[1R-(1α,2β,5α)]-1-(isopropyl)-2-methoxy-4-methylcyclohexane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 103 with and without metabolic activation

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

In vitro Micrunucleus test (OECD 487) and Chromosome Aberration test (OECD 473): overall conclusion: negative with and without metabolic activation in human lymphocytes and in reconstructed human skin model

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 June 2020 to 14 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human Peripheral Blood Lymphocytes (HPBL), primary culture

For lymphocytes:
- Sex, age and number of blood donors: one male, age 24

MEDIA USED
Peripheral blood lymphocytes were cultured in complete medium (RPMI 1640 containing 15 % heat inactivated fetal bovine serum, 2 mM L glutamine, 100 units penicillin and 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood per 5 mL of complete medium with 2% phytohemagglutinin in centrifuge tubes. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1 % CO2 in air) for 44-48 hours.

Cytokinesis block (if used):

Cytochalasin B (cytoB) was dissolved in DMSO to a stock concentration of 2 mg/mL. It was used at 6 µg/mL concentration to block cytokinesis.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg bw, five days before sacrifice. The S9 (Lot No. 4203, Exp. Date: 04 Feb 2022) was purchased commercially from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at 60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2 aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Test concentrations with justification for top dose:

The test concentrations were chosen based on a preliminary toxicity test.
The following doses were chosen:
without S9-mix, treatment time 4 h, recovery time 20 h: 14.6, 29.2, 48.7, 60.9, 76.2, 95.2, 119, 170 µg/mL
without S9-mix, treatment time 24 h, recovery time 0 h: 14.6, 29.2, 48.7, 60.9, 76.2, 95.2, 119, 170 µg/mL
with S9-mix, treatment time 4 h, recovery time 20 h: 14.6, 29.2, 48.7, 60.9, 76.2, 95.2, 119, 170 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: Ethanol was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. In the solubility test conducted at BioReliance, the test substance formed a clear solution in ethanol at a concentration of approximately 500 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

vinblastine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1

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


TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h and 24 h without metabolic acivation), 4 h (with metabolic activation)
- Harvest time after the end of treatment: The 4 hours-treatment time cultures were harvested after a 20 hours-recovery time. The 24 hours-treatment time cultures were harvested directly after treatment.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- cytokinesis block: Cells were collected after being exposed to cyto B for 24 hours (± 30 minutes), 1.5 to 2 normal cell cycles, to ensure identification and selective analysis of micronucleus frequency in cells that have completed one mitosis evidenced by binucleated cells (Fenech and Morley, 1986). The cyto B exposure time for the 4 hour treatment in the non-activated and the S9-activated studies was 20 hours (± 30 minutes).
- Methods of slide preparation and staining technique used including the stain used:
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), capped and the slides were prepared immediately after harvest. To prepare slides, the cells were collected by centrifugation and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the preliminary toxicity test, at least 500 cells, when possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1000 cells (500 cells per culture), when possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus scoring, at least 2000 binucleated cells from each concentration (1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.
- Criteria for scoring micronucleated cells:
Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
• the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- CBPI (cytokinesis block poliferation index) >= 50% relative to the vehicle control

Evaluation criteria:

The test substance is considered to have induced a positive response if
• at least one of the test concentrations exhibits a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase is concentration-related (p ≤ 0.05), and
• results are outside the 95% control limit of the historical negative control data.

The test substance is considered to have induced a clear negative response if none of the criteria for a positive response are met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p > 0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

primary culture, other: Human Peripheral Blood Lymphocytes (HPBL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 60.9 µg/mL onwards the CBPI was >= 50 % relative to the vehicle control

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH of the highest dose of test substance in treatment medium was 7.5.
- Data on osmolality: The osmolality of the test substance doses in treatment medium was considered acceptable (<120% of vehicle).
- Precipitation: precipitation was observed from test substance concentrations of 119 µg/mL onwards.

Table 1: MICRONUCLEUS ASSAY IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION, 4-HOUR TREATMENT, 24-HOUR HARVEST

	Treatment	Replicate	Total #	Count per total cells			CBPI^1	Cytotoxicity^2
	Condition	Culture	of Cells	Cells with # of nuclei
	µg/mL		Counted	1	2	>2
	Ethanol	A	500	125	345	30	1.810
		B	500	123	349	28
	test substance
	14.6	A	500	122	348	30	1.766	5%
		B	500	164	314	22
	29.2	A	500	150	318	32	1.773	5%
		B	500	130	349	21
	48.7	A	500	145	342	13	1.709	12%
		B	500	174	311	15
	60.9	A	500	306	190	4	1.392	52%
		B	500	307	192	1
	76.2	A	500	387	110	3	1.224	72%
		B	500	394	104	2
	95.2	A	500	364	133	3	1.335	59%
		B	500	305	194	1
	119 p	A	500	358	137	5	1.251	69%
		B	500	396	104	0
	170 p	A	500	355	143	2	1.281	65%
		B	500	366	134	0
	MMC, 0.4	A	500	250	250	0	1.498	39%
		B	500	255	242	3
	MMC, 0.5	A	500	272	226	2	1.456	44%
		B	500	274	226	0
	^1CBPI = Cytokinesis-Block Proliferation Index
^2Relative to vehicle control
p: Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

