3-(methylthio)propionaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (according to OECD 471, Ames test): negative in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA with and without metabolic activation

Gene mutation in mammalian cells in vitro (OECD 476, Mouse lymphoma assay): positive in mouse lymphoma L5178Y cells with and without metabolic activation, genotoxic effect attributed to impurity acrolein

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

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 July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophan (trp) reversion system measures his-→ his+ and trp-→ trp+ reversions, respectively.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/ß-naphtoflavone.

Test concentrations with justification for top dose:

In a pre-experiment a concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since toxic effects were observed in experiment I, eight concentrations were tested in experiment II. 5000 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations were tested in experiment II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.

Experiment 1 (plate incorporation test) and Experiment 2 (preincubation test): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate, with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent has been chosen according to its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene dissolved in DMSO; for TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA; +S9 // 4-nitro-o-phenylene-diamine, 4-NOPD dissolved in DMSO; for TA 1537, TA 98; -S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 min
- Exposure duration/duration of treatment: 48 hours at 37 C ± 1.5° C in the dark

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Number of revertant colonies

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration. An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviation were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains at higher concentrations with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Ames test:
- Signs of toxicity , Individual plate counts , Mean number of revertant colonies per plate and standard deviation : Refer to Table no. 1 and 2 under "Any other information on results incl. tables".

HISTORICAL CONTROL DATA
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Table 1:

Summary of Experiment I
Metabolic Activation	Test Group	Dose level per plate	Revertant Colony Count (Mean)
			TA1535	TA1537	TA98	TA100	WP2uvRA
without activation	DMSO		20	10	24	138	39
	Untreated		18	13	28	146	43
	MMP	3 µg	16	8	25	127	40
		10 µg	19	11	20	139	40
		33 µg	17	10	30	129	41
		100 µg	19	9	28	121	41
		333 µg	21	9	33	121	39
		1000 µg	23	12	21	138	54
		2500 µg	8	6	10	1	41
		5000 µg	1P	1P	1P	0P	2P
	NaN3	10 µg	1612			2374
	4-NOPD	10 µg			378
	4-NOPD	50 µg		79
	MMS	2.0 µL					1085
with activation	DMSO		15	11	37	127	49
	Untreated		19	16	34	128	56
	MMP	3 µg	16	15	36	127	47
		10 µg	19	10	39	126	47
		33 µg	15	11	37	134	48
		100 µg	19	10	41	126	55
		333 µg	19	13	38	107	52
		1000 µg	20	13	29	99	48
		2500 µg	20	8	23	96	52
		5000 µg	2P	3P	1P	1P	34P
	2-AA	2.5 µg	362	369	3793	4827
	2-AA	10.0 µg					222

P: Precipitate, NaN3: sodium azide, 2 -AA: 2- aminoanthracene, 4 -NOPD: 4 -nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

Table 2:

Summary of Experiment II
Metabolic Activation	Test Group	Dose level per plate	Revertant Colony Count (Mean)
			TA1535	TA1537	TA98	TA100	WP2uvRA
without activation	DMSO		10	8	24	93	41
	Untreated		14	16	21	105	41
	MMP	3 µg	10	7	18	93	38
		10 µg	8	9	23	100	39
		33 µg	12	10	27	98	35
		100 µg	13	10	27	105	42
		333 µg	14	8	30	120	44
		1000 µg	13	4	16	116	51
		2500 µg	3	1	7	2	6
		5000 µg	0P	0P	5	0P	0P
	NaN3	10 µg	1302			2050
	4-NOPD	10 µg			538
	4-NOPD	50 µg		119
	MMS	2.0 µL					736
with activation	DMSO		13	13	30	94	45
	Untreated		12	15	40	90	48
	MMP	3 µg	17	17	32	95	49
		10 µg	14	11	30	101	44
		33 µg	13	13	27	94	54
		100 µg	15	12	32	103	53
		333 µg	12	12	29	93	41
		1000 µg	15	14	35	90	43
		2500 µg	9	11	3	6	46
		5000 µg	0P	0P	0P	0P	0P
	2-AA	2.5 µg	305	349	3950	2831
	2-AA	10.0 µg					225

