2-Propanol, 1-butoxy-3-(2-propen-1-yloxy)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mouse Lymphoma Assay:

increases in the mutant frequency at the TK +/- locus in L5178Y cells, consequently it is considered to be mutagenic in the presence of metabolic activation in this assay.

Bacterial Reverse Mutation Assay:

no increase in the number of revertant colonies, no a dose-related response was observed at any doses in any strains, with or without metabolic activation.

Chromosomal Aberration Test in Cultured Mammalian Cells:

considered non-clastogenic to cultured mammalian cells under the present test conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 April 2012 - 16 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance: Liquid (specific gravity: 0.95)
Batch: AB20
Purity: 92%
Storage Conditions: Room temperature in the dark

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Preliminary test (dose range finder): 0 (solvent control), 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate
Main test: 0 (solvent control), 156, 313, 625, 1250, 2500, 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on information from the sponsor it was noted that the test material was insoluble in water. A solubility test was conducted in DMSO (50 mL) and Acetone (100 mL). The test material was soluble in each solvent and neither solution gave signs of reaction (evolution of gas or an exothermic reaction). DMSO was therefore selected as the solvent.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

Used for TA100, TA98, and WP2 uvrA in the absence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Used for TA1535 in the absence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl

Remarks:

Used for TA1537 in the absence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Used for TA100, TA98, and TA1537 in the presence of S9 mix

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Used for TA1535 and WP2 uvrA in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation)

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Two plates for each dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The number of revertant colonies were counted and compared with the number of colonies on the control plates. A significant increase in the number was judged as a two-fold increase in the number of colonies relative to the negative control, and a dose-response with reproducibility was considered to indicate a positive result.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition was observed at the highest test concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition was observed at the highest test concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

From the results described above, it is concluded that the test item is not mutagenic in the bacterial reverse mutation assay carried out under the experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 March 2017- 04 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation.

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Physical state/Appearance: Brown liquid
Batch: 15B26
Purity: 81.7%
Expiry Date: 30 April 2017
Storage Conditions: Room temperature, in the dark, under N2

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: TK+/- 3.7.2c mouse lymphoma cell line
- Cell cycle length, doubling time or proliferation index: approximately 12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium with 5% CO2

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

125 to 1800 μg/mL: 4-hour exposure group in the absence of metabolic activation
1.95 to 125 μg/mL: 4-hour exposure group in the presence of metabolic activation
31.25 to 1000 μg/mL: 24-hour exposure group in the absence of metabolic activation
The maximum dose levels in the Mutagenicity Test were limited by test item-induced toxicity.

Vehicle / solvent:

Solvent (DMSO)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable):
for the 4-hour exposure groups in both the absence and presence of metabolic activation: 1 x 106 cells/mL in 10 mL aliquots in R10 medium
for the 24-hour exposure group in the absence of metabolic activation: 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks

DURATION
- Exposure duration: 4 or 24 hours at 37°C
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION:
- Method: Suspension Growth, Relative Suspension Growth, Relative Total Growth, Viability

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 5 x 105 cells/mL

Rationale for test conditions:

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Statistics:

The colony-forming units over the wells is described by the Poisson distribution.
The day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other:

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

See Table 1.
There was evidence of marked toxicity following exposure to the test item in all three of the exposure groups (4-hour exposure -S9, 4-hour exposure+S9, 24-hour exposure-S9).
There was also evidence of reductions in viability (%V) in the 4-hour exposure groups in both the absence and presence of metabolic activation, indicating that residual toxicity had occurred.
The excessive toxicity observed at and above 750 μg/mL in the 24-hour exposure group in the absence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance.
The toxicity observed at 1800 μg/mL in the 4-hour exposure group in the absence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis.
No precipitate of the test item was observed at any of the dose levels.
Statistically significant and dose-related (linear-trend) increases in mutant frequency were observed in the 4-hour exposure group in the presence of metabolic activation.