 

 

Table 2: MICRONUCLEUS ASSAY IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION, 4-HOUR TREATMENT, 24-HOUR HARVEST

	Treatment	Replicate	Total #	Count per total cells			CBPI^1	Cytotoxicity^2
	Condition	Culture	of Cells	Cells with # of nuclei
	µg/mL		Counted	1	2	>2
	Ethanol	A	500	172	307	21	1.699
		B	500	183	284	33
	test substance
	14.6	A	500	172	315	13	1.661	5%
		B	500	196	288	16
	29.2	A	500	216	277	7	1.585	16%
		B	500	216	274	10
	48.7	A	500	237	253	10	1.523	25%
		B	500	260	230	10
	60.9	A	500	267	226	7	1.483	31%
		B	500	258	241	1
	76.2	A	500	334	164	2	1.334	52%
		B	500	336	162	2
	95.2	A	500	380	119	1	1.240	66%
		B	500	381	119	0
	119 p	A	500	476	24	0	1.045	94%
		B	500	479	21	0
	170 p	A	0	0	0	0	0.000	-
		B	0	0	0	0
	CP, 2.5	A	500	276	224	0	1.464	34%
		B	500	263	234	3
	CP, 5	A	500	285	213	2	1.425	39%
		B	500	293	206	1
	CP, 7.5	A	500	344	155	1	1.343	51%
		B	500	315	184	1
	^1CBPI = Cytokinesis-Block Proliferation Index
^2Relative to vehicle control
p: Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

 

 

Table 3: MICRONUCLEUS ASSAY IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION, 24-HOUR TREATMENT, 24-HOUR HARVEST

	Treatment	Replicate	Total #	Count per total cells			CBPI^1	Cytotoxicity^2
	Condition	Culture	of Cells	Cells with # of nuclei
	µg/mL		Counted	1	2	>2
	Untreated	A	500	146	295	59	1.807
		B	500	174	258	68
	Ethanol	A	500	211	247	42	1.705	13%
		B	500	177	272	51
	test substance
	14.6	A	500	259	214	27	1.594	16%
		B	500	201	272	27
	29.2	A	500	298	187	15	1.458	35%
		B	500	285	189	26
	48.7	A	500	334	163	3	1.344	51%
		B	500	325	175	0
	60.9	A	500	407	92	1	1.155	78%
		B	500	440	59	1
	76.2	A	500	465	34	1	1.055	92%
		B	500	481	19	0
	95.2	A	0	0	0	0	0.000	-
		B	0	0	0	0
	119 p	A	0	0	0	0	0.000	-
		B	0	0	0	0
	170 p	A	0	0	0	0	0.000	-
		B	0	0	0	0
	VB, 5 ng/mL	A	500	177	264	59	1.688	2%
		B	500	245	204	51
	VB, 7.5 ng/mL	A	500	151	267	82	1.849	-20%
		B	500	149	284	67
	VB, 10 ng/mL	A	500	347	141	12	1.324	54%
		B	500	355	131	14
	^1CBPI = Cytokinesis-Block Proliferation Index
^2 Cytotoxicity of vehicle is relative to untreated control; Cytotoxicity of test substance is relative to vehicle control.
p: Visible precipitate was observed in the treatment medium at the conclusion of the treatment period.

 

 

Table 4: MICRONUCLEUS ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES
IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION,

MICRONUCLEUS ASSAY: 4-HOUR TREATMENT, 24-HOUR HARVEST

				Percentage of	Average Percent
	Treatment	Replicate	Total #	Micronucleated	Micronucleated
	µg/mL	Culture	of Cells	Binucleated Cells	Binucleated
			Counted	per culture	Cells per Dose
	Ethanol	A	1000	0.30%	0.25%
		B	1000	0.20%
	test substance
	29.2	A	1000	0.20%	0.20%
		B	1000	0.20%
	48.7	A	1000	0.30%	0.30%
		B	1000	0.30%
	60.9	A	1000	0.20%	0.25%
		B	1000	0.30%
	MMC, 0.5	A	1000	3.50%	3.55%**
		B	1000	3.60%
** p £ 0.01, Fisher's exact test, relative to the solvent control.

 

 

Table 5: MICRONUCLEUS ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES
IN THE PRESENCE OF EXOGENOUS METABOLIC ACTIVATION

MICRONUCLEUS ASSAY: 4-HOUR TREATMENT, 24-HOUR HARVEST

				Percentage of	Average Percent
	Treatment	Replicate	Total #	Micronucleated	Micronucleated
	µg/mL	Culture	of Cells	Binucleated Cells	Binucleated
			Counted	per culture	Cells per Dose
	Ethanol	A	1000	0.20%	0.25%
		B	1000	0.30%
	test substance
	29.2	A	1000	0.30%	0.35%
		B	1000	0.40%
	60.9	A	1000	0.30%	0.30%
		B	1000	0.30%
	76.2	A	1000	0.50%	0.45%
		B	1000	0.40%
	CP, 7.5	A	1000	2.00%	1.95%**
		B	1000	1.90%
** p £ 0.01, Fisher's exact test, relative to the solvent control.

