3-mercaptopropionic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3-Mercaptopropionic acid (3-MPA) was negative in a complete battery of in-vitro genotoxicity assays.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 2008 to 28 April 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

minor deviations; absence of clastogenic control (-S9) is not essential for the interpretation of the study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

a clastogenic positive control was not used -S9; not essential for the interpretation of the study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2000

Deviations:

yes

Remarks:

a clastogenic positive control was not used -S9; not essential for the interpretation of the study

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from the livers of male Sprague-Dawley rats weighing ~250 g. These had each orally received three consecutive daily doses of phenobarbitone/ß-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4.

Test concentrations with justification for top dose:

Exp. 1 : +/- S9: 66.31, 132.63, 265.25, 530.5, 705.75, and 1061 µg/ml;
Exp 2:
4 hr w/ S9: 66.31, 132.63, 265.25, 530.5, 705.75, and 1061 µg/ml;
24 hr w/o S9: 16.5, 33, 66, 99, 132, and 198 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: serum-free culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9

Migrated to IUCLID6: 400 / 150 µg/mL (4 / 24 h exposure)

Positive control substance:

cyclophosphamide

Remarks:

with S9

Migrated to IUCLID6: 2 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 4 µg/mL 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.The IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set at 126E-06 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126E-06 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible
increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically
significant.

Statistics:

Linear trend analysis.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

10 mM (1061 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

other: The positive controls caused an increase in mutation frequency, but no clastogenic positive control (e.g., MMS) was used in the absence of S9.

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the 4-hour exposure groups, both in the absence and presence of metabolic activation (S9), there was evidence of modest reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. However, a significant reduction in %RSG values of cells treated with test material was observed in the 24-hour exposure group in the absence of S9. No precipitate of the test material was observed at any of the dose levels. In the subsequent mutagenicity test the maximum dose level used was the 10 mM limit dose for the 4-hour exposure groups. However, for the 24-hour exposure group, the maximum dose level was limited by test material induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: controls were within historical reference range

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the
L5178Y cell line at the TK +/- locus.  The positive control materials induced marked increases in the mutant frequency
indicating the satisfactory performance of the test and the activity of the metabolizing system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency
at any dose level, either with or without metabolic activation, in either the first or the second experiment,
using a dose range that included the 10 mM dose in the 4-hour exposure groups and also a dose level that induced the optimum level of toxicity in the 24-hour exposure group.

The proportion of small colonies was not increased, indicating non-clastogenicity of the test material.

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

Concentration [µg/ mL]	Relative Total Growth	Mutants per 1E+06 cells	Quotient small / large colonies
0	1.00	108.23	0.47
66.31	0.94	94.81	0.43
132.63	0.89	98.63	0.31
265.25	0.64	99.72	0.26
530.5	0.52	81.07	0.43
795.75	0.53	64.61	0.26
1061	0.49	90.63	0.40
EMS, 400	0.36	903.71	0.47

EMS:  Ethyl methane sulphonate

 

 

Table 2: Experiment 1- 4 h exposure - With Metabolic Activation

Concentration [µg/ mL]	Relative Total Growth	Mutants per 1E+06 cells	Quotient small / large colonies
0	1.00	95.59	0.44
66.31	0.82	107.45	0.33
132.63	0.78	97.39	0.38
265.25	0.67	72.72	0.37
530.5	0.75	86.28	0.33
795.75	0.76	110.04	0.37
1061	0.80	112.26	0.39
CP, 2	0.23	941.38	0.77

CP: cyclophosphamide

 

 

Table 3: Experiment 2 - 24 h exposure - Without Metabolic Activation

Concentration [µg/ mL]	Relative Total Growth	Mutants per 1E+06 cells	Quotient small / large colonies
0	1.00	102.67	0.28
16.5	1.54	65.04	0.31
33	1.26	91.52	0.21
66	1.15	76.53	0.22
99	0.94	78.61	0.27
132	0.34	105.28	0.14
198	0.11	89.77	0.15
EMS, 150	0.34	788.05	0.27

EMS: Ethyl methane sulphonate

 

 

Table 4: Experiment 2 - 24 h Exposure - With Metabolic Activation

Concentration [µg/ mL]	Relative Total Growth	Mutants per 1E+06 cells	Quotient large / small colonies
0	1.00	95.61	0.21
66.31	0.94	94.73	0.29
132.63	0.70	100.86	0.21
265.25	0.57	84.05	0.17
530.5	0.54	95.55	0.25
795.75	0.49	71.98	0.27
1061	0.53	114.54	0.24
CP, 2	0.24	480.62	0.60

CP: cyclophosphamide

Conclusions:

MPA was non-mutagenic to L5178Y cells under the conditions of the test. The proportion of small colonies was not increased, indicating non-clastogenicity of the test material.

Executive summary:

The mutagenic potential of 3-MPA was investigated in a mouse lymphoma assay according to OECD TG 476 (TK assay). Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2¢ mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9).
In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 66.31 to 1061 ug/ml in both the absence and presence of metabolic activation for the first experiment. For the second experiment the dose range was 8.25 to 264 ug/ml in the absence of
metabolic activation and 66.31 to 1061 ug/ml in the presence of metabolic activation.

