102.7	13.0	13.7	8.3	10.0	43.7	45.0
	MF	0.83	0.77	1.21	1.13	0.89	0.91	0.71	0.83	1.01	0.98
NPD (4µg)	Mean	428.0	--	--	--	--	--	--	--	--	--
	MF	22.14	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2456.0	--	2473.3	--	215.3	--	208.0	--	--
	MF	--	109.97	--	26.88	--	16.15	--	18.91	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	254.0
	MF	--	--	--	--	--	--	--	--	--	5.60
SAZ (2µg)	Mean	--	--	1057.3	--	1217.3	--	--	--	--	--
	MF	--	--	13.11	--	83.00	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	429.3	--	--	--
	MF	--	--	--	--	--	--	37.88	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1093.3	--
	MF	--	--	--	--	--	--	--	--	25.23	--

 

Table 2: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	18.7	22.7	94.0	99.0	16.3	12.7	12.7	12.3	43.3	46.7
	MF	1.00	1.01	1.06	0.94	1.23	0.97	1.41	1.00	0.98	1.01
DMSO control	Mean	17.3	20.3	--	105.0	--	13.3	11.7	13.3	--	45.3
	MF	0.93	0.91	--	1.00	--	1.03	1.30	1.08	--	0.98
Distilled water control	Mean	18.7	22.3	89.0	105.0	13.3	13.0	9.0	12.3	44.0	46.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	17.7	16.7	63.0	77.7	12.0	10.7	7.3	11.7	19.3	42.0
	MF	0.95	0.75	0.71	0.74	0.90	0.82	0.81	0.95	0.44	0.91
1581	Mean	17.7	21.0	92.7	96.3	12.7	13.7	12.7	12.0	44.7	49.3
	MF	0.95	0.94	1.04	0.92	0.95	1.05	1.41	0.97	1.02	1.06
500	Mean	18.3	20.7	95.0	95.0	12.0	13.7	13.0	12.0	46.0	49.0
	MF	0.98	0.93	1.07	0.90	0.90	1.05	1.44	0.97	1.05	1.06
158.1	Mean	19.0	23.0	95.0	99.3	12.3	12.7	11.7	12.0	42.7	49.0
	MF	1.02	1.03	1.07	0.95	0.93	0.97	1.30	0.97	0.97	1.06
50	Mean	17.3	22.0	84.3	107.3	12.3	12.7	11.3	13.0	41.0	49.0
	MF	0.93	0.99	0.95	1.02	0.93	0.97	1.26	1.05	0.93	1.06
15.81	Mean	19.0	20.7	100.0	102.7	11.0	13.7	10.3	11.7	44.0	47.7
	MF	1.02	0.93	1.12	0.98	0.83	1.05	1.15	0.95	1.00	1.03
5	Mean	17.7	20.7	87.0	105.3	14.0	13.3	10.7	11.0	42.7	49.0
	MF	0.95	0.93	0.98	1.00	1.05	1.03	1.19	0.89	0.97	1.06
NPD (4µg)	Mean	410.7	--	--	--	--	--	--	--	--	--
	MF	23.69	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2453.3	--	2449.3	--	218.3	--	234.0	--	--
	MF	--	120.66	--	23.33	--	16.38	--	17.55	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	246.0
	MF	--	--	--	--	--	--	--	--	--	5.43
SAZ (2µg)	Mean	--	--	1138.7	--	1189.3	--	--	--	--	--
	MF	--	--	12.79	--	89.20	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	412.0	--	--	--
	MF	--	--	--	--	--	--	35.31	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1060.0	--
	MF	--	--	--	--	--	--	--	--	24.09	--

 

Conclusions:

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 Assay 1.