P: Precipitate, NaN3: sodium azide, 2 -AA: 2- aminoanthracene, 4 -NOPD: 4 -nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

Conclusions:

Interpretation of results: negative with and without metabolic activation, no mutagenic potential was detected in the bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

yes

Remarks:

not tested in TA102 or E. coli WP2 uvrA; 2-aminoanthracene as sole positive control for all strains, no historical control data, no justification for lack of a confirmatory experiment, not tested up to cytotoxic/precipitation limit concentrations

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The S. typhimurium histidine (his) reversion system measures his- --> his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

Species / strain / cell type:

other: S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arochlor 1254.

Test concentrations with justification for top dose:

0.01, 0.0316, 0.1 and 0.316 µg/plate for all strains with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-anthramine (2-AM), +S9, 2.5 µg/plate for all strains

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: triplicates in a single experiment

METHOD OF TREATMENT: pre-incubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h at 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
- Method: Number of revertant colonies

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.1 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A dose-rage finding pre-incubation test was performed with tester strain TA100 with concentrations up to 10 µg/plate in the presence and absence of metabolic activation.
In a pre-incubation assay, four log dilutions of the test substance, to a maximum concentration of 10 µl/plate, were tested. The pre-test was performed with strain TA100 in a concentration range-finding test, with and without metabolic activation. A concentration-related increase in toxicity was observed at 0.1 µg/plate and above.

STUDY RESULTS
- No concentration-related increase in mean histidine revertant colonies/plate was observed for any strain in the absence or presence of metabolic activation.
- Concentration-response relationship: There was no dose-response relationship observed for any test concentration with and without metabolic activation.
- Signs of toxicity: Cytotoxicity was evident by a significant reduction in colonies at 0.316 µl/plate for strain TA1537 in the absence of activation, for all concentrations of test substance strain TA1537 in the presence of activation and for all concentrations of the test substance in strain TA100 in the absence of metabolic activation.
- Concurrent vehicle negative and positive control data: All positive and negative controls gave the expected results and were consistent with those reported in the literature. Historical control data were not reported.
- Mean number of revertant colonies per plate and standard deviation: Refer to Table no. 1 under "Any other information on results incl. tables".

Table 1: Experimental results

Pre-incubation test
Strain	TA 98		TA 100		TA 1535		TA 1537		TA 1538
Metabolic activation	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9	without S9	with S9
Vehicle control
DMSO mean	25	41	97	103	14	28	13	29	21	32
± SD	± 4.6	± 6.6	± 12.8	± 7.2	± 3.1	± 12.6	± 4.5	± 6.4	± 7.8	± 5.1
Test item [µg/plate]
0.01 mean	16	31	76	99	19	24	14	15	15	26
± SD	± 4.2	± 4.4	± 5.5	± 5.9	± 2.5	± 2.9	± 3.8	± 4.2	± 5.5	± 7.0
0.0316 mean	19	31	65	97	16	20	13	19	16	28
± SD	± 4.7	± 4.0	± 6.7	± 17.8	± 6.0	± 7.0	± 3.0	± 6.0	± 3.8	± 4.0
0.1 mean	15	31	58	100	22	18	11	17	17	24
± SD	± 4.5	± 4.0	± 6.4	± 5.8	± 5.3	± 5.3	± 3.2	± 0.6	± 1.2	± 5.9
0.316 mean	29	34	65	109	22	20	7	13	13	24
± SD	± 2.3	± 4.0	± 15.0	± 7.0	± 1.2	± 0.6	± 2.6	± 3.8	± 3.5	± 1.5
Positive control
mean	314	1012	1160	2413	1248	479	1256	435	507	1785
± SD	± 10	± 116	± 194	± 352	± 173	± 58	± 89	± 21	± 104	± 269

