Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA 102 with and without metabolic activation

HPRT (OECD 476): negative in V79 cells with and without metabolic activation

MNT (OECD 487): negative in human lymphocytes with and without metabolic activation

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:

Feb 2006

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:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(Commission Directive 2000/32/EC)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation in TA100

Experiment 1 and 2:
0, 50, 150, 500, 1500, 5000 µg/plate with and without metabolic activation in all strains

Vehicle / solvent:

- Vehicle/solvent used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene (2AA); 1,8-Dihydroxyanthraquinone (DAN)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for experiment 1 and 2

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Rationale for test conditions:

The test conditions were chosen according to OECD 471.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results was considerd first, statistical methods, as recommended by the UKEMS (Kirkland D.J., Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press, 1989) were also used as an aid to evaluation, however, statistical significance was not the only determining factor for a positive response. A test material was considered non-mutagenic (negative) in the test system if the above criteria were not met.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

greasy precipitate ≥ 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A greasy precipitate was observed at and above 1500 μg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The dose range of 50 to 5000 µg/plate for both main experiments was determined in a preliminary toxicity assay in S. typhimurium strain TA 100.

HISTORICAL CONTROL DATA: Positive and vehicle controls were within the normal range of historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.

Spontaneous mutation rates for the negative controls were considered to be acceptable. The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial stains, with any dose of the test substance, either with or without metabolic activation.

Table 2. Results of Experiment 1

EXPERIMENT 1
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	114 ± 7.4	23 ± 4.0	332 ± 14.1	21 ± 5.6	16 ± 3.5
50	109 ± 19.5	22 ± 0.6	293 ± 39.7	24 ± 6.7	13 ± 3.0
150	99 ± 3.8	21 ± 4.6	256 ± 41.6	22 ± 6.8	15 ± 3.6
500	96 ± 17.7	21 ± 4.7	265 ± 9.3	19 ± 5.5	12 ± 2.3
1500	105 ± 22.7 P	20 ± 4.4 P	242 ± 34.1 P	20 ± 2.1 P	13 ± 4.6 P
5000	85 ± 11.0 P	23 ± 1.2 P	264 ± 33.0 P	21 ± 1.2 P	12 ± 2.3 P
ENNG	479 ± 50.8	382 ± 25.7
MMC			1739 ± 6.5
4NQO				275 ± 16.8
9AA					756 ± 230.1
S9-Mix	With
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	88 ± 6.4	14 ± 0.0	328 ± 14.2	32 ± 5.0	19 ± 3.5
50	93 ± 6.5	14 ± 1.7	336 ± 30.1	32 ± 1.7	18 ± 4.7
150	89 ± 18.6	10 ± 2.3	285 ± 23.1	30 ± 5.5	20 ± 2.1
500	88 ± 12.5	13 ± 4.2	308 ±19.0	28 ± 5.5	14 ± 6.7
1500	83 ± 7.6 P	10 ± 2.3 P	296 ± 27.1 P	23 ± 5.9 P	11 ± 0.6 P
5000	89 ± 9.6 P	13 ± 2.5 P	286 ± 11.0 P	26 ± 2.3 P	12 ± 3.5 P
2AA	2762 ± 93.6	174 ± 42.2			210 ± 19.6
DAN			1010 ± 101.7
BP				158 ± 36.7
SC = Solvent Control (DMSO) P = Precipitate Positive Controls: ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine; MMC: Mitomycin C; 4NQO: 4-Nitroquinoline-1-oxide; 9-AA: 9-aminoacridine; 2AA: 2-Aminoanthracene; DAN: 1,8-Dihydroxyanthraquinone; BP: Benzo(a)pyrene

