2-piperidinoethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- OECD 471 - Bacterial Reverse Mutation Assay (GLP): negative

- OECD 476 - in vitro Mammalian Cell Gene Mutation Assay (GLP): negative

- OECD 487 - In vitro Mammalian Cell Micronucleus Test (GLP): negative

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:

Sep 2018

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:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: B1056-070518
- Content: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI); water content: 0.1 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions is guaranteed until 07 May 2019 as indicated by the sponsor, and the sponsor holds this responsibility
- Stability of the test substance in the solvent/vehicle: Due to the use of water as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in ultrapure water. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in ultrapure water

Target gene:

his, trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (supplemented with cofactors) derived from phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (SPT; 1st Experiment)
0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (PIT; 2nd Experiment; conducted, because noo mutagenicity was observed in the standard plate test)

3 test plates per dose or per control

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (60 μg/plate (E.coli) or 2.5 µg/plate (S.typhimurium), in DMSO; with S9 mix); N-methyl-N'-nitro-N-nitrosoguanidine (5 μg/plate, in DMSO; without S9 mix); 4-nitro-o-phenylenediamine (10 μg/plate, in DMSO; without S9 mix)

Remarks:

Sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at testing: approx. 10^9 cells per mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours

DETERMINATION OF CYTOTOXICITY
- Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Evaluation criteria:

Acceptance criteria
The experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

decrease in the number of revertants in the preincubation assay without S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

decrease in the number of revertants in the preincubation assay without S9 mix at 2500-5000 µg/plate and with S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

decrease in the number of revertants in the preincubation assay with and without S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

decrease in the number of revertants in the preincubation assay without S9 mix at 1000-5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Precipitation: No precipitation of the test substance was found with and without S9 mix.

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT)

33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A bacteriotoxic effect was only observed in the preoncubation test depending on the strain and test conditions from about 1000 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Nov 2018 - 29 Mar 2019

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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland Pfalz

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: B1056-070518
- Date of production: 07 May 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period was guaranteed until 07 May 2020 as indicated by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle: Good solubility of the test substance in culture medium. Due to the use of culture medium (Ham´s F12) as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in culture medium (Ham’s F12). The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was pipetted thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance solutions were prepared immediately before administration.

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Due to the pH shift observed at the dose group 1300.0 μg/mL (stock solution) in culture medium in the pre-test, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32% (w/v) HCl. No test substance precipitation in culture medium occurred up to the highest applied concentration by the end of treatment in the absence and presence of S9 mix.

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO (Chinese hamster ovary) cell line; source not specified

For cell lines:
- Absence of Mycoplasma contamination: Each batch of cells used for mutagenicity testing was checked for mycoplasma contamination.
- Number of passages if applicable: At least 2 passages were performed before cells were taken for the experiment.
- Methods for maintenance in cell culture: Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm2 plastic flasks).
- Cell cycle length, doubling time or proliferation index: doubling time of about 12 - 16 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes. During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pre-treatment with "HAT" medium.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:

Culture medium:
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS).

Treatment medium (without S9 mix):
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) FCS.

Treatment medium (with S9 mix):
Ham's F12 medium containing stable glutamine and hypoxanthine.

Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10^-3 mg/mL)
- aminopterin (0.18 x 10^-3 mg/mL)
- thymidine (3.88 x 10^-3 mg/mL)
- 10% (v/v) FCS

Selection medium ("TG" medium): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with:
- 6-thioguanine (10 μg/mL)
- 10% (v/v) FCS

All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

- source of S9 : prepared at the testing facility
- method of preparation of S9 mix: The S9 fraction was prepared according to Ames et al.. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days. 24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used.
The concentrations of the cofactors in the S9 mix were:
− MgCl2 8 mM
− KCl 33 mM
− glucose-6-phosphate 5 mM
− NADP 4 mM
− phosphate buffer (pH 7.4) 15 mM
The phosphate buffer was prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.
- concentration or volume of S9 mix and S9 in the final culture medium: 20% S9 mix; 2% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): metabolic activity confirmed by the increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA

Test concentrations with justification for top dose:

The doses/concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test (experimental conduct with records and documentation in general accordance with the GLP principles).
In the pre-test for toxicity based on the purity and the molecular weight of the test substance 1300.0 μg/mL (approx. 10 mM) was used as top concentration both with and without S9 mix at 4-hour exposure time. The pre-test was performed following the method described for the main experiment. The relative survival (RS) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. A slight pH shift was observed in the stock solution prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32% (w/v) HCl. In the pre-test the pH value and the osmolality of the cell cultures were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. No test substance precipitation in culture medium occurred up to the highest applied concentration by the end of treatment in the absence and presence of S9 mix. After 4 hours treatment in the absence of S9 mix, no cytotoxicity was observed as indicated by a reduced RS of about or below 20% of control up to the highest applied concentration. In contrast, in the presence of S9 mix, a slightly reduced relative RS was observed after treatment with 650.0 μg/mL

Based on the data and the observations from the pre-test and taking into account the current guidelines, the following doses were selected in this study.