 

 

 

Table 6: MICRONUCLEUS ANALYSIS OF HUMAN PERIPHERAL BLOOD LYMPHOCYTES
IN THE ABSENCE OF EXOGENOUS METABOLIC ACTIVATION

MICRONUCLEUS ASSAY:  24-HOUR TREATMENT, 24-HOUR HARVEST

			Percentage of	Average Percent
Treatment	Replicate	Total #	Micronucleated	Micronucleated
µg/mL	Culture	of Cells	Binucleated Cells	Binucleated
		Counted	per culture	Cells per Dose
Untreated	A	1000	0.20%	0.20%
	B	1000	0.20%
Ethanol	A	1000	0.30%	0.30%
	B	1000	0.30%
test substance
14.6	A	1000	0.50%	0.45%
	B	1000	0.40%
29.2	A	1000	0.50%	0.45%
	B	1000	0.40%
48.7	A	1000	0.40%	0.30%
	B	1000	0.20%
VB, 10 ng/mL	A	1000	2.90%	3.40%**
	B	1000	3.90%
** p £ 0.01, Fisher's exact test, relative to the solvent control.

 

Conclusions:

The test substance did not induce micronuclei in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.

Executive summary:

The test substance was evaluated for its potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Ethanol was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.1703 to 1703 µg/mL (10 mM), which was the limit dose for this assay. Cytotoxicity [>= 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses >= 170.3 µg/mL in all three exposure groups.  At the end of the treatment period, visible precipitate was observed at doses >= 170.3 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 14.6 to 170 µg/mL for all three exposure groups. In the micronucleus assay, cytotoxicity (>=50% CBPI relative to the vehicle control) was observed at doses >= 60.9 µg/mL in the non-activated 4-hour exposure group; at doses >= 76.2 µg/mL in the S9-activated 4-hour exposure group; and at doses >= 48.7 µg/mL in the non-activated 24-hour exposure group. At the end of the treatment period, visible precipitate was observed at doses >= 119 µg/mL in all three exposure groups. The doses selected for evaluation of micronuclei were 29.2, 48.7, and 60.9 µg/mL for the non‑activated 4-hour exposure group; 29.2, 60.9, and 76.2 µg/mL for the S9-activated 4‑hour exposure group; and 14.6, 29.2, and 48.7 µg/mL for the non‑activated 24-hour exposure group. Neither statistically significant nor dose‑dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data. These results show that the test substance does not induce micronuclei in the presence and absence of the exogenous metabolic activation system. 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 Mar - 7 Apr 2001

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:

adopted in July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Verkehr Baden-Württemberg, Stuttgart, Germany

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Experiment 1
15, 50, 150, 500 and 1500 µg/plate without metabolic activation for TA1535, TA102, TA98 and TA1537
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA100
15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for all strains

Experiment 2
15, 50, 150, 500 and 1500 µg/plate without metabolic activation for TA100
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA1535, TA102, TA98 and TA1537
15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA98
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA100, TA1535, TA102 and TA1537

Vehicle / solvent:

- Vehicle/solvent used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene (2-AA)

Remarks:

+S9: 2-AA (0.8 µg/plate, TA98, TA100; 0.9 µg/plate, TA102, TA1535; 1.7 µg/plate, TA1537); -S9: NaN3 (0.7 µg/plate, TA100, TA1535); 2-NF (2.5 µg/plate, TA98); 9-AA (50 µg/plate, TA1537); Mitomycin C (0.15 µg/plate, TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or diminution of the background lawn

Statistics:

X²-test was used to estimate the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level. Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred up to the highest investigated dose.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and positive control data were between the minimum and maximum value of the historical control data of the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix the test substance was bacteriotoxic in the strains TA98, TA100, and TA1537 at 5000 µg/plate. In the presence of S9 mix the test substance was bacteriotoxic in the strains TA98 and TA100 at 5000 µg/plate.

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9 mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
–	0	92 ± 7	26 ± 7	328 ± 28	24 ± 3	11 ± 4
–	0 (EtOH)	76 ± 8	21 ± 4	326 ± 12	29 ± 4	12 ± 3
–	15	-	20 ± 4	289 ± 22	22 ± 5	8 ± 3
–	50	70 ± 8	22 ± 6	247 ± 19	32 ± 3	9 ± 2
–	150	66 ± 5	21 ± 5	331 ± 8	30 ± 3	8 ± 5
–	500	66 ± 10	20 ± 4	326 ± 11	31 ± 5	5 ± 2
–	1500	74 ± 12	18 ± 7	311 ± 38	28 ± 5	9 ± 4
–	5000	46 ± 6 T	-	-	-	-
Positive controls, –S9 mix	Name	NaN3	NaN3	MMC	2-NF	9-AA
	Concentrations (μg/plate)	0.7	0.7	0.15	2.5	50
	Mean No. of colonies/plate (average of 3 ± SD)	429 ± 70	835 ± 19	967 ± 67	724 ± 73	200 ± 11
+	0	92 ± 11	12 ± 2	241 ± 29	19 ± 4	12 ± 3
+	0 (EtOH)	94 ± 4	31 ± 3	248 ± 4	18 ± 2	12 ± 3
+	50	95 ± 6	29 ± 7	257 ± 39	24 ± 3	12 ± 4
+	150	94 ± 8	27 ± 1	268 ± 21	19 ± 5	10 ± 4
+	500	90 ± 4	28 ± 4	261 ± 37	20 ± 3	11 ± 2
+	1500	86 ± 10	26 ± 5	254 ± 10	17 ± 2	12 ± 4
+	5000	72 ± 10 T	24 ± 5	227 ± 26	11 ± 5 T	11 ± 3
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	0.8	0.9	0.9	0.8	1.7
	Mean No. of colonies/plate (average of 3 ± SD)	593 ± 57	198 ± 24	535 ± 25	532 ± 57	108 ± 5