Table 3. Results of Experiment 2

EXPERIMENT 2
S9-Mix	Without
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	92 ± 13.9	25 ± 5.3	293 ± 33.0	21 ± 1.0	13 ± 5.9
50	98 ± 17.1	19 ± 5.2	321 ± 17.6	14 ± 5.7	13 ± 3.8
150	103 ± 5.3	19 ± 2.6	284 ± 8.5	11 ± 3.5	11 ± 2.6
500	91 ± 13.1	9 ± 3.5	272 ± 16.2	12 ± 4.0	8 ± 1.5
1500	78 ± 7.4 P	12 ± 4.2 P	288 ± 22.5 P	9 ± 0.6 P	11 ± 1.5 P
5000	83 ± 14.2 P	14 ± 5.1 P	274 ± 7.5 P	8 ± 2.0 P	17 ± 2.3 P
ENNG	673 ± 76.4	859 ± 123.0
MMC			1341 ± 95.8
4NQO				128 ± 28.4
9AA					323 ± 32.5
S9-Mix	With
Test substance (µg/plate)	TA 100	TA 1535	TA 102	TA 98	TA 1537
SC	84 ± 12.7	11 ± 2.1	310 ± 30.7	15 ± 4.2	11 ± 3.5
50	84 ± 9.8	17 ± 2.0	315 ± 10.1	17 ± 1.2	10 ± 3.1
150	81 ± 4.0	9 ± 4.0	283 ± 16.5	20 ± 6.1	7 ± 2.1
500	61 ± 7.8	11 ± 0.0	291 ± 20.4	17 ± 5.1	8 ± 1.0
1500	57 ± 5.5 P	10 ± 4.2 P	268 ± 12.1 P	14 ± 2.6 P	11 ± 2.9 P
5000	59 ± 7.0 P	10 ± 2.3 P	272 ± 12.6 P	14 ± 3.6 P	10 ± 3.6 P
2AA	1536 ± 162.9	174 ± 52.5			568 ± 46.2
DAN			733 ± 22.6
BP				173 ± 17.7
SC = Solvent Control (DMSO) P = Precipitate Positive Controls: ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine; MMC: Mitomycin C; 4NQO: 4-Nitroquinoline-1-oxide; 9-AA: 9-aminoacridine; 2AA: 2-Aminoanthracene; DAN: 1,8-Dihydroxyanthraquinone; BP: Benzo(a)pyrene

Conclusions:

Under the conditions of the Ames assay the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.

Executive summary:

A bacterial gene mutation assay (Ames) with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2016). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 50 to 5000 µg/plate were selected for the incubation with and without metabolic activation in both experiments. Precipitation occurred at and above 1500 µg/plate. The test substance was not bacteriotoxic at any dose and strain. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tested strains in the presence and absence of metabolic activation. The vehicle and positive control data were in the range of the historical control data of the laboratory. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains in the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May - 11 Jul 2016

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:

adopted 28 Jul 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Kanpoan No. 287 - Environment Protection Agency, Eisei No. 127 - Ministry of Health & Welfare, Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry“

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank's salts, 10% FBS (except during 4 h treatment), neomycin (5 µg/mL) and amphotericin B (1%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-Experiment:
4 h treatment: 17.8, 35.6, 71.3, 142.6, 285.1, 570.3, 1140.5 and 2281 µg/mL with and without metabolic activation

Main experiment:
4 h treatment: 0.063, 0.13, 0.25, 0.50, 1.0, 2.0 and 4.0 µg/mL without metabolic activation
4 h treatment: 1.0, 2.0, 4.0, 8.0 16.0, 24.0 and 32.0 µg/mL with metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO (0.5% (v/v))
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative low toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

EMS: 300 µg/mL (4 h, -S9); DMBA: 2.3 µg/mL (4 h, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h exposure with and without S9 mix
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: duplicate cultures per concentration level

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the cloning efficiency below 50%

Evaluation criteria:

A test substance is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test substance producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.