Conclusions:

Interpretation of results: negative
The test substance did not show mutagenic activity in Salmonella typhimurium TA1535, TA1537, TA1538, TA98 or TA100 with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

yes

Remarks:

limited information on experimental details and raw data of individual replicates

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

mouse lymphoma thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y mouse lymphoma cells, clone 3.7.2c

For cell lines:
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The cells were cultivated in Fisher's medium supplemented with 31 µg penicillin/mL, 50 µg streptomycin sulfate/mL, 0.1% pluronic F68, 0.22 mg sodium pyruvate/mL, 25 mM HEPES buffer. In the absence of metabolic activation 10% heat-inactivated horse serum were added, in the absence of metabolic activation 5% heat-inactivated horse serum were added.

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Arochlor 1254.

Test concentrations with justification for top dose:

Experiment 1:
Without S9 mix: 0.0001, 0.01, 0.0178, 0.0316 and 0.0562 µL/mL (4 h)
With S9 mix: 0.0562, 0.1, 0.178, 0.316 and 0.562 µL/mL (4 h)

Experiment 2:
Without S9 mix: 0.001, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035 and 0.04 µL/mL (4 h)
With S9 mix: 0.01, 0.05, 0.10, 0.15, 0.20 and 0.25 µL/mL (4 h)

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: hycanthone (-S9), 7.5 and 10.0 µg/mL

Details on test system and experimental conditions:

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h with and without S9 mix

FOR GENE MUTATION:
- Expression time: 48 h
- Selection time: 14 days
- Fixation time: 16 days
- Selection agent: 1 µg/mL trifluorothymidine (TFT)
- Method to enumerate numbers of viable and mutants cells: relative total growth

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative cloning efficiency, relative total growth

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
- Number and size distribution of small and large colonies and total number of colonies
- Mutant frequency

Evaluation criteria:

Colonies in the mutant count and cloning efficiency dishes were counted, and the colonies in the solvent and positive control mutant count plates were sized by hand using a dissecting microscope to enumerate mutant colonies.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.02 µL/mL, 0.15 µL/mL

Vehicle controls validity:

valid

Remarks:

cloning efficiency was below acceptability criteria in the first test; vehicle controls were valid in the second test

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: In the main mutagenicity study no precipitation was observed, neither in the presence nor in the absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the range-finding assay, both in the presence and absence of metabolic activation, there were concentration-related decreases in cell survival. No cells were present in cultures exposed to >0.1 µL/mL in the absence of metabolic activation or in cultures exposed to >1.0 µL/mL with metabolic activation. Slight precipitate was seen without activation at >0.316 µL/mL and with metabolic activation at >0.1 pl/ml. Based on these findings, the first mutagenesis assay was conducted using a concentration range of 0.0001-0.0562 µL/mL without metabolic activating and 0.0562-0.562 µL/mLl with metabolic activation.

STUDY RESULTS:
In the first mouse lymphoma assay, there was a concentration-dependent increase in mutant frequencies, as well as a concentration-dependent increase in induced mutant frequencies (please refer to Table 1 under “Any other information on results incl. tables”). However, no concentration was tested that yielded survival in the 10-20% range. In addition, the average cloning efficiencies of the solvent controls were 62.7% without S9 mix and 55.4% with S9 mix, which were below the acceptability criteria of 70%. Mutant frequencies obtained with the solvent control were high, especially in the presence of metabolic activation, suggesting the preparation may have been mutagenic. For this reason, the mutagenicity assay was repeated.
In the second assay, average cloning efficiencies of solvent controls and spontaneous mutant frequencies were within the expected range. In addition, there was a dose-related decrease in relative total growth and a concentration-related increase in mutant frequency, with and without metabolic activation (please refer to Table 2 under “Any other information on results incl. tables”). The test item induced clear concentration-related increases in induced colonies up to cytotoxic concentrations (relative total growth values ≥ 10%). Further, the test item induced clear increases in small colonies (< 1 mm in diameter) and only relatively slight increases in large colonies, both with and without metabolic activation.
In both experiments, positive controls showed the expected results and were within the range of historical control data (data not provided in publication).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a first assay with concentrations of 0.0001 to 0.0562 µL/mL  without metabolic activation and 0.0562 to 0.562 µL/mL with metabolic activation no concentration tested yielded survival in the range of 10 to 20%. Aaverage cloning efficiencies of the solvent controls were below the acceptability criteria. Therefore a second assay with concentrations of 0.001 to 0.04 µL/mL without S9  and 0.01 to 0.25 µL/mL with S9 was conducted. The cloning efficiencies of the solvent control in the second test were considered acceptable.