Remarks on result:

other: In the preliminary cytotoxicty test

Table 1: Results

Concentration (µg/mL)		4-Hours-S9			Concentration (µg/mL)		4-Hours+S9
		%RSG	RTG	MF§			%RSG	RTG	MF§
0		100	1.00	144.49	0		100	1.00	153.11
125	Ø	97			1.95	Ø	91
250	Ø	97			3.91	Ø	95
500		86	0.85	173.28	7.81		81	0.66	181.62
1000		66	0.73	132.37	15.63		85	0.76	172.61
1200		47	0.50	154.19	31.25		72	0.64	202.21
1400		35	0.35	177.48	62.5		54	0.44	369.38*
1600		21	0.16	178.11	93.75		33	0.23	772.44*
1800	X	3	0.03	214.12	125		19	0.12	944.69*
MF threshold for a positive response = 270.49					MF threshold for a positive response = 279.11
Positive control					Positive control
EMS 400		74	0.72	1546.46	CP 1.5		76	0.49	1269.64

Concentration (µg/mL)		24-Hours-S9
		%RSG	RTG	MF§
0		100	1.00	122.09
31.25		91	0.99	138.96
62.5		76	0.80	132.66
125		62	0.63	129.31
250		49	0.62	166.33
375		32	0.47	126.82
500		17	0.29	154.36
750	Ø	4
1000	Ø	1
MF threshold for a positive response = 248.09
Positive control
EMS 150		38	0.31	1993.24

$	=	Cellcounts(x105 cells/ml).Setup onpreviousdayto 2 x105cells/mlunlessotherwise statedin parenthesis.
%RSG	=	Relative Suspension Growth
RTG	=	Relative Total Growth
§ or #	=	Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis
A,B	=	Replicate cultures
CP	=	Cyclophosphamide
EMS   MF§	=   =	Ethylmethanesulphonate   5-TFT resistantmutants/106 viable cells2daysafterexposure
Ø	=	Not plated due to toxicity or surplus to requirements
X	=	Treatment excluded from test statistics due to toxicity
*	=	p<0.05
***	=	p<0.001

Conclusions:

The test item induced increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor, consequently it is considered to be mutagenic in the presence of metabolic activation in this assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 28 July 2015 and 15 October 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)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Molecular formula: C10H20O3 (main components)
Molecular weight: 188.26 (main components)
Water Solubility: Soluble in water
Lot No. 14F03
Purity: 91.8% (with no adjustment for purity because the test substance is a mixture)
Expiration date: 7 July 2016
Supplier: Study sponsor
Storage conditions: At room temperature in the dark

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell name: CHL/IU
Rationale for selection of the cells: The cell line was selected considering the cell growth rate, chromosomal stability in a serially subcultured line, ease of observation of chromosomal specimens, and sensitivity to the known mutagens.

Metabolic activation:

not specified

Test concentrations with justification for top dose:

The highest dose of the test substance was set at 5000 μg/mL, and lower doses were set in a twofold dilution series (8 doses in total: 5000, 2500, 1250, 625, 313, 156, 78.1, and 39.1 μg/mL).

Vehicle / solvent:

Dimethyl sulfoxide
Rationale for selection of the vehicle: In the preliminary investigation of preparation of the test substance conducted by the test facility, the test substance was insoluble (at 50 mg/mL), and not homogeneously suspended in physiological saline. While in DMSO and acetone, the test substance was dissolved at 500 mg/mL. No exothermic reaction, foaming, or discoloration was noted in any preparations. Accordingly, DMSO was selected as the vehicle (negative control substance) of the test substance.

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

Test series:
The preliminary test was performed in three test series: short-term treatment (− S9), short-term treatment (+ S9), and 24-hour continuous treatment.

Test groups:
The highest dose of the test substance was set at 5000 μg/mL, and lower doses were set in a twofold dilution series (8 doses in total: 5000, 2500, 1250, 625, 313, 156, 78.1, and 39.1 μg/mL). In addition, a negative control group was used for each test series. Two plates were used at each concentration, each of which was marked with an identification number.

Cell seeding:
Five milliliters of the cell suspension of a density 0.4 × 104 cells/mL was seeded in each 60-mm culture plate and the plate was incubated in a CO2 incubator (MCO-175; Panasonic Healthcare Corporation) set at 5.0% CO2 and 37.0°C.