A positive response is described as follows:
A test substance is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 4 µg/mL without metabolic activation in culture I

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test substance.
- Precipitation: Phase separation was observed in the pre-experiment at 35.6 µg/mL and above with and without metabolic activation. In the main experiment phase separation occurred at 16 µg/mL and above with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed in order to determine the concentration range for the mutagenicity experiments. The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test substance concentrations between 17.8 µg/mL and 2281 µg/mL were used. Phase separation of the test substance as well as severe cytotoxicity was noted at 35.6 µg/mL and above with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% occurred at the maximum concentration without metabolic activation in culture I only. The cytotoxicity was exceedingly severe, leading to a relative cell density of just 4.2% of the solvent control at the end of treatment. The parallel culture did not yet show any relevant cytotoxicity at this test point. This difference is most likely caused by minor fluctuations in cell density during treatment. Substances binding to proteins or lipids tend to be less toxic at higher cell densities or in the presence of an exogenous source of proteins as with metabolic activation. No relevant cytotoxity was noted up to and including phase separating concentrations with metabolic activation.

Table 2: Main experiment - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Rel. cloning efficiency	Rel. cell density	Rel. adjusted cloning efficiency	Mutant colonies per 10^6cells
Culture I
0 (DMSO)	100.0	100.0	100.0	15.9
0.063	130.7	74.9	97.9	Culture was not continued^#
0.13	124.3	73.0	90.7	21.8
0.25	109.8	71.1	78.0	12.5
0.50	100.7	78.7	79.3	21.7
1.0	103.3	69.0	71.3	15.7
2.0	129.7	73.4	95.2	13.2
4.0	-	4.2	Culture was not continued^##
EMS, 300	102.3	90.0	92.1	95.1
Culture II
0 (DMSO)	100.0	100.0	100.0	18.5
0.063	102.9	92.5	95.2	Culture was not continued^#
0.13	93.4	112.2	104.8	Culture was not continued^#
0.25	93.6	83.5	78.1	20.7
0.50	82.3	80.8	66.5	9.3
1.0	102.6	103.4	106.1	23.2
2.0	94.3	93.0	87.7	16.7
4.0	95.1	86.7	82.4	10.4
EMS, 300	87.2	81.0	70.6	161.5

DMSO: dimethyl sulfoxide

EMS: ethylmethane sulfonate

#: culture was not continued since a minimum of only four concentrations required

##: culture was not continued due to exceedingly severe cytotoxic effects

 

Table 3: Main experiment - 4 h exposure - With Metabolic Activation

Concentration [µg/mL]	Rel. cloning efficiency	Rel. cell density	Rel. adjusted cloning efficiency	Mutant colonies per 10^6cells
Culture I
0 (DMSO)	100.0	100.0	100.0	16.3
1.0	Culture was not continued^#
2.0	116.9	66.5	77.8	Culture was not continued^#
4.0	114.2	65.0	74.3	16.7
8.0	113.5	66.3	75.2	12.0
16.0^PS	110.5	66.0	72.9	10.4
24.0^PS	109.4	81.5	89.1	17.7
32.0^PS	84.8	52.8	44.8	20.9
DMBA, 2.3	116.4	65.4	76.2	94.5
Culture II
0 (DMSO)	100.0	100.0	100.0	20.0
1.0	Culture was not continued^#
2.0	105.7	84.8	89.6	Culture was not continued^#
4.0	95.9	89.4	85.7	12.1
8.0	111.9	85.9	96.1	18.3
16.0^PS	110.6	78.0	86.2	16.7
24.0^PS	96.2	77.8	74.8	24.3
32.0^PS	67.6	58.7	39.7	19.6
DMBA, 2.3	96.0	87.0	83.5	154.9

DMSO: dimethyl sulfoxide

DMBA: 7,12-dimethylbenzanthracene

PS: phase separation

#: culture was not continued since a minimum of only four concentrations required

Conclusions:

Under the experimental conditions of the gene mutation assay the test substance did not induce gene mutations at the HPRT locus in V79 cells with and without metabolic activation.