1st Experiment
with and without S9 mix
81.3; 162.5; 325.0; 650.0; 1300.0 μg/mL

2nd Experiment
with S9 mix
250.0; 500.0; 750.0; 1000.0; 1300.0 μg/mL

Vehicle / solvent:

Due to the good solubility of the test substance in culture medium, culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative controls, with and without S9 mix, were treated with culture medium without test substance in parallel to the other treatment groups.

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x10^6 cells in 40 mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 - 24 h
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 - 9 days
- Selection time (if incubation with a selective agent): 6 – 7 days
- Selective agent: 6-thioguanine (10 μg/mL); incubation for 6 – 7 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cloning efficiency (CE) (pre-experiment); Relative survival after treatment (Cloning efficiency 1 [CE1] adjusted by cell loss); Cloning efficiency 2 (CE2; viability); Relative survival (RS)

The cloning efficiency (CE, %) was calculated for each test group as follows:
total number of colonies in the test group
CEabsolute = ————————————————————— x 100
total number of seeded cells in the test group

CEabsolute of the test group
CErelative = ————————————————— x 100
CEabsolute of the negative control

The number of colonies in every petri dish was counted and recorded. Using the formula above the values of absolute cloning efficiencies (CEabsolute, CE1 absolute and/or CE2 absolute) were calculated. Based on these values the relative cloning efficiencies (CErelative, CE1 relative and/or CE2 relative) of the test groups were calculated and reported as a percentage of the respective CEabsolute value of the corresponding negative control (negative control = 100%).

In addition, with regard to cell loss while exposure period, relative survival (RS) was calculated based on CE of cells plated immediatedly after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative controls.
number of cells at the end of treatment
Adjusted CE = —————————————————— x CE1
number of seeded cells

adjusted CE of the test group
RS = ———————————————————— x 100
adjusted CE of the negative control


METHODS FOR MEASUREMENTS OF GENOTOXICIY
The number of colonies in each flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10^6 cells seeded.
The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:
total number of mutant colonies
MFuncorr. = —————————————–— x 10^6
number of seeded cells

The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):
MFuncorr.
MFcorr. =——————— x 100
CE2 absolute

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
- absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix)
- Background mutant frequency in the negative/vehicle controls should be within historical negative control data range (95% control limit).
- positive controls both with and without S9 mix should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase in mutant frequencies compared with the concurrent negative control

The test substance is considered to be clearly positive if all following criteria are met:
- statistically significant increase in mutant frequencies
- dose-related increase in mutant frequencies
- corrected mutation frequencies exceeds both the concurrent negative/vehicle control value and the range of historical negative control data (95% control limit)

The test substance is considered to be clearly negative if all following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of historical negative control data (95% control limit)

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction.
However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: Osmolality and pH values of the cell cultures were not influenced by test substance treatment.
- Possibility of evaporation from medium: not specified
- Precipitation and time of the determination: In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed macroscopically up to the highest applied test substance concentration.

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 table 2
- Negative (solvent/vehicle) historical control data: see table 3

Table 1: Summary of results

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						RS [%]	CE2[%]
1	4	Negative control	-	n.d.	0.71	100.0	100.0
		81.3	-	-	n.c.	187.1	n.c.
		162.5	-	-	0.68	159.0	104.3
		325.0	-	-	1.52	143.7	93.8
		650.0	-	-	1.79	191.2	92.7
		1300.0	-	-	0.55	97.8	86.3
		Positive control^1	-	n.d	210.16^S	107.7	60.7
1	4	Negative control	+	n.d.	0.25	100.0	100.0
		81.3	+	-	n.c.	94.9	n.c.
		162.5	+	-	1.07	88.5	92.8
		325.0	+	-	1.48	97.0	83.8
		650.0	+	-	2.23^S	82.6	89.1
		1300.0	+	-	3.40^S	87.4	65.9
		Positive control^2	+	n.d.	193.36^S	43.2	63.7
2	4	Negative control	+	n.d.	3.35	100.0	100.0
		250.0	+	-	n.c.	93.7	n.c.
		500.0	+	-	2.05	97.2	95.5
		750.0	+	-	2.56	99.4	87.4
		1000.0	+	-	1.33	83.7	105.3
		1300.0	+	-	2.26	70.5	111.2
		Positive control^2	+	n.d.	179.65^S	69.8	64.5