In Assay 2 inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in Salmonella typhimurium TA100, TA1537 strains and Escherichia coli strain without metabolic activation at 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 substancehad 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:

experimental study

Adequacy of study:

key study

Study period:

12 June 2020 to 17 March 2021

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:

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

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (Hprt) enzyme locus in CHO K1 Chinese hamster ovary cells

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: Sub-line (K1) of Chinese hamster ovary cell line CHO.
The CHO K1 cell line was 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:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: yes: 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:
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.
Four types of Ham's F12 medium were prepared as follows
1) Final concentration in F12-1:
Foetal bovine serum (FBS, heat inactivated): 1 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *0.01 mL/mL
2)Final concentration in F12-5:
Foetal bovine serum (FBS, heat inactivated): 5 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL
3)Final concentration in F12-10:
Foetal bovine serum (FBS, heat inactivated): 10 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL
4)Final concentration in F12-SEL**:
Foetal bovine serum (FBS, heat inactivated): 10 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL

*: Standard content of the antibiotic-antimycotic solution is 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B.
**: Hypoxanthine-free Ham’s F-12 medium was used for preparation of the selection culture medium

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.

- source of S9 : Male Wistar rats (331-421 g animals were 7-9 weeks old at the initiation (Test Facility internal code: E13090) and 444-628 g animals were 17-20 weeks old at the initiation (Test Facility internal code: E13142)) 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 (Test Facility internal code: E13090) and 02 September 2019 (Test Facility internal code: E13142).
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) and 05 September 2019 (Test Facility internal code: E13142, Expiry date: 05 September 2021).

- method of preparation of S9 mix:
The S9-mix was prepared as follows:
a) Concentration of the stock solution:
HEPES* 20 mM
KCl 330 mM
MgCl 2 50 mM
NADP** 40 mM
D-Glucose 6 phosphate (Monosodium salt) 50 mM
F12-10 -
S9 fraction -
b) Concentration in the mix:
HEPES* 0.2 mL/mL
KCl 0.1 mL/mL
MgCl2 0.1 mL/mL
NADP** 0.1 mL/mL
D-Glucose 6 phosphate (Monosodium salt) 0.1 mL/mL
F12-10 0.1 mL/mL
S9 fraction 0.3 mL/mL

*HEPES = N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**NADP= β-Nicotinamide-adenine dinucleotide-phosphate
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 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 (Test Facility internal code: E13090) and 24.5 g/L (Test Facility internal code: E13142). The sterility of the preparation was confirmed. 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.

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:
Assay 3 repeated: 5-hour treatment in the presence and absence of S9-mix:: 2000, 1000, 500, 250, 125 and 62.5 µg/mL
Assay 2: 5-hour treatment in the presence of S9-mix and 24-hour treatment in the absence of S9-mix: 2000, 1000, 500, 250, 125 and 62.5 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
Vehicle of the positive control: Dimethyl sulfoxide (DMSO)

- Justification for choice of solvent/vehicle: Based on the available information results of a trial formulation performed at the Test Facility, distilled water was selected as vehicle for this study in agreement with the Sponsor. The test item was soluble at 200 mg/mL concentration in distilled water. This vehicle (solvent) is compatible with the survival of the cells and the S9 activity.

- Justification for percentage of solvent in the final culture medium:

Untreated negative controls:

yes

Remarks:

to demonstrate that the selected vehicle (solvent) had no mutagenic effects.

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Remarks:

Positive control solutions were freshly prepared at the beginning of the experiments in the testing laboratory in a sterile hood and were filtered sterile using a 0.22 µm syringe filter before use.

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate cultures (in the main tests)
cytotoxicity (plate for survival): triplicate
mutagenicity: for selection of mutants 5 replicate plates; for viability (CE) 3 replicate plates.
- Number of independent experiments: 2 (Assay 3 repeated and Assay 2)

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Assay 3: 5 hours exposure (with and without metabolic activation)
Assay 2: 5 hours exposure with metabolic activation and 24 hours exposure without metabolic activation
- Harvest time after the end of treatment (start of plating for survival):
5 hours exposure experiments: 5 h exposure + 19 h incubation= 24 h
24 hours exposure experiment: 24 h exposure + 0 h incubation= 24h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
Assay 3 and Assay 2: 7 days expression
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells):
Survival: 6 days ((1 d treatment or 5 h treatment and 19 h incubation; 5 d colony growing)
Viability: 13 days (1 d treatment or 5 h treatment and 19 h incubation; 7 d expression; 5 d colony growing)
Mutagenicity: 15 days ((1 d treatment or 5 h treatment and 19 h incubation; 7 d expression; 7 days colony growing in selective medium)
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. 6-thioguanine, 10 µg/mL, 7 days colony growing
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
viability: adjust cell number to 4x10E5 cells/mL, then diluted to 40 cells/mL
mutant phenotype: adjust cell number to 4x10E5 cells/mL