Treatment with test preparations:
The test preparations were treated by the procedures described below. The negative control substance was treated by the same method.
a Short -term treatment (− S9)
On the third day after cell seeding, culture solution in the plate was removed and 3 mL of fresh medium was added to the plate, and subsequently 0.03 mL of the test preparation was added to the plate. The plate was incubated for 6 hrs. After 6-hrs incubation, the fluid in the plate was removed, and the cells were rinsed with PBS (−). Then, 5 mL of fresh medium was added to the plate, and the plate was incubated for an additional 18 hrs.

b Short -term treatment (+ S9 )
On the third day after cell seeding, the culture solution in the plate was removed and 2.5 mL of fresh medium and 0.5 mL of S9 mix (final concentration of S9: approximately 5 vol%) were added to the plate. Subsequently, 0.03 mL of the test preparation was added to the plate, and the plate was incubated for 6 hrs. After 6-hrs incubation, the fluid in the plate was removed, and the cells were rinsed with PBS (−). Then, 5 mL of fresh medium was added to the plate and incubated for an additional 18 hrs.

c 24 -hour continuous treatment
On the third day after cell seeding, the culture solution in the plate was removed and 5 mL of fresh medium was added to the plate. Subsequently, 0.05 mL of the test preparation was added to the plate, and the plate was incubated for 24 hrs.

Observation for precipitation of the test substance:
The culture solution in the plate was visually observed for precipitation of the test substance at the beginning and end of treatment with the test preparations.

Observation for the effects of the test preparations on the pH of the culture solution:
The culture solution in the plate was visually observed for color change at the beginning and end of treatment with the test preparations. As no color change was noted, treatment with the test preparations was considered to have no effects on the pH of the culture solution.

Measurement of the cell growth rate and calculation of the giving giving 50% inhibition of cell growth (IC 50 )
After the end of incubation, the fluid in the plate was removed and the cells were rinsed with PBS (−). The cells were then treated with 0.5 mL of 0.02% EDTA-0.25% trypsin, and 4.5 mL of fresh medium was added to the cells. The cell suspension was pipetted to suspend the cells, and the number of cells in each plate was counted with a hemocytometer. Then the percentage of the number of cells (cell growth rate) was calculated with the number in the negative control group defined as 100%. In addition, the average was calculated for each test group. In the preliminary test, cell growth was inhibited by 50% or more in all test series and the IC50 values were 1694.4 μg/mL following short-term treatment (− S9), and 1231.1 μg/mL following 24-hour continuous treatment. Following short-term treatment (+ S9), severe cytotoxicity was noted even at the lowest dose (39.1 μg/mL) and the cell growth rate was 0%. In addition, it was not possible to calculate IC50 values. Accordingly, for short-term treatment (+ S9), it was determined to conduct the preliminary test 2 (cell growth inhibition test 2) at lower doses.

Preliminary test 2 (cell growth inhibition test 2):
Test series:
The preliminary test 2 was performed in short-term treatment (+ S9).

Test groups:
Based on the results of the preliminary test, the highest dose of the test substance was set at 50 μg/mL, and lower doses were set in a twofold dilution series (8 doses in total: 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, and 0.391 μg/mL). In addition, a negative control group was used for each test series.
Two plates were used at each concentration, each of which was marked with an identification number.

Cell seeding:
According to the section "Cell seeding"

Treatment with test preparations:
According to the section "Short-term treatment (+S9)"

Observation for precipitation of the test substance:
According to the section "Observation for precipitation of the test substance"

Observation for the effects of test preparations on the pH of the culture solution:
According to the section "Observation for the effects of the test preparations on the pH of the culture solution"

Measurement of the cell growth rate and calculation of the concentration giving 50% inhibition of cell growth (IC 50 )
According to the section "Measurement of the cell growth rate and calculation of the concentration giving 50% inhibition of cell growth (IC50)"
following short-term treatment (+S9) was 19.9 μg/mL.

Main test (chromasomal aberration test)
Test series:
The main test was performed in three test series: short-term treatment (− S9), short-term treatment (+ S9), and 24-hour continuous treatment.

