O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is negative for causing gene mutation in bacteria cells. A substance of similar structure caused no gene mutations in mammalian cells in vitro. The positive clastogenic finding in vitro could not be confirmed in vivo. The substance is therefore considered to be not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 19, 1989 - May 29, 1989

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:

1983

Deviations:

yes

Remarks:

(no statistics)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Target gene:

Salmonella typhimurium: His (-/-)
E. Coli: Tryp (-/-)

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Concentrations in the main test (with metabolic activation): 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 mL
Concentrations in the main test (without metabolic activation): 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 mL
Concentration range in the preliminary toxicity test: 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 µg/0.1 mL

Vehicle / solvent:

Acetone (test article); DMSO (positive controls)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with and without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION (in agar, plate incorporation):

DURATION
- Incubation period: about 48 h at 37 ± 1.5°C in darkness

NUMBER OF REPLICATIONS:
- 3 Petri dishes per strain and per group per experiment

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration
level for one or more of the following strains: TA 98, TA 1535, TA 1537, TA 1538 and E. coli WP2uvrA,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100. Generally a concentration-related effect should be demonstrable

Statistics:

not performed

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:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

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:

E. coli WP2 uvr A

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: at the concentrations of 500 µg/0.1 mL and above the test substance precipitated in soft agar

RANGE-FINDING/SCREENING STUDIES:
Nine concentrations ranging from 20 to 5000 ug/0.1 mL were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained, the highest concentration suitable for the mutagenicity test was found to be 5000 ug/0.1 mL.

Experiment I

	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2uvrA
Dose (µg/0.1 ml)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	13	28	117	153	8	13	4	17	7	19	22	29
5	17	32	118	149	16	13	7	14	10	16	24	28
10	14	29	114	146	9	3	3	13	9	17	18	26
50	24	36	128	136	14	16	6	18	5	21	19	17
100	16	23	114	141	16	13	6	14	5	16	17	19
500	19	29	124	153	14	11	9	16	5	16	18	24
1000	15	32	126	137	15	13	7	15	8	18	19	26
5000	15	27	120	136	13	12	4	13	9	18	23	27
positive controls:
solvent control	14	23	100	139	8	14	7	14	8	19	17	21
concentration A	139	385	481	428	199	163	21	127	200	140	362	609
concentration B	222	376	754	542	313	94	149	124	358	123	674	718

Experiment II

	TA 98		TA 100		TA 1535		TA 1537		TA 1538		WP2uvrA
Dose (µg/0.1 ml)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	17	28	135	185	14	9	6	10	7	20	24	34
5	15	26	118	186	15	10	4	18	5	23	21	33
10	11	28	120	188	10	9	8	17	3	15	27	27
50	22	27	105	156	9	8	6	11	11	14	25	22
100	14	21	108	145	19	8	5	17	5	15	19	29
500	23	29	124	133	9	11	8	14	11	16	25	24
1000	14	29	113	153	14	9	3	14	5	19	23	28
5000	20	24	123	133	17	12	5	15	9	19	24	35
positive controls:
solvent control	13	25	104	135	11	12	3	16	6	22	18	24
concentration A	137	455	318	289	219	144	30	191	189	179	392	498
concentration B	268	429	388	461	410	160	171	140	265	157	562	441

The slight increase in the number of back-mutant colonies observed with strain TA 1535 in the first experiment without microsomal activation at the concentrations of 5 and 100 µg/0.1 ml could not be confirmed in the second experiment and is therefore attributed to spontaneously occurring back-mutants.