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 5 plates at 4x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 10E6 clonable cells.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA
The assay was considered valid if all of the following criteria were met (based on the relevant guidelines):
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the historical control data.
2. The positive control chemicals induce a statistically significant increase in mutant frequency and should be within the historical data for positive controls.
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.
EVALUATION CRITERIA
The test item was considered to be mutagenic in this assay if the following criteria were 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.
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:

Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 5 plates at 4x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 10E6 clonable cells.
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:

CHO K1

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Assay 3: 5 h exposure

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO K1

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Assay 2: 5 h exposure

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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO K1

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Assay 2: 24 h exposure

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
- Data on pH: In Assay 2 and 3, there were no large changes in pH after treatment in any cases.
- Data on osmolality: In Assay 2 and 3, there were no large changes in osmolality after treatment in any cases.
- Precipitation and time of the determination: In Assay 2 and 3, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation.
- 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).
Six 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.
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : No dose response to the treatment was observed (a trend analysis showed no effect of treatment).
- Statistical analysis; p-value if any

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency
Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
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.

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”

Historical Control Data

(updated on 17 October 2017 using data of GLP studies)

	Mutation frequency (Number of 6-TG resistant mutants per 106 clonable cells)
	Untreated control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	18.3	20.7	19.0
standard deviation	15.1	16.4	17.2
minimum	5.1	5.5	3.3
maximum	64.1	55.5	58.0
n	27	13	14
	DMSO control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	21.8	18.9	18.4
standard deviation	15.9	11.6	14.4
minimum	5.4	6.5	6.8
maximum	57.3	47.4	48.5
n	29	13	14
	Distilled water / Water based vehicle control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	11.5	9.1	15.5
standard deviation	3.8	3.4	5.6
minimum	6.1	5.2	9.2
maximum	15.8	11.6	20.1
n	6	3	3
	Positive controls
	DMBA	EMS	EMS
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	905.2	445.6	1176.6
standard deviation	562.7	118.6	610.9
minimum	141.2	239.6	363.1
maximum	2119.4	636.6	2449.8
n	27	13	14

 

Conclusions:

In conclusion, no mutagenic effect of Exodiss IB was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.

Executive summary:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the Hprt locus to evaluate the potential of the registered substance 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.

In Assay 1, 5-hour treatment was performed with and without metabolic activation (in the presence and absence of S9 mix). In the assay (with and without metabolic activation) the number of the mutation frequencies in case of the negative (vehicle) controls and the positive controls were lower than the number of the mutation frequencies of the general historical control range. Therefore, the acceptance criteria of the assay were not met according to the Study Plan. The Assay 1 was declared invalid and was repeated in an additional experiment (Assay 3) with the same experimental conditions to meet the acceptability criteria. The data of the invalid experiment is not reported, but all the relevant documentation will be kept and archived in the raw data binder.

Distilled water 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 3 repeated

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

2000, 1000, 500, 250, 125 and 62.5 µg/mL

Assay 2

5-hour treatment in the presence of S9-mix and 24-hour treatment in the absence of S9-mix: 2000, 1000, 500, 250, 125 and 62.5 µg/mL

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 2 and 3, no insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.

In Assay 2 and 3, in the presence and absence of S9-mix (5-hour or 24-hour treatment), no cytotoxicity of the test item was observed at any examined concentrations.

Statistically significant increase in the mutation frequency (at p<0.05 or p<0.01 level) was only observed in Assay 3 at concentration levels of 1000, 500, 125 µg/mL and at the negative control for the positive control (DMBA), although the observed values were within the general historical control range. Furthermore, the observed mutant frequencies were 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. In the absence of metabolic activation, no statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data. In overall, this experiment was concluded as negative.