Test groups:
a Test substance
Based on the results of the preliminary test and the preliminary test 2, dose levels for the chromosomal aberration test were set as shown below, including the highest dose that causes cell growth inhibition by 50% or more in all test series.
Short-term treatment (−S9): 750, 1000, 1250, 1500, 1750, and 2000 μg/mL
Short-term treatment (+S9): 2.5, 5, 10, 15, 20, and 25 μg/mL
24-hour continuous treatment: 250, 500, 750, 1000, 1250, and 1500 μg/mL

b Control substance
A negative control group and positive control groups shown in the table below were set for each test series.
Test series Positive control substance Dose (μg/mL)
Short-term treatment (− S9) MMC 0.1
Short-term treatment (+ S9) BP 10
24-hour continuous treatment MMC 0.05

c Number of plates
Two plates were used for each group. Each plate was marked with an identification number.

Cell seeding:
According to the section "Cell seeding"

Treatment with Test Preparations:
a Short-term treatment (− S9 )
According to the section "Short-term treatment (− S9)"
b Short-term treatment (+ S9)
According to the section "Short-term treatment (+ S9)"
c 24 -hour continuous treatment
According to the section "4-hour continuous treatment"

Observation for precipitation of the test substance:
According to the section "Observation for precipitation of the test substance"

Observation for the effects of test preparations on the pH of the culture solution:
According to the section "Observation for the effects of the test preparations on the pH of the culture solution"

Measurement of the cell growth rate:
The cell growth rate was calculated by the method described in the section"Measurement of the cell growth rate and calculation of the concentration giving 50% inhibition of cell growth (IC50)" using the cell suspension obtained in the section "Observation for the effects of test preparations on the pH of the culture solution". However, in the main test, IC50 value was not calculated, and the cell growth rate in the positive control group was not measured.

Preparation of chromosome specimens:
Two hrs before the end of incubation, colcemid (Lot No. 1621913, GIBCO) was added to each plate at a final concentration of 0.2 μg/mL. At the end of incubation, the fluid in each plate was collected into respective centrifuge tube. The plate was treated with 0.02% EDTA (0.5M EDTA: Lot No. 1045847, GIBCO)-0.25% trypsin (2.5% trypsin: Lot No. 1620318, GIBCO) to detach the cells. The resultant cell suspension was collected into the above centrifuge tube and centrifuged at 1,000 rpm for 5 min. After the supernatant was removed, 0.075 mol/L potassium chloride (Lot No. 310U1825; Kanto Chemical Co., Inc.) was added to the cells, and the mixture was allowed to stand for 15 min at 37ºC with gentle pipetting while the cells swelled. After iced Carnoy’s fixative (mixture of methanol and acetic acid in a ratio of 3:1. Methanol: Lot No. 702B1158; Kanto Chemical Co., Inc. Acetic acid: Lot No. ECN4685; Wako Pure Chemical Industries, Ltd.) was added to the mixture to fix the cells, the mixture was centrifuged at 1,000 rpm for 5 min, from which the supernatant was removed, and then another Carnoy’s fixative was added to fix the cells. After these procedures were repeated three times, the cell suspension was dropped onto a glass slide, which was then allowed to air-dry. Two chromosome specimens were prepared from each plate. Each slide was stained with 3% Giemsa solution (Giemsa solution: Lot No. KR896; Wako Pure Chemical Industries, Ltd. Instant phosphate buffer [pH 6.8]: Lot No. E406; LSI Medience Corporation) for 20 min. After washing with water and air-dried, the slide was sealed with a mounting agent (Malinol: Lot No. 1300901; Muto Pure Chemicals Co., Ltd.).

Selection of doses for observation:
In the section "Measurement of cell growth rate", dose levels were set as follows including the highest dose that reduces the cell growth rate to less than 50% of that in the negative control group: 1500, 1750, and 2000 μg/mL for short-term treatment (−S9); 10, 15, and 20 μg/mL for short-term treatment (+S9); and 500, 700, and 1000 μg/mL for 24-hour continuous treatment.

Confirmation of selected speecimens:
Confirmation was conducted in the specimens of the doses selected in the section "Selection of doses for observation" and those in the control groups. It was confirmed that at least approximately 50 metaphase cells were obtained from each specimen prepared.

Coding of chromosome specimens:
Among the specimens of the doses selected for observation, one specimen per plate was selected and coded.