Executive summary:

The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (2016). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. A pre-experiment was performed in order to determine the concentration range for the mutagenicity experiments. The pre-experiment was performed in the absence and presence (4 h treatment) of metabolic activation. Test substance concentrations between 17.8 and 2281 µg/mL (equal to 2 mg/L) were used. Phase separation of the test substance as well as severe cytotoxicity were noted at 35.6 μg/mL and above with and without metabolic activation. Based on the results of the pre-experiment, cells were exposed to the test substance for 4 h with and without metabolic activation up to concentrations of 32 and 4 µg/mL, respectively. Relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% occurred only in one culture at the maximum concentration of 4 µg/mL without metabolic activation. No relevant cytotoxicity was noted up into the phase separating concentration range with metabolic activation. No relevant and reproducible increase in mutant colony numbers/10E06 cells was observed in the main experiment up to the maximum concentration. The mutation frequency remained within the 95% confidence interval. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Jun - 29 Aug 2016

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 26 September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Cell proliferation: Blood was collected from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (22 years old) for Experiment I and from a male donor (32 years old) for Experiment II. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemagglutinin (PHA) to the culture medium for a period of 48 h.
- Type and identity of media: 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% fetal bovine serum, 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U./mL)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Without S9 mix:
Experiment I (4 hours exposure): 1.5, 2.6, 4.5, 8.0*, 13.9*, 24.4*, 42.6, 74.6, 131, 229, 400 and 700 µg/mL
Experiment II (20 hours exposure): 3.5, 6.1, 10.7*, 18.7*, 32.7*, 57.1 and 100 µg/mL

With S9 mix:
Experiment I (4 hours exposure): 1.5, 2.6, 4.5, 8.0, 13.9*, 24.4*, 42.6*, 74.6, 131, 229, 400 and 700 µg/mL

* evaluated for cytogenetic damage

Vehicle / solvent:

- Vehicle/solvent used: DMSO (final concentration in cell culture medium was 0.5% (v/v))
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative low toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcin

Remarks:

-S9: MMC (1.0 µg/mL), demecolcin (75 ng/mL); +S9: CPA (15 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 40 h; 20 h treatment: 40 h

ACTIN POLYMERISATION INHIBITOR (cytogenetic assays): cytochalasin B, 4 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Two parallel cultures in 2 independent experiments

NUMBER OF CELLS EVALUATED: 1000 binucleated cells per culture

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: cytokinesis-block proliferation index (CBPI)

Evaluation criteria:

The micronucleus assay was considered acceptable if it meets the following criteria:
- The concurrent solvent control was within the laboratory historical solvent control data range.
- The concurrent positive controls induces responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
- Cell proliferation criteria in the solvent control were considered to be acceptable.
- All experimental conditions described were tested unless one exposure condition resulted in a clearly positive result.
- The quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
- The criteria for the selection of top concentration were consistent.

A test substance was classified as non-clastogenic and non-aneugenic if:
- None of the test substance concentrations exhibited a statistically significant increase compared with the concurrent solvent control.
- There was no concentration-related increase.
- The results in all evaluated test substance concentrations were within the range of the laboratory historical solvent control data.

A test substance was classified as clastogenic and aneugenic if:
- At least one of the test substance concentrations exhibited a statistically significant increase compared with the concurrent solvent control.
- The increase was concentration-related in at least one experimental condition.
- The results were outside the range of the laboratory historical solvent control data.

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:

cytotoxicity

Remarks:

Exp. I: -S9: at 24.4 µg/mL; Exp. II: -S9: at 32.7 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: No relevant influence on osmolarity or pH value was observed.
- Other confounding effects: In Experiment I, phase separation of the test substance in the culture medium was observed at 24.4 µg/mL and above in the absence of metabolic activation and at 74.6 µg/mL and above in the presence of metabolic activation at the end of treatment. In addition, phase separation occurred in Experiment II in the absence of metabolic activation at 57.1 µg/mL and above at the end of treatment.

RANGE-FINDING/SCREENING STUDIES:
With regard to the solubility and cytotoxicity properties of the test substance, 700 µg/mL was applied as top concentration for treatment of the cultures in the preliminary test. Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I in the absence of metabolic activation, clear cytotoxicity was observed at the highest evaluated concentration, which showed phase separation. In the presence of metabolic activation, no cytotoxicity was observed up to the highest evaluated concentration. However, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment II in the absence of metabolic activation, clear cytotoxicity was observed at the highest evaluated concentration.