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all evaluated 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 evaluated assays. Six 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 Exodiss IB was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June 2020 to 9 March 2021

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)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Cultured peripheral human lymphocytes. Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years).
- The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2019) are presented below:
Dose-range finding study: age 28, AGT = 13.3 h
First cytogenetic assay: age 26, AGT = 13.2 h
Second cytogenetic assay: age 25, AGT = 13.0 h

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)
- Whether blood from different donors were pooled or not: Not specified. (Immediately after blood collection lymphocyte cultures were started.)
- Mitogen used for lymphocytes: 0.1 mL (9 mg/mL) Phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
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.
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 51 - 95%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium, was used as solvent for the positive controls.

Cytokinesis block (if used):

Cytochalasin B (Sigma; 5 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat S9 homogenate 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).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life Technologies).
The above solution was filter (0.22 μm)-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.
- concentration or volume of S9 mix and S9 in the final culture medium : 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 (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Not specified.

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 2000 µg/mL or showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control. A concentration of 2000 µg/mL showed no precipitation in the culture medium and was used as the highest concentration of the test item.
-In the dose-range finding test, blood cultures were treated with 62.5, 125, 250, 500, 1000 and 2000 µg test item/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
-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: 500, 1000, 1500 and 2000 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).
The following dose levels were selected for scoring of micronuclei:
Without and with S9-mix: 500, 1000 and 2000 µg/mL culture medium(3 hours exposure time, 27 hours harvest time).
-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: 100, 500, 1000, 1500, 1750 and 2000 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).
Dose levels of 1750 and 2000 µg/mL exceeded the prescribed cytotoxicity levels and were not selected for the scoring of micronuclei.
The following dose levels were selected for the scoring of micronuclei:
Without S9-mix : 100, 1000 and 1500 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium (culture medium, Life Technologies, Bleiswijk, The Netherlands).
Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium, was used as solvent for the positive controls.

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 medium

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Remarks:

Vehicle of the positive controls: Hanks’ Balanced Salt Solution (HBSS) without calcium and magnesium

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments: 2 (First and Second cytogenetic assay)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium (RPMI 1640 medium)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
First cytogenetic assay (in the absence and presence of S9-fraction): 3 hours exposure
Second cytogenetic assay (in the absence of S9-mix): 24 hours exposure
- Harvest time after the end of treatment (sampling/recovery times):
First cytogenetic assay (in the absence and presence of S9-fraction): 27 hours harvest time
Second cytogenetic assay (in the absence of S9-mix): 24 hours harvest time

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.
Cytochalasine B (Sigma; 5 μg/mL) 24 hours 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 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the 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 (Merck)/ether (Merck) 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 min with 6.7% (v/v) Giemsa (Merck) 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 in 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): To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) 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.
- 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).
- Any supplementary information relevant to cytotoxicity:
A minimum of 500 cells (with a maximum deviation of 5%) 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).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}
(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)
CBPI =
Total number of cells

t = test item or control treatment culture
c = vehicle control culture
Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The highest concentration analyzed was the recommended 2000 µg/mL or were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
During or after exposure of the stimulated human lymphocytes to the test item, cells were cultured to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells. Micronuclei are small particles consisting of acentric chromosome fragments (clastogenic event) or whole chromosomes (aneugenic event leading to chromosome loss), which are unable to migrate to the poles during the anaphase stage of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm.

Evaluation criteria:

ACCEPTABILITY 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).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was 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,
one-sided, 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.

Key result

Species / strain:

lymphocytes: cultured human peripheral lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Second cytogenetic assay: 24 h exposure, 24 h harvest

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Dose levels of 1750 and 2000 µg/mL exceeded the prescribed cytotoxicity levels and were not selected for the scoring of micronuclei.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: cultured human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

First cytogenetic assay: 3 h exposure, 27 h harvest

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded. The pH of a concentration of 2000 µg/mL was 8.0 (compared to 7.7 in the solvent control).
- Data on osmolality: The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded. The osmolarity of a concentration of 2000 µg/mL was 294 mOsm/kg (compared to 285 mOsm/kg in the solvent control).
- Precipitation and time of the determination: A concentration of 2000 µg/mL showed no precipitation in the culture medium and was used as the highest concentration of the test item.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. The test item was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma; 5 µg/mL) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was 2000 µg/mL. This is the highest dose level recommended in the guideline for testing.
After 3 hours exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of the test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 2000 µg/mL or showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.
In the dose-range finding test, blood cultures were treated with 62.5, 125, 250, 500, 1000 and 2000 µg test item/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.