Observation of chromosome specimens:
Using a microscope (BX51TF, Olympus Corporation) with a total magnification of 1000×, 100 metaphase cells per plate (200 metaphase cells per dose) were selected and observed. The specimens were assessed for chromosomal aberrations according to the classification systems described below. Cells with 25 ± 2 chromosomes were observed for structural aberration.
a Structural aberration
• Chromatid break (ctb) Distinctive discontinuity (break) in the chromatid; either a discontinuity wider than the chromatid or a break off the major axis of the chromatid
• Chromatid exchange (cte) At least two breaks in the chromatid exchanged with each other (combined)
• Chromosome break (csb) Break at the same position in a pair of chromatids and judged according to the criteria for chromatid break
• Chromosome exchange (cse) Exchange at the same position in a pair of chromatids toward the same direction
• Others Other structural aberrations include fragmentation (defined as breaks or gaps affecting almost all metaphase cells with no exchange-type aberration)

b Gap
Unstained area (no stain) in chromatids or chromosomes with a width less than that of the chromatid.

c Numerical aberration
• Polyploid (poly) Cells with 36 or more chromosomes (triploid, tetraploid, etc.); i.e. having three, four or more times as many chromosomes as the normal number of chromosomes (25 ± 2), respectively.
• Others Other numerical aberrations include endoreduplication (defined as double chromosomes not separated but arranged in parallel). These aberrations differ from polyploid.

Calculation of observation results:
The numbers of cells with structural aberrations and those with numerical aberrations meeting each definition described below and their total sum were calculated by plate and by dose level. In addition, the incidence (%) of the cells with aberrations was calculated for each dose level, as the percentage of the number of cells with aberrations relative to the total number of cells observed (number of metaphase cells). A cell with more than one structural aberrations was counted as one, and gap was not included in the structural aberrations.

a Structural aberrations
• ctb: number of cells with chromatid breaks
• cte: number of cells with chromatid exchanges
• csb: number of cells with chromosome breaks
• cse: number of cells with chromosome exchanges
• others: number of cells with other structural aberrations
• total: number of cells with any structural aberrations

b Gap
• gap: number of cells with gaps

c Numerical aberration
• others: number of cells with other numerical aberrations
• total: number of cells with any numerical aberrations

Validity criteria for the test:
The validity of the test was verified when the two criteria shown below were met. The present test results of all test series met both of them.
a Both the incidences of cells with structural and numerical aberrations in the negative control group are < 5% in all test series.
b The incidences of structural aberrations of chromosomes in the positive control group are ≥ 10% in all test series.

Evaluation of test results:
The results were determined to be negative when both the incidences of cells with structural and numerical aberrations were < 5%. When either or both of them were ≥ 5% but < 10%, a confirmatory test was conducted, and the results were determined to be equivocal when good reproducibility was obtained. The results were determined to be positive when either or both incidences were ≥ 10% and good reproducibility or dose-dependent increase in the incidences was observed.

Evaluation criteria:

Evaluation of test results:
The results were determined to be negative when both the incidences of cells with structural and numerical aberrations were < 5%. When either or both of them were ≥ 5% but < 10%, a confirmatory test was conducted, and the results were determined to be equivocal when good reproducibility was obtained. The results were determined to be positive when either or both incidences were ≥ 10% and good reproducibility or dose-dependent increase in the incidences was observed.

Statistics:

Statistical analysis was not conducted.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary test:
Precipitation of the test substance and effects on the pH of the culture solution were not observed at the start or end of treatment at any doses in any test series.

Cell growth was inhibited by 50% or more in all test series and the IC50 values were 1694.4 μg/mL for short-term treatment (− S9), and 1231.1 μg/mL for 24-hour continuous treatment. For short-term treatment (+ S9), severe cytotoxicity was noted even at the lowest dose (39.1 μg/mL) and the cell growth rate was 0%, and it was not possible to calculate IC50 values. Accordingly, for short-term treatment (+ S9), it was determined to conduct the preliminary test 2 (cell growth inhibition test 2) at lower doses.

Preliminary test 2:
Precipitation of the test substance and effects on pH of the culture solution were not observed at the start and end of treatment at any doses.

Cell growth was inhibited by 50% or more and the IC50 value was 19.9 µg/mL.

Main test:
Precipitation of the test substance and effects on pH of the culture solution were not observed at the start and end of treatment at any doses.