The maximum dose level used was the 10 mM limit dose for the 4-hour exposure groups. However, for the 24-hour exposure group, the maximum dose level was limited by test material induced toxicity. No precipitate of test material was observed at any of the dose levels.

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment, using a dose range that included the 10 mM limit dose in the 4-hour exposure groups and also a dose level that induced the optimum level of toxicity in the 24-hour exposure group.
The test material was considered to be non-mutagenic to L5178Y cells under the  conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March 2008 to 27 April 2008

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 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S.typhimurium)
trp operon (E. coli)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from the livers of male Sprague-Dawley rats weighing ~250 g. These had each orally received three consecutive daily doses of phenobarbitone/ß-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4.

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Migrated to IUCLID6: 3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA-

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Migrated to IUCLID6: 80 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Migrated to IUCLID6: 0.2 µg/plate for TA98

Positive control substance:

other: 2-Aminoanthracene, 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA-

Remarks:

with metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Migrated to IUCLID6: 5 µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

Statistics:

n.a.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test material caused no visible reduction in the growth of the bacterial background lawn at any test concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: within historical reference range

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Experiment 1 - Without Metabolic Activation

S9 Mix	Test substance concentration (µg/ plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA^–	TA98	TA1537
–	0	103	20	28	24	11
–	50	103	20	22	19	7
–	150	93	30	23	21	13
–	500	88	24	23	17	12
–	1500	84	25	17	18	10
–	5000	100	23	24	10	14
Pos controls ‑S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Conc. (µg/plate)	3	5	2	0.2	80
	No. of revertants per plate	413	172	212	146	862

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

 

 

Table 2: Experiment 1 - With Metabolic Activation

S9 Mix	Test substance concentration (µg/ plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA^–	TA98	TA1537
+	0	83	11	18	29	12
+	50	75	15	28	27	13
+	150	83	13	28	22	9
+	500	92	11	25	20	8
+	1500	79	13	24	21	5
+	5000	70	0 V	25	15	5
Pos controls +S9	Name	2AA	2AA	2AA	BP	2AA
	Conc. (µg/plate)	1	2	10	5	2
	No. of revertants per plate	1481	211	496	187	249

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

V: Very weak bacterial background lawn

 

Table 3: Experiment 2 - Without Metabolic Activation

S9 Mix	Test substance concentration (µg/ plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA^–	TA98	TA1537
–	0	108	21	24	19	16
–	50	103	25	25	24	17
–	150	103	26	21	14	14
–	500	108	24	23	13	16
–	1500	107	26	25	18	16
–	5000	82	25	23	15	14
Pos controls ‑S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Conc. (µg/plate)	3	5	2	0.2	80
	Avg. no. of revertants per plate	473	449	998	110	847

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

 

 

Table 4: Experiment 2 - With Metabolic Activation

S9 Mix	Test substance concentration (µg/ plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
+	0	113	14	32	32	18
+	50	110	18	27	26	14
+	150	106	13	30	24	13
+	500	100	21	24	21	13
+	1500	80	16	25	19	10
+	5000	70	13	19	20	16
Pos controls +S9	Name	2AA	2AA	2AA	BP	2AA
	Conc. (µg/plate)	1	2	10	5	2
	No. of revertants per plate	1672	232	558	189	227

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

Conclusions:

Interpretation of results: negative with and without metabolic activation

MPA is negative in the Ames test, with and without metabolic activation.

Executive summary:

The mutagenic potential of 3-MPA was investigated in a bacterial reverse mutation assay according to OECD TG 471. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA’ were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 pg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. For Salmonella strain TA1535, dosed in the presence of S9, there was a discrepancy in toxic response with weakened lawns noted at 5000 pg/plate in Experiment 1 but no toxicity observed in Experiment 2. This discrepancy in toxic response was considered spurious and of no biological relevance. The test material was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
The test material was considered to be non-mutagenic under the conditions of this test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 April 2007 to 17 July 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: not applicable, primary culture
- Periodically checked for Mycoplasma contamination: not applicable, primary culture
- Periodically checked for karyotype stability: not applicable, primary culture
- Periodically "cleansed" against high spontaneous background: not applicable, primary culture

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from the livers of male Sprague-Dawley rats weighing ~250 g. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and ß-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.

Test concentrations with justification for top dose:

4-h exposure: 33.1 to 1060 µg/mL
24-h exposure: 33.1 to 530 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Migrated to IUCLID6: 0.4 and 0.2 µg/mL for 4- and 24-h exposure

Positive control substance:

cyclophosphamide

Remarks:

with S9

Migrated to IUCLID6: 5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4, 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h


SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): 5% Gurrs Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 200 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


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

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear concentration-relationship. For modest increases in aberration frequency a concentration response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

Fisher's Exact test.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

10 mM

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The concentration range for the Preliminary Toxicity Test was 16.5 to 1060 µg/mL. The maximum concentration was the 10 mM concentration. No precipitate of the test material was observed at the end of the exposure period in any of the three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1060 µg/mL in the 4(20)-hour exposures both in the presence and absence of metabolic activation (S9). The maximum concentration with metaphases present in the 24-hour continuous exposure was 1060 µg/mL, though it should be noted that frequency of metaphases was very low. The test material induced some evidence of toxicity.