STUDY RESULTS
See under “Any other information on results incl. tables”

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:
Distribution historical positive control data from experiments performed between June2017 and June 2020.
*Binucleated cells, -S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 46.9
SD: 20.1
n: 92
Lower Control Limit (95% Control Limits): 7
Upper Control Limit (95% Control Limits): 86
*Binucleated cells, -S9 mix, 24 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 42.4
SD: 17.3
n: 94
Lower Control Limit (95% Control Limits): 8
Upper Control Limit (95% Control Limits): 76
*Binucleated cells, +S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 34.1
SD: 13.8
n: 98
Lower Control Limit (95% Control Limits): 7
Upper Control Limit (95% Control Limits): 61
SD = Standard deviation
n = Number of observations

- Negative (solvent/vehicle) historical control data:
Distribution historical negative control data from experiments performed between June 2017 and June 2020.
*Binucleated cells, -S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 6.1
SD: 4.1
n: 89
Lower Control Limit (95% Control Limits): -2
Upper Control Limit (95% Control Limits): 14
*Binucleated cells, -S9 mix, 24 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 5.8
SD: 4.1
n: 87
Lower Control Limit (95% Control Limits): -2
Upper Control Limit (95% Control Limits): 14
*Binucleated cells, +S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 6.4
SD: 4.1
n: 91
Lower Control Limit (95% Control Limits): -2
Upper Control Limit (95% Control Limits): 14
SD = Standard deviation
n = Number of observations

Table 1. Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with Exodiss IB in the Dose-range Finding Test

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0	273	189	38	1.53	0
62.5	245	212	43	1.60	-12
125	280	179	41	1.52	2
250	346	132	22	1.35	34
500	315	154	31	1.43	18
1000	308	167	32	1.46	14
2000	311	157	32	1.44	17

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time

 

Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0	312	170	19	1.42	0
62.5	281	193	36	1.52	-25
125	273	186	43	1.54	-31
250	311	163	26	1.43	-4
500	307	168	35	1.47	-12
1000	257	206	37	1.56	-35
2000	324	155	21	1.39	5

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0	321	144	35	1.43	0
62.5	292	173	35	1.49	-14
125	364	111	25	1.32	25
250	329	142	34	1.42	3
500	371	107	22	1.30	29
1000	391	100	9	1.24	45
2000	413	82	5	1.18	57

Note: All calculations were performed without rounding off.

Table 2. Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with Exodiss IB in the First Cytogenetic Assay
Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration µg/mL	CBPI	Mean CBPI	% cytostasis
0	1.83 - 1.92	1.87	0
500	1.78 - 1.83	1.80	8
1000	1.72 - 1.87	1.80	9
1500	1.76 - 1.83	1.79	9
2000	1.72 - 1.84	1.78	10
0.25 MMC-C	1.49 - 1.60	1.55	37
0.38 MMC-C	1.48 - 1.51	1.49	44
0.1 Colch	1.12 - 1.13	1.13	86

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time

Concentration µg/mL	CBPI	Mean CBPI	% cytostasis
0	1.77 - 1.85	1.81	0
500	1.80 - 1.86	1.83	-2
1000	1.77 - 1.78	1.77	5
1500	1.76 - 1.88	1.82	-1
2000	1.70 - 1.85	1.77	5
15 CP	1.29 - 1.31	1.30	63
17.5 CP	1.25 - 1.25	1.25	69

Note: All calculations were performed without rounding off.