NaN3: Sodium azide

2-NF: 2-nitrofluorene

9-AA: 9-aminoacridine

MMC: Mitomycin C

2-AA: 2-aminoanthracene

EtOH: Ethanol

T: bacteriotoxic

Table 2. Test results of experiment 2 (plate incorporation).

With or without S9 mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
–	0	80 ± 5	14 ± 5	316 ± 23	52 ± 7	13 ± 3
–	0 (EtOH)	77 ± 6	8 ± 2	294 ± 11	37 ± 4	9 ± 3
–	15	71 ± 7	-	-	-	-
–	50	63 ± 3	8 ± 4	311 ± 35	38 ± 5	7 ± 3
–	150	72 ± 7	7 ± 2	256 ± 21	29 ± 6	10 ± 3
–	500	68 ± 10	5 ± 3	253 ± 21	19 ± 3	7 ± 3
–	1500	66 ± 114	4 ± 2	266 ± 16	18 ± 7	6 ± 2
–	5000		6 ± 4	221 ± 5	17 ± 3 T	4 ± 3 T
Positive controls, –S9 mix	Name	NaN3	NaN3	MMC	2-NF	9-AA
	Concentrations (μg/plate)	0.7	0.7	0.15	2.5	50
	Mean No. of colonies/plate (average of 3 ± SD)	625 ± 52	451 ± 37	807 ± 21	589 ± 45	115 ± 10
+	0	97 ± 8	11 ± 3	279 ± 48	24 ± 2	17 ± 4
+	0 (EtOH)	84 ± 3	18 ± 3	314 ± 6	27 ± 1	12 ± 3
+	15				26 ± 2
+	50	87 ± 12	17 ± 3	313 ± 14	23 ± 2	9 ± 4
+	150	73 ± 2	15 ± 2	346 ± 29	27 ± 5	13 ± 3
+	500	78 ± 8	18 ± 6	287 ± 10	19 ± 3	14 ± 4
+	1500	82 ± 4	16 ± 5	288 ± 11	19 ± 3	12 ± 4
+	5000	67 ± 4	13 ± 4	249 ± 41	19 ± 3	11 ± 2
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	0.8	0.9	0.9	0.8	1.7
	Mean No. of colonies/plate (average of 3 ± SD)	334 ± 64	145 ± 7	613 ± 24	727 ± 42	114 ± 17

NaN3: Sodium azide

2-NF: 2-nitrofluorene

9-AA: 9-aminoacridine

MMC: Mitomycin C

2-AA: 2-aminoanthracene

EtOH: Ethanol

T: bacteriotoxic

Conclusions:

Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA100, TA1535, TA102, TA98 and TA1537) tested up to the limit dose with and without metabolic activation.

Executive summary:

The mutagenicity of the test item was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system.

The test item was dissolved in ethanol and tested in concentrations of 15 to 5000 µg per plate in the absence and presence of S9. In the absence of S9-mix test item was bacteriotoxic towards the strains TA98, TA100, and TA1537 at 5000 µg/plate. In the presence of S9-mix the test item was bacteriotoxic towards the strains TA98 and TA100 at 5000µg/plate. Precipitation of the test compound an the plates was not observed.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, the test item did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 Mar - 21 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Cell proliferation: Blood samples were drawn from one healthy non-smoking male donor (31 years old) not receiving medication. Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemagglutinin (PHA) to the culture medium for a period of 48 hours. The cell harvest time point was approximately 1.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.
- Type and identity of media: DMEM/F12, mixture 1:1, supplemented with 200 mM GlutaMax, 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:

Pre-Experiment:
4 h treatment: 11.0, 19.3, 33.8, 59.2, 103.6, 181.3, 317.2, 555.1, 971.4 and 1700 µg/mL with and without S9 mix

Since the cultures fulfilled the requirements for cytogenetic evaluation and increases in chromosomal aberrations were observed in the presence of S9 mix, this preliminary test was designated Main Experiment and no further experiment was performed.

Main Experiment:
11.0, 19.3 and 33.8 µg/mL were selected for scoring of chromosome aberrations without S9 mix
181.3, 317.2 and 555.1 µg/mL were selected for scoring of chromosome aberrations with S9 mix

Vehicle / solvent:

- Vehicle/solvent used: ethanol (final concentration in the culture medium was 0.5%)
- 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

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

ethylmethane sulfonate (EMS): 825 µg/mL (4 h, -S9); cyclophosphamide (CPA): 15 µg/mL (4 h, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.2 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 replications in 1 experiment

NUMBER OF CELLS EVALUATED: 150 well-spread metaphases were evaluated per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− 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
The test item is then considered unable to induce chromosomal aberrations in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− 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
When all of the criteria are met, the test item is then considered able to induce chromosomal aberrations in this test system.