Conclusions:

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The test substance was tested in the Salmonella typhimurium reverse mutation assay with strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100 and TA98) and in Escherichia coli WP2UvrA in two independent experiments. These tests were following GLP guidelines and were based on the OECD testing guideline 471. In a dose range finding test, the test article was tested up to concentrations of 5000 µg/0.1 ml in the absence and presence of S9-mix. At this dose level no toxicity was observed. In the mutation assays, the test substance was tested up to concentrations of 5000 µg/0.1 ml in the absence and presence of S9-mix. The test article precipitated at concentrations of 500 µg/0.1 ml and above. No decrease in the number of revertants was observed. No dose-related increase in the number of revertant both in the absence and presence of S9-metabolic activation was observed. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell chromosome aberration assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Doubling time: about 15 h at 37°C
- Source: Dainippon Pharmaceutical Co.,Ltd. on January 14,1992
- Medium: Eagle's MEM medium (Nissui pharmaceutical Co.,Ltd. Lot No. 3161304 for chromosome aberration test, 323306 for additional test) supplemented with 10% calf serum (heat inactivate at 56°C; for 30 min, Irvine Scientific Lot No. 311702)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

S9:(Lot No. RAA-298; Kikkoman Co.), prepared from the livers of male 7-week old Sprague-Dawley rats, treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Positive controls:
- MMC: 0.05 µg/mL in physiological saline
- BP: 20 µg/mL in DMSO

Test article:
- 156 µg/mL, 313 µg/mL, 625 µg/mL, 1250 µg/mL, 2500 µg/mL, 5000 µg/mL

Vehicle / solvent:

acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(acetone)

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: continuous treatment (without S9) for 24 h and 48 h, puls treatment for 6 h with/without metabolic activation
- Expression time (cells in growth medium): 18 h (pulse treatment), 24 h and 48 h (continous treatment)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (for 6 h pulse treatment and 24 h continuous treatment, respectively) and 48 h (48 h continuous treatment)

SPINDLE INHIBITOR (cytogenetic assays): colcemid, added 2 h before harvest
STAIN (for cytogenetic assays): giemsa

NUMBER OF REPLICATIONS: 2 experiments à 2 plates per concentration

NUMBER OF CELLS EVALUATED: 200 per dose per experiment

SCORING OF SLIDE PREPARATION:
Metaphase cells were examined under a microscope for structural and numerical abnormalities. In the case of numerical abnormalities, only polyploidy cells (more than 37 chromosomes ) were scored for these abnormalities. Structural abnormalities of chromosome were classified as gap (included chromosome type and chromatid type), chromatid break (ctb), chromosome break (csb), chromatid exchange (ete), chromosome exchange (cse), and other (fragmentation and so on). Observations of slides were carried out by the plural number of observers. The cell having at least one aberration was recorded as an aberrant cell.

Evaluation criteria:

Numbers of cells with chromosome aberration and polyploidy cells in the groups treated by the test substance are compared with those of the control groups for each dose. In principle, the test substance is judged to be clastogenic or aneugenic, if significant increase in aberration frequency, dose response and reproducibility of the result could be confirmed. The final judgment was decided as follows;
Negative (-): less than 5%
Inconclusive (+/-): from 5% to less than 10%
Positive (+): from 10% to less than 20%
Positive (++): from 20% to less than 50%
Positive (+++): 50% or more
The final evaluation was made on the basis of the total incidence of aberrant cells in which also cells with only gap were included. Gap is a clear existing discontinuity, but the distal part of the chromatid or chromosome showed no dislocation.

Statistics:

no data

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: due to the insolubility of the test article in aqueous media (water), emulsification occurs upon addition of its acetone solution to the cell culture during test substance treatment. This is discussed as possible reason as to why a definite dose reponse was not observed in the study.

- Significant increase in chromosome aberration for 24 h and 48 h continuous treatment and for 6 h pulse treatment without S9 Mix

- The major types of structural aberrations were chromatid break and chromatid exchange.

Conclusions:

The test substance is considered to be clastogenic under the experimental condition of this analysis.