The cell growth rates decreased to 50% or less at 2000 µg/mL of short-term treatment (-S9), 20 µg/mL and higher doses of short-term treatment (+S9), and at 1000 µg/mL and higher doses of 24-hour continuous treatment.

Based of the results of cell growth rates, 1500, 1750, and 2000 µg/mL for short-term treatment (-S9), 10, 15, and 20 µg/mL for short-term treatment (+S9), and 500, 750, and 1000 µg/mL for 24-hour continuous treatment were selected, and the selected speciments and control specimens were used for confirmation of selected speciments. As the results, at least 50 metaphase cells were obtained at all doses in all test series; therefore, observation of speciments was subsequently conducted.

As the results of the observation of specimens, the incidences of structural and numerical aberrations of chromosomes were less than 5% at all doses of short-term treatment (− S9), short-term treatment (+ S9), and 24-hour continuous treatment.
In the positive control group, the incidences of structural aberrations of chromosomes were 40.5% following short-term treatment (− S9), 46.0% following short-term treatment (+ S9), and 50.5% following 24-hour continuous treatment.

Conclusions:

The clastogenicity of PX-1E-PA-B was investigated in vitro using Chinese hamster lung cells (CHL/IU).

As the results of the main test, the incidences of structural and numerical aberrations of chromosomes were less than 5% at all doses in all test series, and the results were determined to be negative. The results of precipitation of the test substance and the effects on the pH of the culture solution were similar between the preliminary test or preliminary test 2 and the main test, which indicated good reproducibility.

In the positive control group, the incidences of structural aberrations of chromosomes were clearly positive in all test series, demonstrating that the cell line used in this study had appropriate sensitivity.
On the basis of the above results, PX-1E-PA-B was considered non-clastogenic to cultured mammalian cells under the present test conditions.

Executive summary:

The potential of PX-1E-PA-B to induce clastogenicity was investigated in vitro using Chinese hamster lung cells (CHL/IU). The test was conducted in three test series: short-term treatment without S9 (− S9), short-term treatment with S9 (+ S9), and 24-hour continuous treatment.

In the preliminary test (cell-growth inhibition test, doses of the test substance: 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL), precipitation of the test substance and effects on the pH of the culture solution were not detected at any doses in any test series.

Cell growth was inhibited by 50% or more in all test series and the concentrations giving 50% inhibition of cell growth (IC₅₀) were 1694.4 μg/mL following short-term treatment (-S9) and 1231.1 μg/mL following 24 -hour continuous treatment. Following short-term treatment (+S9), severe cytotoxicity was noted even at the lowest dose (39.1 μg/mL) and the cell growth rate was 0% and it was not possible to calculate IC₅₀ values. Accordingly, for short-term treatment (+S9), the preliminary test 2 (cell growth inhibition test 2: doses of the test substance, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5, 25 and 50 μg/mL), the IC50 value was 19.9 μg/mL.

Based on the results of the preliminary test and the preliminary test 2, doses for the main test (chromosomal aberration test) were set at 750, 1000, 1250, 1500, 1750, and 2000 μg/mL for short-term treatment (− S9), 2.5, 5, 10, 15, 20, and 25 μg/mL for short-term treatment (+S9), and 250, 500, 750, 1000, 1250, and 1500 μg/mL for 24-hour continuous treatment.

Specimens were observed at the doses selected based on the measurements of cell growth rates: 1500, 1750, and 2000 μg/mL for short-term treatment (− S9), 10, 15, and 20 μg/mL for short-term treatment (+ S9), and 500, 750, and 1000 μg/mL for 24-hour continuous treatment. As the results, the incidences of structural and numerical aberrations of chromosomes were less than 5% at all doses in all test series, and these results were determined to be negative. The results of the effects on cell growth in all test series, precipitation of the test substance, and the effects on the pH of the culture solution were consistent with those in the preliminary test, which indicated good reproducibility.

In the positive control group, the incidences of structural aberrations of chromosomes were clearly positive in all test series, demonstrating that the cell line used in this study had appropriate sensitivity.

On the basis of the above results, PX-1E-PA-B was considered non-clastogenic to cultured mammalian cells under the present test conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

According to the CLP Regulation (Table 3.5.1) substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.