COMPARISON WITH HISTORICAL CONTROL DATA: within historical reference range

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.  All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.  The test material was moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a concentration range that included a concentration level that induced approximately 50% mitotic inhibition.

MPA produced some evidence of toxicity at 1060 µg/mL in the preliminary  toxicity test, as well as in the chromosome aberration test.

Table 1: Experiment 1 - 4 h treatment, 20 h fixation - Without Metabolic Activation

Concentration [µg/ mL]	Mitotic index [%]	Polyploid cells [%]	Aberrant cells (%)
			incl. gaps	excl. gaps
0	100	0.0	0.5	0.0
265	91	0.0	1.5	0.5
530	75	0.5	0.0	0.0
1060	71	0.0	2.0	1.5
MMC, 0.4	60	0.0	35.3	33.3***

MMC: Mitomycin C

*** p<0.001

 

Table 2: Experiment 1- 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration [µg/ mL]	Mitotic index [%]	Polyploid cells [%]	Aberrant cells (%)
			incl. gaps	excl. gaps
0	100	0.0	1.0	0.5
265	100	0.0	0.5	0.5
530	69	0.0	0.5	0.5
1060	70	0.0	1.5	1.5
CP, 5	30	0.0	38.8	33.0***

CP: Cyclophosphamide

*** p<0.001

 

Table 3: Experiment 2- 20 h treatment, 20 h fixation - Without Metabolic Activation

Concentration [µg/ mL]	Mitotic index [%]	Polyploid cells [%]	Aberrant cells (%)
			incl. gaps	excl. gaps
0	100	0.5	0.5	0.5
66.25	104	0.0	2.0	0.5
132.5	82	0.0	2.5	0.5
265	36	0.0	3.0	2.0
MMC, 0.4	50	0.0	17.0	16.0***

MMC: Mitomycin C

*** p<0.001

 

 

Table 4: Experiment 2- 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration [µg/ mL]	Mitotic index [%]	Polyploid cells [%]	Aberrant cells (%)
			incl. gaps	excl. gaps
0	100	0.0	0.0	0.0
265	178	0.0	2.0	1.5
530	142	0.0	0.5	0.5
1060	161	0.0	2.5	0.5
CP, 5	54	0.0	28.0	24.0***

CP: Cyclophosphamide

*** p<0.001

Conclusions:

Interpretation of results: negative with and without metabolic activation

The test material is non-clastogenic to human lymphocytes in vitro, with and without metabolic activation.

Executive summary:

The clastogenic potential of 3-MPA was investigated in a chromosome aberration assay according to OECD TG 473.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to three concentration levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a concentration range that included a concentration level that induced approximately 50% mitotic inhibition.

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay

The mutagenic potential of 3-MPA was investigated in a bacterial reverse mutation assay according to OECD TG 471. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA’ were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 pg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. For Salmonella strain TA1535, dosed in the presence of S9, there was a discrepancy in toxic response with weakened lawns noted at 5000 pg/plate in Experiment 1 but no toxicity observed in Experiment 2. This discrepancy in toxic response was considered spurious and of no biological relevance. The test material was, therefore, tested up to the maximum recommended dose level of 5000 pg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
The test material was considered to be non-mutagenic under the conditions of this test.

 

Mutation assay in mammalian cells

The mutagenic potential of 3-MPA was investigated in a mouse lymphoma assay according to OECD TG 476 (TK assay). Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2¢ mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9).
In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and was 66.31 to 1061 ug/ml in both the absence and presence of metabolic activation for the first experiment. For the second experiment the dose range was 8.25 to 264 ug/ml in the absence of
metabolic activation and 66.31 to 1061 ug/ml in the presence of metabolic activation.

The maximum dose level used was the 10 mM limit dose for the 4-hour exposure groups. However, for the 24-hour exposure group, the maximum dose level was limited by test material induced toxicity. No precipitate of test material was observed at any of the dose levels.

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment, using a dose range that included the 10 mM limit dose in the 4-hour exposure groups and also a dose level that induced the optimum level of toxicity in the 24-hour exposure group.
The test material was considered to be non-mutagenic to L5178Y cells under the  conditions of the test.

 

Chromosome aberration assay in mammalian cells

The clastogenic potential of 3-MPA was investigated in a chromosome aberration assay according to OECD TG 473.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to three concentration levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a concentration range that included a concentration level that induced approximately 50% mitotic inhibition.

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

 

 

Justification for classification or non-classification

Based on the available data, the substance does not require classification for mutagenicity according to regulation (EC) 1272/2008