Statistics:

Statistical significance will be confirmed by using the Fisher’s exact test (p < 0.05) using the validated R Script FisherMidP_V1.rnw for those values that indicate an increase in the number of cells with chromosomal aberrations compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at 317.2 and 555.1 µg/mL following 4 h treatment with S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 971.4 µg/mL and above following 4 h treatment with S9 mix

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 was observed.
- Precipitation: No precipitation of the test item in the culture medium was observed.
- Other confounding effects: Phase separation was observed at 33.8 µg/mL and above in the absence of S9 mix and at 555.1 µg/mL and above in the presence of S9 mix at the end of treatment.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. With regard to the molecular weight of the test item, 1700 µg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 11.0 to 1700 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Since the cultures fulfilled the requirements for cytogenetic evaluation and increases in chromosomal aberrations were observed in the presence of S9 mix, this preliminary test was designated Main Experiment and no further experiment was performed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the absence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In the presence of S9 mix no cytotoxicity was observed up to the highest evaluated concentration.

Table 1. Results of the chromosomal aberration test.

Test item	Concentration [µg/mL]	Mitotic Index (% of control)	Aberrant cells per 150 metaphase chromosome spreads
			No. of cells with aberrations (+ gaps)	No. of cells with aberrations (- gaps)
Exposure period 4 h, fixation time 22 h, without S9 mix
Ethanol	0.5% (v/v)	100	2.3	2.3
EMS	825	58.4	18.3	18.0^S
Test substance	11.0	109.3	2.0	1.7
	19.3	98.6	2.3	1.7
	33.8^PS	105.1	3.0	3.0
Exposure period 4 h, fixation time 22 h, with S9 mix
Ethanol	0.5% (v/v)	100	2.0	1.7
CPA	15	25.9	24.0	23.7^S
Test substance	181.3	97.0	1.7	1.7
	317.2^#	78.5	6.0	5.5^S
	555.1^PS	74.9	8.0	6.7^S

EMS: Ethylmethane sulfonate

CPA: Cyclophosphamide

S: Aberration frequency statistically significant higher than corresponding control values

PS: Phase separation occurred at the end of treatment

#: Evaluation of 300 metaphases per culture

In the absence of S9 mix, no statistically significant or biologically relevant increases in the number of cells carrying structural chromosome aberrations were observed.

 

In the presence of S9 mix statistically significant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with 317.2 and 555.1 µg/mL (5.5 and 6.7% aberrant cells, excluding gaps). These values exceed the laboratory historical solvent control data (0.0 – 3.5% aberrant cells, excluding gaps) and dose-dependency was observed.

 

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

 

Either EMS or CPA were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

Under the experimental conditions of the in vitro chromosome aberration test, the test item induced structural chromosomal aberrations in human lymphocytes with metabolic activation. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the presence of S9 mix, when tested up to phase separating concentrations.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Mar - 05 June 2015

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, 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 and 24 h treatment: 13.3, 26.6, 53.1, 106.3, 212.5, 425, 850 and 1700 µg/mL without metabolic activation
4 h treatment: 13.3, 26.6, 53.1, 106.3, 212.5, 425, 850 and 1700 µg/mL with metabolic activation

Experiment 1:
4 h treatment: 3.3, 6.5, 13, 19.5, 26, 39, 52 and 78 µg/mL without metabolic activation
4 h treatment: 26.6, 53.1, 106.3, 212.5, 318.8 and 425 µg/mL with metabolic activation

Experiment 2:
24 h treatment: 3.1, 6.3, 12.5, 25, 50, 100, 150 and 200 µg/mL without metabolic activation
4 h treatment: 53.1, 106.3, 212.5, 325, 375, 425 and 475 µg/mL with metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: ethanol (0.5% (v/v))
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-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:

ethylmethane sulfonate (EMS): 0.15 mg/mL (4 and 24 h, -S9); 7,12-dimethylbenzanthracene (DMBA): 1.1 and 2.2 µg/mL (4 h, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
1st experiment: 4 h exposure with and without S9 mix
2nd experiment: 4 h exposure with S9 mix and 24 h 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: duplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency I or cell density below 50%

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations of the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

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 26 and 50 µg/mL following 4 h treatment without S9 in experiment I and II, respectively; at 425 µg/mL following 4 h treatment with S9

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 item.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 and 24 h) prior to removal to the test item.
Pre-Experiment:
No phase separation or precipitation was noted at the end of treatment.
Main Experiment:
Phase separation was noted at the two highest analysable concentrations of experiment II 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 (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 13.3 µg/mL and 1700 µg/mL (equal to a molar concentration of approx. 10 mM) were used. The highest concentration of the pre-experiment was chosen with regard to the purity (99.9%) and the molecular weight (170.3 g/mol) of the test item. Strong cytotoxic effects were observed at 850 µg/mL and above without metabolic activation and at 425 µg/mL and above with metabolic activation following 4 h treatment indicated by a completely inhibited cell growth. Following 24 h treatment without metabolic activation relevant cytotoxicity was noted at 850 µg/mL and above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cultures at 425 µg/mL with metabolic activation were not continued based on exceedingly severe cytotoxicity. In experiment II without metabolic activation, the cultures at 50 to 200 µg/mL were not continued due to exceedingly severe toxic effects. In experiment II with metabolic activation the cultures at 475 µg/mL were not continued for the same reason.