Table 1: Experimental results, first mouse lymphoma assay

Test group	RSG (%)	RTG (%)	MF x 10^6	IMF x 10^6
Without metabolic activation, 4 h exposure
DMSO control
10	88.8	90.9	81	-
10	111.2	108.6	82	-
Test item [µL]
0.0001	88.1	97.7	92	11
0.0010	75.7	98.4	87	6
0.0100	71.4	88.8	63	-
0.0178	52.1	62.7	107	26
0.0316	33.7	37.6	116	35
0.0562	0.8	-	-	-
HC 7.5 µg	26.9	14.5	685	604
HC 10.0 µg	23.5	12.1	690	609
With metabolic activation, 4 h exposure
DMSO control
10	112.3	104.0	291	-
10	87.7	94.2	363	-
Test item [µL]
0.05620	86.0	94.7	361	34
0.10000	80.5	91.3	364	37
0.17800	76.2	72.9	381	54
0.31600	52.9	55.7	356	29
0.56200	14.9	6.7	596	269
CP 2.5 µg	50.2	19.2	862	535
CP 3.75 µg	47.2	2.7	977	650

HC: Hycanthone, CP: Cyclophosphamide, RSG: relative suspension growth, RTG: relative total growth, MF: mutant frequency, IMF: induced mutant frequency

Table 2: Experimental results, second mouse lymphoma assay

Test group	RSG (%)	RTG (%)	MF x 10^6	IMF x 10^6	Colony formation MF x 10^6
					small	large	total
Without metabolic activation, 4 h exposure
DMSO control
10	102.0	105.2	70	-	31	34	70
10	98.0	94.9	58	-	28	30	58
Test item [µL]
0.0010	89.7	101.0	56	-	24	39	56
0.0100	81.0	106.5	77	13	43	34	77
0.0150	61.0	71.0	94	30	69	25	94
0.0200	57.9	47.7	117	53	86	30	117
0.0250	45.0	35.2	144	80	118	26	144
0.0300	28.0	17.4	174	110	123	51	174
0.0350	17.7	8.0	281	217	217	64	281
0.0400	7.7	2.4	545	482	427	115	545
HC 7.5 µg	46.1	33.0	296	233	203	93	296
HC 10.0 µg	50.4	27.0	289	225	211	78	289
With metabolic activation, 4 h exposure
DMSO control
10	88.2	97.2	43	-	22	20	43
10	111.8	100.4	47	-	22	24	47
Test item [µL]
0.010	99.9	91.4	41	-	15	26	41
0.050	89.2	81.9	26	-	16	10	26
0.100	68.4	48.3	67	23	52	15	67
0.150	45.9	27.0	85	40	59	26	85
0.200	23.5	11.5	228	184	194	35	228
0.250	10.2	2.4	647	602	538	109	647
CP 2.5 µg	97.7	40.2	221	176	177	44	221
CP 3.75 µg	61.1	14.5	317	272	269	48	317

HC: Hycanthone, CP: Cyclophosphamide, RSG: relative suspension growth, RTG: relative total growth, MF: mutant frequency, IMF: induced mutant frequency

Conclusions:

Interpretation of results: positive with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genotoxicity in somatic cells in vivo (OECD 474, Micronucleus test): negative for clastogenicity in CD-1 mice