Executive summary:

This study was designed to assess the mutagenicity of the test item in the metaphase chromosome analysis ofthe Chinese hamster lung cell line (CHL/IU). The test substance increased the frequency of structural chromosome aberrations in tlie continuous and pulse treatment. The increase in frequency of numerical chromosome aberrations was not observed with both continuous and pulse treatments. All negative (solvent) controls gave frequencies of aberrations within the range of negative values expected for the CHL/IU. The positive controls gave highly significant increases in the frequency of aberrations indicating the satisfactory performance of the test. The test substance is considered to be clastogenic under the experimental condition of this analysis.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-2011

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay (HPRT assay)

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/b-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

19.5, 39.1, 78.1, 156.3, 1250.0, 2500.0, 5000.0 mg/L (experiment 1; 4h, without S9)
25, 50, 100, 625, 1250, 2500, 5000 mg/L (experiment 2; 24h, without S9)
19.5, 39.1, 78.1, 156.3, 312, 1250, 2500 mg/L (experiment 1; 4h, with S9)
25, 50, 100, 200, 400, 600, 800 mg/L (experiment 2; 4h, with S9)

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9:300 μg/mL ethyl methanesulfonate, with S9: 20 μg/mL methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24h
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 5 - 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: Population doubling time 12 - 16h

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 15.95 mutants per 10exp6 clonable cells
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data given in Appendix to study report)
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dos-response relationship.

Statistics:

Due to the negative findings, a statistical evaluation was not carried out.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in the 1st and 2nd Experiment in the presence of S9 mix at least in the highest applied concentrations. In both experimental parts in the absence of metabolic activation no distinct cytotoxicity was obtained.

In detail, with S9 mix, there was a decrease in the number of colonies from 625 μg/mL (CE1 relative: 8.3%) onward in the 1st Experiment and from 400 μg/mL (CE1 relative: 17.6%) onward in the 2nd Experiment. The cell densities were distinctly reduced.

Osmolarity and pH values were not influenced by test substance treatment.
In this study, under all test conditons, test substance precipitation in culture medium at the end of treatment was observed at about 50 μg/mL and above.

Summary of Results

						Cytotoxicity***
Exp	Exposure period [h]	Test groups [μg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	CE1 [%]	CE2 [%]
1	4	Vehicle control^1	-	-	2.03	100.00	100.00
		19.5	-	-	3.62	101.20	101.80
		39.1	-	-	2.74	108.40	106.60
		78.1	-	+	0.34	107.10	116.50
		156.3	-	+	0.00	84.80	95.30
		1250.0	-	+	0.00	62.30	115.90
		2500.0	-	+	0.00	45.30	105.40
		5000.0	-	+	3.58	50.80	115.80
		Positive control^2	-	-	97.16	83.90	127.00
2	24	Vehicle control^1	-	-	0.00	100.00	100.00
		25.0	-	-	1.40	101.60	101.90
		50.0	-	+	7.92	96.70	94.20
		100.0	-	+	1.84	88.40	102.90
		625.0	-	+	0.90	68.60	105.40
		1250.0	-	+	3.86	58.60	113.30
		2500.0	-	+	0.00	40.30	106.60
		5000.0	-	+	11.87	60.30	114.70
		Positive control^2	-	-	740.49	95.10	50.80
1	4	Vehicle control^1	+	-	4.49	100.00	100.00
		19.5	+	-	5.03	95.30	90.70
		39.1	+	-	3.62	84.80	91.20
		78.1	+	+	0.82	101.70	90.00
		156.3	+	+	2.21	93.40	89.20
		312.5	+	+	3.55	35.20	93.10
		625.0	+	+	2.30	8.30	106.20
		1250.0	+	+	0.70	3.60	108.90
		Positive control^3	+	-	61.64	80.10	87.20
2	4	Vehicle control^1	+	-	2.95	100.00	100.00
		25.0	+	-	0.00	100.90	94.30
		50.0	+	+	0.84	100.70	99.00
		100.0	+	+	4.18	96.90	93.80
		200.0	+	+	1.17	64.10	101.60
		400.0	+	+	n.c.^1	17.6	n.c.^1
		600.0	+	+	n.c.^1	8.5	n.c.^1
		800.0	+	+	n.c.^1	6.6	n.c.^1
		Positive control^3	+	-	51.66	86.70	96.60

* Precipitation in culture medium at the end of exposure period

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

*** Cloning efficiency related to the respective vehicle control

n.c.¹Culture was not continued due to strong cytotoxicity

¹DMSO 1% (v/v)