Table 1: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Rel. cloning efficiency I	Rel. cell density	Rel. cloning efficiency II	Mutant colonies per 10^6cells	Induction factor
Culture I
0 (DMSO)	100.0	100.0	100.0	19.3	1.0
3.3	96.5	Culture was not continued^#
6.5	86.4	Culture was not continued^#
13	79.6	Culture was not continued^#
19.5	59.4	96.0	99.0	14.5	0.7
26	30.2	85.2	99.7	22.3	1.2
39	22.5	83.6	102.3	9.9	0.5
52	7.7	77.5	96.8	21.4	1.1
78	0.0	86.9	98.5	30.3	1.6
EMS, 150	96.2	100.9	100.4	104.3	5.4
Culture II
0 (DMSO)	100.0	100.0	100.0	31.7	1.0
3.3	96.2	Culture was not continued^#
6.5	87.1	Culture was not continued^#
13	65.0	Culture was not continued^#
19.5	52.9	50.8	101.1	21.6	0.7
26	34.3	56.9	98.9	12.7	0.4
39	18.2	55.1	97.1	25.4	0.8
52	6.6	35.1	97.6	27.2	0.9
78	0.0	41.6	98.2	34.4	1.1
EMS, 150	102.9	94.8	99.3	128.6	4.1

DMSO: Dimethyl sulfoxide

EMS: Ethylmethane sulfonate

#: Culture was not continued since a minimum of only four analysable concentrations is required.

##: Culture was not continued due to exceedingly severe cytotoxic effects.

 

Table 2: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [µg/mL]	Rel. cloning efficiency I	Rel. cell density	Rel. cloning efficiency II	Mutant colonies per 10^6cells	Induction factor
Culture I
0 (DMSO)	100.0	100.0	100.0	8.5	1.0
26.6	102.8	103.7	104.6	13.0	1.5
53.1	107.8	100.5	108.7	11.4	1.3
106.3	82.6	97.9	110.8	13.7	1.6
212.5	90.6	89.3	111.2	7.8	0.9
318.8	83.7	105.5	101.1	7.0	0.8
425	87.3	Culture was not continued^##
DMBA, 1.1	93.4	78.1	103.7	260.3	30.5
Culture II
0 (DMSO)	100.0	100.0	100.0	15.9	1.0
26.6	86.7	108.1	79.3	24.9	1.6
53.1	84.4	99.1	98.5	12.2	0.8
106.3	72.9	96.4	87.5	8.8	0.6
212.5	77.9	106.9	93.1	9.3	0.6
318.8	78.5	40.1	102.5	11.0	0.7
425	62.8	Culture was not continued^##
DMBA, 1.1	73.7	98.5	101.9	199.6	12.6

DMSO: Dimethyl sulfoxide

DMBA: 7,12-dimethylbenzanthracene

#: Culture was not continued since a minimum of only four analysable concentrations is required.

##: Culture was not continued due to exceedingly severe cytotoxic effects.

 

Table 3: Experiment II - 24 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Rel. cloning efficiency I	Rel. cell density	Rel. cloning efficiency II	Mutant colonies per 10^6cells	Induction factor
Culture I
0 (DMSO)	100.0	100.0	100.0	16.5	1.0
3.1	105.3	55.8	103.9	13.3	0.8
6.3	87.7	41.9	100.7	14.2	0.9
12.5	94.0	43.8	100.7	11.9	0.7
25	82.1	45.3	104.1	6.9	0.4
50	0.6	4.1	Culture was not continued^##
100	1.5	1.8	Culture was not continued^##
150	1.0	2.8	Culture was not continued^##
200	1.0	0.5	Culture was not continued^##
EMS, 150	93.5	69.1	101.1	296.5	18.0
Culture II
0 (DMSO)	100.0	100.0	100.0	14.9	1.0
3.1	97.1	69.5	101.8	13.7	0.9
6.3	89.0	55.0	99.8	23.3	1.6
12.5	86.0	56.7	99.4	13.9	0.9
25	82.7	58.7	99.4	12.1	0.8
50	0.2	6.4	Culture was not continued^##
100	1.0	Culture was not continued^##
150	0.7	Culture was not continued^##
200	1.3	Culture was not continued^##
EMS, 150	97.8	100.0	100.4	316.2	21.2

DMSO: Dimethyl sulfoxide

EMS: Ethylmethane sulfonate

#: Culture was not continued since a minimum of only four analysable concentrations is required.

##: Culture was not continued due to exceedingly severe cytotoxic effects.