Genotoxicity in somatic cells in vivo (OECD 474, Micronucleus test): positive for clastogenicity C57BL mice, genotoxic effect attributed to impurity acrolein

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994-06-20 to 1994-06-24

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: TSCA Test Guidelines, Federal Register, Vol. 50, #188, Part 798

Version / remarks:

adopted 27 Sep 1985

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

yes

Remarks:

no information on generation of the test atmosphere, analytical determination of impurities in the atmosphere, no historical control data, bone marrow toxicity / exposure not evident

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 23.8 - 33.0 g (males) and 21.9 - 26.5 g (females)
- Housing: individually in stainless steel cages
- Diet: Purina Rodent Laboratory Chow, ad libitum
- Water: ad libitum
- Acclimatisation period: up to 7 days

Route of administration:

inhalation

Vehicle:

air

Details on exposure:

Groups of 5 animals of each sex were exposed nose only, 1 h on two consecutive days to atmospheres containing 37.4, 88.5, 155.6 ppm. Analytical determination of the test compound by GC was mentioned, but the records were not included in the report.

Duration of treatment / exposure:

2 days

Frequency of treatment:

1 h exposure per day on 2 consecutive days

Post exposure period:

24 h

Dose / conc.:

50 ppm (nominal)

Remarks:

corresponding to analytical concentrations of 37.4 ppm or 161.6 mg/m3 air

Dose / conc.:

100 ppm (nominal)

Remarks:

corresponding to analytical concentrations of 88.5 ppm or 382 mg/m3 air

Dose / conc.:

150 ppm (nominal)

Remarks:

corresponding to analytical concentrations of 155.6 ppm or 672 mg/m3 air

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: intraperitoneal injection, single application on study Day 2
- Doses / concentrations: 0.4 mg/kg bw

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Dose levels were chosen based on a preliminary range-finding study

TREATMENT AND SAMPLING TIMES:
Two single doses were administered, one on each of two consecutive days. Sampling was performed 24 h after the last dose.

DETAILS OF SLIDE PREPARATION:
2-3 µL of blood per slide was obtained from the mid-ventral tail vein of a mouse and placed on top of the serum. The blood was mixed with the serum and spread on the slide to produce a thin, even film, then the slide was allowed to air dry. Three slides were prepared per mouse, and after the slides were dry, the erythrocytes were fixed by placing the slides in absolute methanol for two minutes and air-dried. The slides were stained for 20 minutes in 5% Giemsa stain in phosphate buffer containing 3% methanol and 3% 0.1M citric acid, rinsed by dipping them in deionized water until clear, and allowed to air dry.

METHOD OF ANALYSIS:
For each animal, 1000 polychromatic and 1000 normochromatic erythrocytes were scored for the presence of micronuclei. In addition, the proportion of polychromatic (immature) among total (polychromatic + normochromatic) erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Sex:

male/female

Genotoxicity:

positive

Toxicity:

no effects

Remarks:

assessed by PCE/NCE ratios

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY:
- Induction of micronuclei: Significant elevation of micronucleated PCEs was observed in males of the low and high dose group, but initially not of the middle dose group. When 1000 additional PCEs were evaluated in the mid dose male mice and the negative control an increase was also observed at this dose level. In female mice the number of micronuclated PCEs was not elevated significantly compared to the negative controls. The result in females was considered equivocal by the authors.
- Ratio of PCE/NCE: A dose related depression in PCE/NCE ratios was seen in female mice, however they were higher than the untreated controls. In male mice no clear dose related depression in PCE/NCE ratios was observed.
- Clinical signs of toxicity: No toxic symptoms were noted in the treated animals.