Table 3. Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Exodiss IB 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	0	1	1
500	8	1	0	1
1000	9	1	0	1
2000	10	1	0	1
0.25 MMC-C	37	4	16	20***
0.38 MMC-C	44	36	31	67***
0.1 Colch	86	22	13	35***

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	1	0	1
500	-2	0	0	0
1000	5	2	0	2
2000	5	1	0	1
15 CP	63	21	20	41***

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

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

Table 4. Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with Exodiss IB 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
0	1.36 - 1.44	1.40	0
100	1.33 - 1.38	1.36	11
500	1.34 - 1.34	1.34	15
1000	1.26 - 1.31	1.29	28
1500	1.19 - 1.20	1.19	52
1750	1.14 - 1.16	1.15	63
2000	1.12 - 1.13	1.13	69
0.15 MMC-C	1.16 - 1.18	1.17	58
0.23 MMC-C	1.08 - 1.13	1.10	74
0.05 Colch	1.00 - 1.00	1.00	100

Note: All calculations were performed without rounding off.

Table 5. Number Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Exodiss IB in the Second Cytogenetic Assay
Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	2	2	4
100	11	0	1	1
1000	28	1	0	1
1500	52	1	0	1
0.15 MMC-C	58	28	20	48***
0.05 Colch	100	0^2	1^2	1

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

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

²⁾    572 and 478 binucleated cells were scored for the presence of micronuclei, respectively.

Conclusions:

In conclusion, this test is valid and the registered substance is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the registered substance 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.

Batch 2002149071 of the test item was a colourless liquid. A correction factor of 2.17 was used to correct for the purity (46.0%; consisting of the active ingredient (41.4%) and impurities). The vehicle of the test item was culture medium.

In the first cytogenetic assay, the test item was tested up to 2000 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. This is the highest dose level recommended in the guideline for testing.

In the second cytogenetic assay, the test item was tested up to 1500 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

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 and cyclophosphamide both 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 item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, this test is valid and the registered substance 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

A 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. The study included 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 Assay 1. In Assay 2 inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in S. typhimurium TA100, TA1537 strains and Escherichia coli strain without metabolic activation at 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 (Orovecz, 2021).

 

An in vitro mammalian cell assay was performed according to OECD Test Guidance No. 476 for the registered substance in CHO K1 Chinese hamster ovary (CHO) cells at the Hprt locus to evaluate the potential to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation and for 24 hours without metabolic activation. In Assay 1, 5-hour treatment was performed with and without metabolic activation (in the presence and absence of metabolic activation). In the assay (with and without metabolic activation) the number of the mutation frequencies in case of the negative (vehicle) controls and the positive controls were lower than the number of the mutation frequencies of the general historical control range. Therefore, the acceptance criteria of the assay were not met according to the Study Plan. The Assay 1 was declared invalid and was repeated in an additional experiment (Assay 3) with the same experimental conditions to meet the acceptability criteria. Distilled water 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:

- 5-hour treatment with/without metabolic activation: 2000, 1000, 500, 250, 125 and 62.5 µg solid fraction of the test item/mL.

- 5-hour treatment with metabolic activation and 24-hour treatment without metabolic activation: 2000, 1000, 500, 250, 125 and 62.5 µg solid fraction of the test item/mL.

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.

No insolubility was detected in the final treatment medium at the end of the treatment with or without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases. Further, no cytotoxicity of the test item was observed at any examined concentrations.

Statistically significant increase in the mutation frequency (at p<0.05 or p<0.01 level) was only observed in Assay 3 at concentration levels of 1000, 500, 125 µg/mL and at the negative control for the positive control (DMBA), although the observed values were within the general historical control range. Furthermore, the observed mutant frequencies were 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. In the absence of metabolic activation, no statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data. In overall, this experiment was concluded as negative.

The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all evaluated 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 evaluated assays. Six 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 the registered subsstance was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values (Orosz, 2021).

 

An in vitro Micronucleus assay according to OECD Test Guidance No. 487 was performed for the registered substance 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 vehicle of the test item was culture medium. In the first cytogenetic assay, the test item was tested up to 2000 µg solid fraction of the test item/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. This is the highest dose level recommended in the guideline for testing. In the second cytogenetic assay, the test item was tested up to 1500 µg solid fraction of the test item/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. 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 and cyclophosphamide both 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 item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. In conclusion, this test is valid and the registered substance is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report (Verbaan, 2021).

 

 

 

Justification for classification or non-classification

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