 

Table 4: Experiment II - 4 h exposure - With Metabolic Activation

Concentration [µg/mL]	Rel. cloning efficiency I	Rel. cell density	Rel. cloning efficiency II	Mutant colonies per 10^6cells	Induction factor
Culture I
0 (DMSO)	100.0	100.0	100.0	12.6	1.0
53.1	96.9	Culture was not continued^#
106.3	90.4	79.9	96.8	11.6	0.9
212.5	90.7	69.7	98.4	12.1	1.0
325	101.5	96.4	100.4	12.2	1.0
375	18.7	88.8	128.4	9.3	0.7
425	87.9	26.2	103.1	9.8	0.8
475	78.7	Culture was not continued^##
DMBA, 2.2	90.3	116.1	101.5	168.2	13.3
Culture II
0 (DMSO)	100.0	100.0	100.0	11.7	1.0
53.1	103.8	99.5	100.1	9.5	0.8
106.3	103.5	133.5	100.2	7.5	0.6
212.5	103.7	88.1	98.3	15.0	1.3
325	101.7	133.8	102.2	12.9	1.1
375	104.1	103.1	100.9	23.6	2.0
425 (PS)	91.6	Culture was not continued^##
475 (PS)	87.0	Culture was not continued^##
DMBA, 2.2	103.1	64.4	101.5	230.5	19.7

DMSO: Dimethyl sulfoxide

DMBA: 7,12-dimethylbenzanthracene

(PS): Phase separation

#: Culture was not continued since a minimum of only four analysable concentrations is required.

##: Culture was not continued due to exceedingly severe cytotoxic effects.

The experimental part without metabolic activation in experiment I was terminated based on exceedingly severe cytotoxicity and repeated. The repeat experiment was again, terminated due to exceedingly severe cytotoxicity and repeated. The experimental part without metabolic activation in experiment II was terminated and repeated twice based on exceedingly severe cytotoxicity following treatment.

 

The recommended cytotoxic range of approx. 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation.

 

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The mutant frequency did not exceed the historical range of solvent controls.

 

A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of experiment II without metabolic activation. This trend however, was reciprocal, going down versus increasing concentrations and consequently without any biological relevance.

 

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.5 up to 31.7 mutants per 10⁶ cells; the range of the groups treated with the test item was from 6.9 up to 34.4 mutants per 10⁶ cells.

 

EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

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

Executive summary:

The study was performed to investigate the potential of MENTHYL METHYL ETHER to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum test item concentration of the pre-experiment (1700 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and phase separation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

The tested concentrations are described in chapter 3.6. The evaluated experimental points and the results are summarised in Table 3.

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.

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 with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2001). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 were exposed to the test substance using the standard plate incorporation method. Test substance concentrations of 15 to 5000 µg/plate were selected for two independent experiments with and without metabolic activation. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control, neither in the presence nor absence of metabolic activation. In absence of metabolic activation the test substance was bacteriotoxic towards the strains TA 98, TA 100, and TA 1537 at 5000 µg/plate. In the presence of metabolic activation the test substance was bacteriotoxic towards the strains TA 98 and TA 100 at 5000 µg/plate. All positive and negative control values were found to be within the respective historical control ranges. 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 (2015). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. Based on the results of a pre-test, cells were exposed for 4 and 24 h to the test substance up to concentrations of 200 µg/mL in the absence and 475 µg/mL in the presence of metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in the first experiment at 26 μg/mL and above without metabolic activation. In the second experiment exceedingly severe cytotoxic effects were noted at 50.0 μg/mL and above without metabolic activation. In the experimental part with metabolic activation relevant cytotoxicity was noted at 425.0 μg/mL in culture I. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency generally did not exceed the historical range of solvent controls. 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 not considered to be mutagenic in this HPRT assay.

 

 Cytogenicity

The clastogenic activity of the test substance was investigated in an in vitro mammalian chromosome aberration test in cultured human lymphocytes performed according to OECD Guideline 473 and GLP (2015). The test substance was dissolved in ethanol. Only one experiment was performed, since the test item was considered to be mutagenic after the first experiment. The exposure period was 4 h with and without S9 metabolic activation. The chromosomes were prepared 22 h after start of treatment with the test substance. In each experimental group two parallel cultures were analyzed. At least 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 1700.0 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test substance. Phase separation was observed at 33.8 μg/mL and above in the absence of S9 mix and at 555.1 μg/mL and above in the presence of S9 mix at the end of treatment. In the absence and presence of S9 mix no cytotoxicity was observed up to the highest applied or evaluated concentration. In the absence of S9 mix, no statistically significant or biologically relevant increases in the number of cells carrying structural chromosome aberrations were observed. In the presence of S9 mix statistically significant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with 317.2 and 555.1 μg/mL (5.5 and 6.7% aberrant cells, excluding gaps). These values exceed the laboratory historical solvent control data (0.0 – 3.5% aberrant cells, excluding gaps) and dose-dependency was observed. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Either ethylmethan sulfonate (825.0 μg/mL) or cyclophosphamide (15.0 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, the test substance induced structural chromosomal aberrations in human lymphocytes in vitro in the presence of metabolic activation under the experimental conditions reported. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test, when tested up to phase separating or the highest evaluable concentrations.