Conclusions:

Interpretation of results: positive. According to the authors an impurity in the sample might have caused the positive result. However, no further evidence was provided for this statement.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-11-18 to 2000-01-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 21 Jul 1997

Deviations:

yes

Remarks:

high mortality at 200 mg/kg bw/day, thus only 2 valid dose levels were used

Qualifier:

according to guideline

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests

Version / remarks:

adopted 1995

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF

Version / remarks:

adopted 1990

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS-42193 Mammalian Eryhtrocyte Micronucleus Test

Version / remarks:

adopted Aug 1997

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Weight at study initiation: 22 - 31 g (males) and 21 - 27 g (females)
- Assigned to test groups randomly: yes
- Housing: in groups of no more than three animals of the same sex in appropriate caging, cleaned and dried before use
- Diet: laboratory chow diet (Special Diets Services Ltd), ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 54 - 61
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 2.5, 5.0 and 10.0 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing formulations were made by mixing the test item with corn oil. Dilutions were made using corn oil. The test article formulations were inverted periodically during dosing and used within approximately 2.75 hours of initial formulation.

Duration of treatment / exposure:

2 days

Frequency of treatment:

administration of 2 doses, 24 h apart

Post exposure period:

24 h

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg bw, single administration

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study was performed to find the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES:
Two single doses were administered, one on each of two consecutive days. Sampling was performed 24 h after the last dose.

DETAILS OF SLIDE PREPARATION:
The cells were centrifuged and the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum and the suspension was dropped on each of two coded slides. Slides were allowed to air-dry and fixed for 5 min in absolute methanol. Finally, the slides were stained with Giemsa (1:6 v/v in distilled water), rinsed, air-dried and cleared in xylene for 3 min.

METHOD OF ANALYSIS:
The proportion of polychromatic (immature, PCE) among total (polychromatic + normochromatic (NCE)) erythrocytes was determined for each animal by counting a total of 1000 erythrocytes (PCE + NCE).
2000 polychromatic erythrocytes were counted per animal to determine the presence of micronuclei.

Evaluation criteria:

A test article is considered as positive in this assay if a statistically significant increase in the frequency of micronucleated PCE occurs at least at one dose, and if the frequency of micronucleated PCE at such a point exceeds the historical vehicle control range.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs at 100 and 200 mg/kg bw/day

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
Dose levels were chosen based on an initial range finding study with groups of 3 male and female mice at a dose range of 50 to 400 mg/kg bw/day, once daily for two consecutive days. Death of all animals occured at dose levels of 300 and 400 mg/kg bw/day. Clinical symptoms were observed from 100 mg/kg bw/day. Consequently doses of 50, 100 and 200 mg/kg bw/day were chosen for the main study.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The test item did not induce a statistically significant increase in the frequency of micronucleated PCEs up to 200 mg/kg bw/day. Group mean frequencies of micronucleated PCE were also similar to that seen in the vehicle controls. The number of micrunucleated PCE of the control animals fell within the historical control rate. The positive control substance induced a statistically significant increase in the frequency of micronucleated PCEs.
- Ratio of PCE/NCE: PCE/NCE ratios of the treated animals were similar to those of the vehicle controls.
- Statistical evaluation: Chi-square test

MORTALITY: Several animals (6/6 males and 2/6 females) of the high dose group died prior to the sampling time.
CLINICAL SIGNS: Clinical symptoms were observed at 100 and 200 mg/kg bw/day. At 100 mg/kg bw/day, lethargy and eye closure were noted. At 200 mg/kg bw/day, the animals showed abnormal breathing, lethargy, eye closure, prostration and abnormal gait.

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

In vitro gene mutation in bacteria:

The test item was investigated for the mutagenic potential in a Bacterial Reverse Mutation Assay (Ames test) performed according to OECD guideline 471 and in compliance with GLP (2020 -5042 -DGM). S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA were exposed to the test in item in triplicates in the presence and absence of metabolic activation (S9 mix).

Based on a plate incorporation pre-experiment with and without metabolic activation, concentrations for the main mutation assay were selected at 3 – 5000 μg/plate. The pre-experiment was reported as experiment I. A pre-incubation test with the same concentrations with and without metabolic activation served as experiment II. Solvent (DMSO) and appropriate positive controls were included. After 48 h of incubation at 37 °C, all plates were inspected for bacterial background lawn and the number of revertant colonies was determined.