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 10⁶ cells corrected with the CE₂ value

*** Cloning efficiency related to the respective negative control

s Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.d. Not determined

1 EMS 400 μg/mL

2 DMBA 1.25 μg/mL

The observed increased number of mutant colonies in the 1st Experiment as compared to the corresponding vehicle control in the presence of S9 mix is considered as artificial and is due to the rather low vehicle control value, since the values were within the 95% control limit of the historical negative control data and the increases could not be reproduced in the 2nd experiment. All obtained values were clearly within the range of the 95% negative control limit.

Table 2: Positive historical control data (period: March 2016 – December 2017)

Without S9 mix 400 µg/mL ethyl methanesulfonate (EMS)
	Corrected Mutant Frequency*
Exposure period	4 hrs
Mean	158.01
Minimum	42.47
Maximum	419.90
Standard Deviation	80.87
95% Lower Control Limit	0.00
95% Upper Control Limit	320.94
No. of Experiments	63
With S9 mix 1.25 µg/mL 7,12-Dimethylbenz[a]anthracene (DMBA)
	Corrected Mutant Frequency*
Exposure period	4 hrs
Mean	125.89
Minimum	21.52
Maximum	270.48
Standard Deviation	56.16
95% Lower Control Limit	13.02
95% Upper Control Limit	238.77
No. of Experiments	69

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Table 3: Negative historical control data (period: March 2016 – December 2017)

	Without S9 mix - all vehicles*	With S9 mix - all vehicles*
Corrected Mutant Frequency**
Exposure period	4 hrs	4 hrs
Mean	2.86	2.93
Minimum	0.00	0.00
Maximum	7.09	9.93
Standard Deviation	1.81	2.24
95% Lower Control Limit	0.00	0.00
95% Upper Control Limit	6.49	7.43
No. of Experiments	69	72

* = culture medium, water 10% (v/v), DMSO 1% (v/v), acetone 1% (v/v)

** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Conclusions:

Under the experimental conditions of this study, the test substance was not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, without and/or with the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance at concentrations up to 1300 µg/mL for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations, demonstrating the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed.

In this study, in both experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug - Sep 2018

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: B1056-070518
- Purity: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI)
- Expiry date: 07 May 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle deionized water over a period of 7 days was verified analytically.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble and stable indeionized water over a period of 7 days.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Stock formulations of the test item and serial dilutions were made in deionized water. The pH in the stock formulations were adjusted with 2 N NaCl to physiological values. The final concentration of deionized water in the culture medium was 10 %. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. All formulations were prepared freshly before treatment and used within two hours of preparation.

FORM AS APPLIED IN THE TEST: diluted in deionized water

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood samples from healthy non-smoking donors (males) not receiving medication
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Sex, age and number of blood donors if applicable: Experiment I: one male donor (34 years old); Experiment II: one male donor (21 years old)
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Whether blood from different donors were pooled or not: no pooling
- Mitogen used for lymphocytes: PHA (3 μg/mL)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Phenobarbital/b-Naphthoflavone induced rat liver S9)

Test concentrations with justification for top dose:

Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test.
With regard to the molecular weight of the test item, 1292 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 8.4 to 1292 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
No cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 1292 μg/mL were chosen as top treatment concentration for Experiment II.

Concentrations applied:
Experiment I: With and without S9 mix; 4 h exposure: 8.4; 14.7; 25.7; 45.0; 78.7; 138; 241; 422; 738; 1292 µg/mL
Experiment II: Without S9 mix; 20 h exposure: 78.7; 138; 241; 422; 738; 1292 µg/mL

Vehicle / solvent:

- deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 10.0% deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine (500 ng/mL; without metabolic activation; continuous treatment)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

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

DURATION
- Preincubation period: 48 h incubation with PHA (mitogen) to stimulate lymphocyte proliferation
- Exposure duration: 4 h (pulse exposure); 20 h (continuous exposure; without S9 mix)
- Expression time (cells in growth medium): 16 h (recovery period; pulse exposure only) + another 20 h after addition of Cytochalasin B
- Fixation time (start of exposure up to fixation of cells): 40 hours