²EMS 300 μg/mL

³MCA 20 μg/mL

Conclusions:

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

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated in this study:

1st Experiment

without S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 1250.0; 2500.0; 5000.0 μg/mL

with S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 312.5; 625.0; 1250.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period): 0; 25; 50; 100; 625; 1250; 2500; 5000 μg/mL

with S9 mix (4-hour exposure period): 0; 25; 50; 100; 200; 400; 600; 800 μg/mL

After an attachment period of 20-24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6-8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. Cytotoxicity indicated by reduced survival (CE1) of below 20% of control was observed in both experiments in the presence of metabolic activation only. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance is considered not to be clastogenic based on the negative result in the in vivo MNT Assay in hamster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene Mutation

In bacteria cells

In a reverse gene mutation assay in bacteria (OECD 471), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium and E. coli WP2uvrA were exposed to the test article (96.2% pure) in acetone at concentrations of 5, 10, 50, 100, 500, 1000 and 5000 (limit concentration) µg/0.1 mL in the presence and absence of mammalian metabolic activation with a plate incorporation method. No dose related increase of revertants was seen. The positive controls induced the appropriate response in the corresponding strains. There was no evidence of induced mutant colonies over background. No cytotoxicity was observed up to the limit concentration. Substance precipitation that occurred from the concentration of 500 µg/0.1 mL and above did not affect scoring integrity. The study is classified as acceptable as it satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity data (bacterial reverse gene mutation) and GLP Guideline.

In mammalian cells

The analogue substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (OECD 476, GLP). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated in this study:

1st Experiment:

Without S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 1 250.0; 2 500.0; 5 000.0 μg/mL

With S9 mix (4-hour exposure period): 0; 19.5; 39.1; 78.1; 156.3; 312.5; 625.0; 1 250.0 μg/mL

2nd Experiment:

Without S9 mix (24-hour exposure period): 0; 25; 50; 100; 625; 1 250; 2 500; 5 000 μg/mL

With S9 mix (4-hour exposure period): 0; 25; 50; 100; 200; 400; 600; 800 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. Cytotoxicity indicated by reduced survival (CE1) of below 20% of control was observed in both experiments in the presence of metabolic activation only. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other.

The analogue substance has a lower degree of alkylation and is slightly better soluble. Neither the analogue substance nor the target substance carry a known structural alert for genotoxicity. For detailed information on read-across, it is referred to the attachment in IUCLID chapter 13.

 

Chromosomal Mutation

In vitro

In a mammalian cell cytogenetic assay, CHO/IU cell cultures were exposed to the test article (96.2% pure) in acetone at concentrations of 156, 313, 625, 1250, 2500 and 5000 (limit concentration) µg/mL with and without metabolic activation. Positive controls induced the appropriate response. There was evidence of structural chromosome aberration induced over background, but without dose-dependency. The test item is a liquid that was diluted in acetone. Upon addition to the cell culture medium, the test item formed droplets in the culture medium. The major types of aberrations were chromatid breaks and chromatid exchanges. There was no evidence of polyploidy induced over background. This study is classified as acceptable as it satisfies the requirement for Test Guideline in vitro mammalian cytogenics (Chromosome Aberration Test), OECD 473 for in vitro cytogenetic mutagenicity data and GLP Guideline. The test article is assumed to be clastogenic in this study.

 

In vivo

In a Chinese hamster bone marrow micronucleus assay, 8 animals/sex/dose/sacrifice time were given a single application of the test substance (96.2% pure) in arachis oil per gavage at a single dose 5000 mg/kg bw. The test substance was tested at an adequate dose based on a preliminary test. Bone marrow cells were harvested at 16, 24 and 48 hours post-treatment. There was no mortality nor were there any signs of systemic toxicity during the study. There was no cytotoxicity as evidenced in the ratio of PCR/NCR in the treated animals compared to controls. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any sampling time. This study is classified as acceptable as it satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data and GLP Guideline.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008.