The test item did not induce a biologically relevant increase in the number of revertant colonies in any of the tested strains either with or without metabolic up to the highest tested concentration in the presence and absence of S9 mix. Positive and vehicle controls confirmed the validity of the test system and demonstrated the functionality of the S9 mix. Cytotoxicity was observed at the higher concentrations (2500 µg/plate). Precipiation of the test material was observed at the top dose of 5000 µg/plate.

Thus, under the experimental conditions chosen, the test item is not mutagenic in bacteria with and without metabolic activation.

In a second Ames test (Ballantyne 2000) the test substance did not show mutagenic activity in Salmonella typhimurium TA1535, TA1537, TA1538, TA98 or TA100 with and without metabolic activation.

In vitro gene mutation in mammalian cells:

The mutagenic potential of the test item in vitro was investigated in a mouse lymphoma assay (94-0198-FKM). In an experimental procedure similar to OECD guideline 476, mouse lymphoma L5178Y TK+/- cells were exposed to the test item (purity of 97.1%), vehicle (DMSO) or positive controls in the presence and absence of metabolic activation (S9 mix). The cells were exposed for 4 h to concentrations in the range of 0.0001 – 0.0562 µL/mL in the absence of S9 mix and to concentrations in the range of 0.0562 – 0.562 µL/mL in the presence of metabolic activation in experiment I. The cells were exposed for 4 h to concentrations in the range of 0.001 – 0.040 µL/mL in the absence of S9 mix and to concentrations in the range of 0.01 – 0.25 µL/mL in the presence of metabolic activation in experiment II.

In experiment I, there was a concentration-dependent increase in mutant frequencies, as well as a concentration-dependent increase in induced mutant frequencies. However, no concentration was tested that yielded survival in the 10-20% range. In addition, the average cloning efficiencies of the solvent controls were 62.7% without S9 mix and 55.4% with S9 mix, which were below the acceptability criteria of 70%. Mutant frequencies obtained with the solvent control were high, especially in the presence of metabolic activation, suggesting the preparation may have been mutagenic. For this reason, the mutagenicity assay was repeated.

In experiment II, average cloning efficiencies of solvent controls and spontaneous mutant frequencies fell within the expected range. In addition, there was a dose-related decrease in relative total growth and a concentration-related increase in mutant frequency, with and without metabolic activation. The test item induced clear concentration-related increases in induced colonies up to cytotoxic concentrations (relative total growth values ≥ 10%). The test item induced clear increases in small colonies (< 1 mm in diameter) and only relatively slight increases in large colonies, both with and without metabolic activation.

Cytotoxicity was observed at concentrations in the range of 0.015 – 0.04 µL/mL. The positive controls (hycanthone without S9 mix and cyclophosphamide with S9 mix) demonstrated the validity of the test. The increase in mutation frequency was almost exclusively attributable to an increase in sigma-colonies indicating that the test substance primarily induced chromosomal aberrations in this test system. Lambda colonies were only increased at the highest concentrations tested (survival rate below 3%). Under the conditions of the test, the experimental findings indicate a mutagenic potential of the test item. However, acrolein is present in the test substance as an impurity (0.05%). It is unclear if acrolein contributes to the genotoxic effects observed in the test.

 

Genetic toxicity in vivo:

Two micronucleus tests in vivo are available.

In a guideline study performed similar to OECD guideline 474 and in compliance with GLP, the test item (99.8% purity) was dissolved in corn oil and administered intraperitoneally to CD1 mice (99-0098-DGM). Based on the results of a preliminary range-finding study, dose levels for the mutagenicity study were selected. Groups of 6 mice/sex received two injections on two consecutive days at doses of 50, 100 and 200 mg/kg bw. A group of 6 mice served as positive control and received 40 mg/kg bw cyclophosphamide. 24 h after the last injection, bone marrow smears were prepared. For each animal, 2000 polychromatic erythrocytes were screened for the presence of micronuclei and 1000 erythrocytes per mouse were scored for the ratio of polychromatic (PCE) to normochromatic (NCE) erythrocytes.