 

In a second cytogenicity study the test substance was evaluated for its potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. The study was conducted according to OECD Guideline 487 with a primary culture of HPBL. The cells were treated for 4 hours in the absence and presence of S9-mix, and for 24 hours in the absence of S9-mix. Ethanol was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.1703 to 1703 µg/mL (10 mM), which was the limit dose for this assay. Cytotoxicity [>= 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses >= 170.3 µg/mL in all three exposure groups.  At the end of the treatment period, visible precipitate was observed at doses >= 170.3 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 14.6 to 170 µg/mL for all three exposure groups. In the micronucleus assay, cytotoxicity (>=50% CBPI relative to the vehicle control) was observed at doses >= 60.9 µg/mL in the non-activated 4-hour exposure group; at doses >= 76.2 µg/mL in the S9-activated 4-hour exposure group; and at doses >= 48.7 µg/mL in the non-activated 24-hour exposure group. At the end of the treatment period, visible precipitate was observed at doses >= 119 µg/mL in all three exposure groups. The doses selected for evaluation of micronuclei were 29.2, 48.7, and 60.9 µg/mL for the non activated 4-hour exposure group; 29.2, 60.9, and 76.2 µg/mL for the S9-activated 4 hour exposure group; and 14.6, 29.2, and 48.7 µg/mL for the non activated 24-hour exposure group. Neither statistically significant nor dose dependent increases in micronuclei induction were observed at any dose in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). The results were within the 95% control limit of the historical negative control data. These results show that the test substance does not induce micronuclei in the presence and absence of the exogenous metabolic activation system.

 

 

Furthermore, an in vitro micronucleus test using Reconstructed skin Micronucleus assay in EpiDermTM as supporting study is available performed according to GLP (2016). The assay design was derived from an expert recommended protocol which was derived from the general OECD Guideline 487. The purpose of this study was to evaluate the genotoxic potential of the test substance by assessing induction of micronuclei in the reconstructed skin micronucleus assay (RSMN) in EpiDermTM. In the preliminary cytotoxicity and the micronucleus assays, EpiDermTM tissues were treated twice, 24 hours apart and tissues were processed at 48 hours (2-day dosing regimen). In the confirmatory micronucleus assay, the tissues were treated three times, 24 hours apart and tissues were processed at 72 hours (3-day dosing regimen). Tissues were treated by application of 10 μL of the test substance/vehicle mixture at the appropriate concentration on the top surface of the EpiDermTM tissue. Acetone was used as the vehicle. Cytotoxicity was assessed by the cytokinesis-block proliferation index (CBPI) and relative viable cell count (RVCC). Based on the results of the preliminary cytotoxicity test, the micronucleus assay was conducted using triplicate tissues at concentrations ranging from 0.5 to 100 mg/mL. Cytotoxicity (CBPI and RVCC relative to vehicle control) was observed at concentrations of 80 and 100 mg/mL. The concentrations selected for scoring micronuclei were 5, 20, and 60 mg/mL. The highest concentration selected for analysis had approximately 50% cytotoxicity based on RVCC. One thousand binucleated cells per tissue were scored for the presence of micronuclei. The percentage of cells with micronucleated binucleated cells in the test substance-treated tissues was not significantly increased relative to the vehicle control at any concentration analyzed. Since the result of the micronucleus assay using a 2-day dosing regimen was negative, a confirmatory assay was conducted with a 3-day dosing regimen at concentrations ranging from 3 to 60 mg/mL using triplicate tissues. In the confirmatory micronucleus assay, cytotoxicity (≥ 50% CBPI relative to vehicle control) was not observed at any concentration, while cytotoxicity based on RVCC was observed at concentrations of 50 and 60 mg/mL. The concentrations selected for scoring micronuclei were 3, 20, and 50 mg/mL. The highest concentration selected for analysis had approximately 50% cytotoxicity based on RVCC. One thousand binucleated cells per tissue were scored for the presence of micronuclei. In the confirmatory micronucleus assay, the percentage of cells with micronucleated binucleated cells in the test substance-treated tissues was not significantly increased relative to the vehicle control at any concentration analyzed. The results for the positive and negative controls indicate that all criteria for a valid assay were met. Based on the findings of this study, the test substance was concluded to be negative for the induction of micronuclei in the reconstructed skin micronucleus assay (RSMN) in EpiDerm™.

 

The studies on cytogenicity were used in an weight-of evidence approach. The MNT studies were clearly negative, and the positive results of the chromosome aberration study were only slightly exceeding the historical control values of the historical solvent control data, only for exposure in presence of metabolic activation. As only three test substance concentrations were included in the chromosome aberration assay, the dose-dependency of the results was not proven with certainty. The overall conclusion for the endpoint cytogenicity is negative.

Justification for classification or non-classification

The available data on genetic toxicity in vitro reveal negative results in the Ames test and the gene mutation test in mammalian cells. Based on the available cytogenicity studies (one chromosome aberration assay in cultured human lymphocytes and two in vitro micronucleus assays, one in primary lymphocytes and one in EpiDermTM tissue) the cytogenicity results were concluded to be negative as well.

Therefore the test substance is not considered to be classified for genotoxicity according to Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.