Table 2: Results of Experiment I.

	Concentration in µg/mL	Proliferation index CBPI	Cytostasis in %	Number of cells with MN in %^1)
Exposure period 4 h, preparation interval 40 h, without S9 mix
DMSO	0.5% (v/v)	1.90	-	0.45
MMC	1.0	1.68	24.6	11.50^S
Test substance	8.0	1.97	n.c.	0.25
	13.9	1.92	n.c.	0.25
	24.4^PS	1.43	52.4	0.60
Exposure period 4 h, preparation interval 40 h, with S9 mix
DMSO	0.5% (v/v)	1.91	-	0.50
CPA	15.0	1.58	36.0	8.50^S
Test substance	13.9	2.02	n.c.	0.95
	24.4	2.00	n.c.	0.30
	42.6	1.93	n.c.	0.40

1) The number of micronucleated cells was determined in a sample of 2000 binucleated cells.

CPA: cyclophosphamide

DMSO:dimethyl sulfoxide

MMC: mitomycin C

n.c.: not calculated as the CBPI is equal or higher than the solvent control value

PS: phase separation occurred at the end of the treatment

S: the number of micronucleated cells is statistically significantly higher than corresponding control values. 

 

Table 3: Results of Experiment II.

	Concentration in µg/mL	Proliferation index CBPI	Cytostasis in %	Number of cells with MN in %^1)
Exposure period 20 h, preparation interval 40 h, without S9 mix
DMSO	0.5% (v/v)	1.85	-	1.10
Demecolcin	75.0	1.57	32.3	4.75^S
Test substance	10.7	1.72	15.5	0.85
	18.7	1.70	17.8	0.55
	32.7	1.35	58.4	0.75

1) The number of micronucleated cells was determined in a sample of 2000 binucleated cells.

DMSO:dimethyl sulfoxide

n.c.: not calculated as the CBPI is equal or higher than the solvent control value

S: the number of micronucleated cells is statistically significantly higher than corresponding control values. 

In the absence and presence of metabolic activation, no relevant increase in the number of micronucleated cells was observed after treatment with the test substance. In Experiment II, however, the value of the solvent control with 1.10% of micronucleated cells slightly exceeded the range of the laboratory historical control data (95% control limit: 0.05 -1.05%). Because this value was well within the data for the minimal and maximal limits (min: 0.05% and max: 1.43%), this was declared as biologically irrelevant. In both experiments, either Demecolcin, MMC or CPA were used as positive controls and showed distinct increases in cells with micronuclei.

Conclusions:

Under the experimental conditions of the in vitro micronucleus assay the test substance did not induce micronuclei in human lymphocytes with and without metabolic activation.

Executive summary:

The potential of the test substance to induce micronuclei was investigated in an in vitro mammalian cell micronucleus test in cultured peripheral human lymphocytes performed according to OECD Guideline 487 and GLP (2016). The test substance was dissolved in DMSO and in total two independent experiments were performed. A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Test substance concentrations ranging from 1.5 to 700 µg/mL were chosen for the evaluation of cytotoxicity and the exposure period was 4 h with and without metabolic activation. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated as Experiment I. In Experiment II, cultures were exposed to the test substance for 20 h without metabolic activation up to a maximum concentration of 100 µg/mL. The cells were prepared 40 h after start of treatment. In each experimental group two parallel cultures were analysed and at least 1000 binucleate cells per culture were evaluated for cytogenetic damage. In Experiment I, phase separation of the test substance was observed at and above 24.4μg/mL and 74.6μg/mL without and with metabolic activation, respectively. In addition, phase separation occurred in Experiment II without metabolic activation at 57.1μg/mL and above. No relevant influence on osmolarity or pH was observed. Relevant toxic effects were observed after 4 hours treatment at 24.4μg/mL and above without metabolic activation and in Experiment II, after 20 h treatment at 32.7 µg/mL without metabolic activation. However, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. No biologically relevant increase in the number of micronucleated cells was observed after treatment with the test substance with and without metabolic activation. In Experiment II, the value of the solvent control (1.10%) slightly exceeded the range of the laboratory’s historical control data (95% control limit: 0.05 – 1.05%). Because this value is well within the data for the minimal and maximal limits (min: 0.05% and max: 1.43%), this was declared as biologically irrelevant. Mitomycin C, demecolcin and cyclophosphamide were used as positive controls and induced statistically significant increases in cells with micronuclei. In conclusion, the test substance did not induce micronuclei in the in vitro micronucleus test in human lymphocytes under the experimental conditions reported. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria

A bacterial gene mutation assay (Ames) with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2016). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 50 to 5000 µg/plate were selected for the incubation with and without metabolic activation in both experiments. Precipitation occurred at and above 1500 µg/plate. The test substance was not bacteriotoxic at any dose and strain. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tested strains in the presence and absence of metabolic activation. The vehicle and positive control data were in the range of the historical control data of the laboratory. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains in the presence and absence of metabolic activation.

Gene mutation in mammalian cells

The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (2016). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. A pre-experiment was performed in order to determine the concentration range for the mutagenicity experiments. The pre-experiment was performed in the absence and presence (4 h treatment) of metabolic activation. Test substance concentrations between 17.8 and 2281 µg/mL (equal to 2 mg/L) were used. Phase separation of the test substance as well as severe cytotoxicity were noted at 35.6 μg/mL and above with and without metabolic activation. Based on the results of the pre-experiment, cells were exposed to the test substance for 4 h with and without metabolic activation up to concentrations of 32 and 4 µg/mL, respectively. Relevant cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% occurred only in one culture at the maximum concentration of 4 µg/mL without metabolic activation. No relevant cytotoxicity was noted up into the phase separating concentration range with metabolic activation. No relevant and reproducible increase in mutant colony numbers/10E06 cells was observed in the main experiment up to the maximum concentration. The mutation frequency remained within the 95% confidence interval. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Cytogenicity in mammalian cells

The potential of the test substance to induce micronuclei was investigated in an in vitro mammalian cell micronucleus test in cultured peripheral human lymphocytes performed according to OECD Guideline 487 and GLP (2016). The test substance was dissolved in DMSO and in total two independent experiments were performed. A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Test substance concentrations ranging from 1.5 to 700 µg/mL were chosen for the evaluation of cytotoxicity and the exposure period was 4 h with and without metabolic activation. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated as Experiment I. In Experiment II, cultures were exposed to the test substance for 20 h without metabolic activation up to a maximum concentration of 100 µg/mL. The cells were prepared 40 h after start of treatment. In each experimental group two parallel cultures were analysed and at least 1000 binucleate cells per culture were evaluated for cytogenetic damage. In Experiment I, phase separation of the test substance was observed at and above 24.4μg/mL and 74.6μg/mL without and with metabolic activation, respectively. In addition, phase separation occurred in Experiment II without metabolic activation at 57.1μg/mL and above. No relevant influence on osmolarity or pH was observed. Relevant toxic effects were observed after 4 hours treatment at 24.4μg/mL and above without metabolic activation and in Experiment II, after 20 h treatment at 32.7 µg/mL without metabolic activation. However, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. No biologically relevant increase in the number of micronucleated cells was observed after treatment with the test substance with and without metabolic activation. In Experiment II, the value of the solvent control (1.10%) slightly exceeded the range of the laboratory’s historical control data (95% control limit: 0.05 – 1.05%). Because this value is well within the data for the minimal and maximal limits (min: 0.05% and max: 1.43%), this was declared as biologically irrelevant. Mitomycin C, demecolcin and cyclophosphamide were used as positive controls and induced statistically significant increases in cells with micronuclei. In conclusion, the test substance did not induce micronuclei in the in vitro micronucleus test in human lymphocytes under the experimental conditions reported. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to cytotoxic or the highest evaluable concentrations.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.