6/6 males and 2/6 females of the high dose group died prior to the sampling time. Clinical symptoms were observed at 100 and 200 mg/kg bw: lethargy and eye closure at 100 mg/kg bw, and abnormal breathing, lethargy, eye closure, prostration and abnormal gait at 200 mg/kg bw.

There were no treatment-related changes in the frequency of micronucleated PCEs. The test item did not induce a statistically significant increase in the frequency of micronucleated PCEs up to 200 mg/kg bw. Group mean frequencies of micronucleated PCE were also similar to that seen in the vehicle controls and fell within the range of historical control data.

The positive control substance induced a statistically significant increase in the frequency of micronucleated PCEs, thus demonstrating the sensitivity of the test system. Under the conditions of the mentioned study, the test item has no genotoxic potential in vivo.

A second, less reliable mouse micronucleus study was conducted following TSCA test guideline, Fed. Reg. 50 § 188 part 798 and GLP using a test substance with purity 97.1 % (94-0214-DGM). Groups of 5 male and 5 female C57BL mice were exposed by inhalation (nose only) to concentrations of 37.4, 88.5 and 155.6 ppm (analytical concentration) for 1 h on 2 consecutive days. Negative controls were exposed to air. 24 h after the last exposure blood was collected from the animals and the peripheral erythrocytes were examined for micronuclei (number of cells evaluated was not specified in the study report). No clinical effects were observed in the exposed animals. A dose-related decrease in the PCE/NCE ratio was reported for female animals only, but the ratio was higher than in controls. A significant increase of the number of micronucleated polychromatic erythrocytes was reported in the high and low dose males, but not in the females. An additional evaluation of 1000 PCEs in the mid dose males resulted also in a significantly increased rate of micronucleated PCEs. Several deficiencies compared with the current OECD guideline were noted. The number of erythrocytes counted per animal is not given and the number of PCEs counted is lower than recommended. There are big inter-individual differences between the number of micronuclei/1000 PCEs in the dose groups. With regard to males of all dose groups, 1 to 3 animals did not have an increased amount of micronucleated PCEs, while the ratio varied between 2.5 and 15 in the other animals. A dose-related increase in micronuclei and statistically significant increase in micronucleated PCEs was observed in low-, mid-, and high-dose males. Considering the deficiencies, the results of this study are regarded as equivocal.

Conclusion:

In conclusion, the available experimental data on gene mutation in vivo and in vitro revealed equivocal results. There was no evidence for genotoxicity in bacteria (Ames test). A positive result for genotoxicity in mammalian cells in vitro was obtained in a mouse lymphoma assay, in which an increase in mutations for sigma colonies was indicative of a clastogenic effect in vitro in the absence of S9 mix. In the presence of S9 mix, significant increases in mutation rates were only observed at highly cytotoxic concentrations. Acrolein is present in the test substance as an impurity, it is unclear if acrolein contributes to the genotoxic effects observed in the test. However, the concentration of acrolein in the batch used in the test is similar to the concentration of acrolein given in the substance composition and the result of the in vitro mouse lymphoma assay can be considered relevant. A guideline-compliant in vivo micronucleus test with test material of an analytical purity of 99% was negative for mutagenicity in vivo, indicating that the clastogenic effects that was demonstrated in vitro did not occur in vivo. In a second micronucleus assay which suffered from a number of deficiencies and inconsistencies and a the test material of reduced purity (97 - 97.8%) revealed an equivocally positive results. It was unclear whether the genotoxic effects can be attributed to the acrolein, which is present in the test substance as an impurity.

Justification for classification or non-classification

The available information on genotoxicity in vitro (Ames test and mouse lymphoma assay) and in vivo (micronucleus test) do not require classification according to Regulation No. (EC) 1272/2008.

No classification for genetic toxicity is warranted according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.