CYTOKINESIS BLOCK: Cytochalasin B (4 μg/mL); duration of cell exposure: 20 h

STAIN (for cytogentic assay): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cultures were harvested by centrifugation (5 min)
- cells were resuspended in approximately 5 mL "saline G" and spun down once again (5 min)
- cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes
- 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added and the cells were resuspended
- after removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold
- slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide
- cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).
- The micronuclei have to be stained in the same way as the main nucleus.
- The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: determination of CBPI (Cytokinesis-block proliferation index) in 500 cells per culture; A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
CBPI = ((Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)) / Total number of cellsNUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate)
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- 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.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: human

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:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no

HISTORICAL CONTROL DATA
- Positive historical control data: see table 1
- Negative (solvent/vehicle) historical control data: see table 1

Table 1: Historical control data

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
No. of experiments	61*	65**
Mean	0.62	0.56
95 % Ctrl limit	0.06 – 1.19	0.00 – 1.11
1x SD (2x SD)	0.28 (0.56)	0.28 (0.56)
Min – Max	0.00 – 1.18	0.05 – 1.10

*   Aqueous solvents – 21 Experiments; Organic solvents – 40Experiments

** Aqueous solvents – 24 Experiments; Organic solvents – 41 Experiments

Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
No. of experiments	80*
Mean	0.73
95 % Ctrl limit	0.08 – 1.38
1x SD (2x SD)	0.33 (0.66)
Min – Max	0.10 – 1.85

                   *   Aqueous solvents – 30 Experiments; Organic solvents – 50 Experiments

Positive Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
	MMC	Demecolcine
No. of experiments	62	65
Mean	14.63	3.68
95 % Ctrl limit	3.92 – 25.34	1.47 – 5.89
1x SD (2x SD)	5.35 (10.70)	1.11 (2.22)
Min – Max	2.60 – 28.50	2.10 – 8.80

 

Positive Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
	CPA
No. of experiments	86
Mean	5.45
95 % Ctrl limit	0.70 – 10.20
1x SD (2x SD)	2.37 (4.74)
Min – Max	2.25 – 13.30

 

Table 2: Summary of results

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells	95% Ctrl limit
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	40 hrs	Solvent control1	1.86		1.10	0.06 – 1.19
		Positive control2	1.73	15.0	13.05S	3.92 – 25.34
		422	1.80	7.6	0.90
		738	1.86	n.c.	1.05
		1292	1.75	12.6	1.10
Exposure period 20 hrs without S9 mix
II	40 hrs	Solvent control1	1.81		0.35	0.00 – 1.11
		Positive control3	1.63	21.4	2.75S	1.47 – 5.89
		422	1.74	8.7	0.25
		738	1.81	n.c.	0.45
		1292	1.84	n.c.	0.50
Exposure period 4 hrs with S9 mix
I	40 hrs	Solvent control1	1.85		0.10	0.08 – 1.38
		Positive control4	1.47	44.4	2.70S	0.70 – 10.20
		422	1.75	12.1	0.40
		738	1.80	6.1	0.30
		1292	1.77	10.0	0.40

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S The number of micronucleated cells is statistically significantly higher than corresponding control values

1 Deion. water 10.0 % (v/v)

2 MMC 1.0 µg/mL

3 Demecolcine 50 ng/mL

4 CPA 17.5 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Executive summary:

The test item was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I
Stimulation period	48 hrs	48 hrs	48 hrs
Exposure period	4 hrs	20 hrs	4 hrs
Recovery	16 hrs	-	16 hrs
Cytochalasin B exposure	20 hrs	20 hrs	20 hrs
Total culture period	88 hrs	88 hrs	88 hrs

In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (1292 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In both independent experiments in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay

In order to assess the mutagenicity of the test substance a bacterial reverse mutation assay (GLP) was conducted according to OECD Guideline 417 using the S. typhimurium strains TA100, TA1535, TA1537 and TA98 and the E. coli strain WP2 uvrA in the Standard plate test and the preincubation test with and without metabolic activation (liver S9 mix from induced rats).

The test substance exhibited no mutagenic activities up to cytotoxic concentrations and brought no gene mutations in these tests.

In vitro gene mutation in mammalian cells

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, without and/or with the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance at concentrations up to 1300 µg/mL for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations, demonstrating the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed.

In this study, in both experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system.

Thus, under the experimental conditions of this study, the test substance was not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

In vitro Mammalian Cell Micronucleus Test

The test substance, dissolved in deionized water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments according to OECD Guideline 487.

In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (1292 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In both independent experiments in the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.