Propanoic acid, 2-hydroxy-, propyl ester, (2S)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data is available for the target substance Propyl (S)-lactate, except one in vitro bacterial reverse mutation assay conducted according to OECD 471. In this study, the target substance was tested negative.

In addition, data from the suitable read-across partners propanol, lactic acid, ethyl (S) lactate and ethylhexyl (S)-lactate were used in a weight-of-evidence approach to assess the genotoxic potential of Propyl (S)-lactate. L-lactic acid, ethyl (S)-lactate and ethylhexyl (S)-lactate were tested negative each in two in vitro genotoxicity tests conducted according to OECD 473 and OECD 476 (nowadays OECD 490). Propanol was tested negative in an in vitro mammalian cell micronucleus test. Based on the available data from suitable read-across partners, the target substance can be considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

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

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
- Effects of pH: At the highest concentration of test substance (0.01 M equal to 901 µg/mL) the pH was 6.84 compared to a pH of 7.31 in the solvent control.
- Effects of osmolality: At the highest concentration of test substance (0.01 M equal to 901 µg/mL) the osmolarity was 0.319 Osm/kg compared to an osmolarity of 0.299 Osm/kg in the solvent control
- Water solubility: miscible
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity data were obtained by treating 8 x 10^6 cells (10^6 cells/mL for 3 hours treatment) or 5 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hours treatment) with 0, 17, 52, 164, 512 and 901 µg of test substance for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix.
After exposure, the cells were separated from treatment solutions centrifugation steps and re-suspended in RPM 1640 medium supplemented with 10% (v/v) inactivated horse serum (R10 medium). Cells were counted with the coulter particle counter.
For determination of the cytotoxicity, the surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted (day 1) and subcultured again for another 24 hours, after which the cells were counted (day 2). The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 105 cells/mL were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

COMPARISON WITH HISTORICAL CONTROL DATA:
Spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control range.

Experiment 1 & 2: For individual results see Tables 3-5 in box 'Any other information on results incl. tables'.

Table 1: Dose-range finding test: Cytotoxicity of L(+)-lactic acid (3 hours treatment)

Dose (µg/mL)	Cell count after 3 hours of treatment (cells/mL x 10^5)	Cell count after 24 hours of subculture (cells/mL x 10^5)	Cell count after 48 hours of subculture (cells/mL x 10^5)	SG(1) x 10^5 cells/mL)	RSG (2) %
without metabolic activation
SC	6.9	5.0	6.9	152	100
17	6.3	5.0	7.4	149	98
52	7.0	5.2	6.9	161	106
164	7.3	5.2	7.1	173	113
512	7.6	5.2	6.8	172	113
901	6.8	5.4	7.0	166	109
with metabolic activation
SC	5.3	4.9	7.8	130	100
17	5.2	5.2	7.5	130	100
52	4.2	5.2	7.6	106	82
164	4.1	5.3	7.2	100	77
512	5.0	5.1	7.5	122	94
901	4.3	5.1	7.4	104	80

Note: all calculations were made without rounding off

SC = solvent control = exposure medium

(1) = suspension growth

(2) relative suspension growth

SG= (Cell count after 3 h treatment) x (Cell count after 24 h subculture)/(Cells subcultured (at t=3 h)(1.25x10^5 c/mL)) x (Cell count after 48 h subculture)/(Cells subcultured (at t=24 h) (1.25 x 10^5 c/mL))

RSG = [SG(test)/SG(control)] x 100

Table 2: Dose-range finding test: Cytotoxicity of L(+)-lactic acid (24 hours treatment)

Dose (µg/mL)	Cell count after 24 hours of treatment (cells/mL x 10^5)	Cell count after 24 hours of subculture (cells/mL x 10^5)	SG(1) x 10^5 cells/mL)	RSG (2) %
without metabolic activation
SC	9.5	5.9	45	100
17	8.9	5.9	42	93
52	9.3	5.7	42	93
164	9.1	5.2	39	85
512	8.8	5.5	39	87
901	7.2	4.6	26	58

Note: all calculations were made without rounding off

SC = solvent control = exposure medium

(1) = suspension growth

(2) relative suspension growth

SG = (Cell count after 24 h treatment) x (Cell count after 24 h subculture)/(Cells subcultured after treatment (1.25 x 10^5 c/mL)

RSG = [SG(test)/SG(control)] x 100

Cytotoxic and mutagenic response of L(+)-lactic acid in the mouse lymphoma L5178Y test system

Abbreviations:

RSG: Relative Suspension Growth

CE: Cloning Efficiency

RS: Relative Survival

RTG: Relative Total Growth

MF: Mutation Frequency per 10^6 Survivors

SC: Solvent Control (= Exposure Medium)

MMS: Methylmethanesulfonate

CP: Cyclophosphamide

Experiment 1

Table 3: 3 h treatment, without metabolic activation

Dose [µg/mL]	RSG [%]	CEday2 [%]	RSday2 [%]	RTG [%]	MF total	MF small	MS large
SC1	100	97	100	100	89	70	16
SC2	100	80	100	100	86	66	18
0.54	107	86	98	105	98	71	25
1.7	118	79	89	106	98	72	23
5.4	126	83	93	117	94	62	28
17	129	77	87	112	122	91	26
52	108	75	84	91	124	97	23
164	112	78	88	99	104	66	34
512	106	72	82	87	147	99	41
901	101	88	99	100	116	89	23
MMS	79	41	47	37	1149	870	191

Table 4: 3 h treatment, with metabolic activation

Dose [µg/mL]	RSG [%]	CEday2 [%]	RSday2 [%]	RTG [%]	MF total	MF small	MS large
SC1	100	68	100	100	51	26	23
SC2	100	64	100	100	55	30	25
0.54	92	78	118	108	53	31	20
1.7	78	111	168	132	26	17	8
5.4	56	93	140	79	38	28	10
17	60	97	146	88	31	22	9
52	65	66	100	65	62	47	14
164	92	74	111	102	53	45	7
512	64	77	116	74	45	12	32
901	93	81	123	114	45	25	19
CP	37	29	43	16	849	647	167

Experiment 2

Table 5: 24 h treatment, without metabolic activation

Dose [µg/mL]	RSG [%]	CEday2 [%]	RSday2 [%]	RTG [%]	MF total	MF small	MS large
SC1	100	98	100	100	57	26	30
SC2	100	102	100	100	50	19	30
0.54	92	84	84	77	63	19	42
1.7	91	86	86	78	71	40	28
5.4	99	89	89	88	65	25	38
17	90	89	89	80	49	11	38
52	86	98	98	84	51	16	34
164	85	88	87	74	72	34	36
512	78	90	90	71	50	22	26
901	64	107	107	68	53	9	43
MMS	80	61	61	49	621	198	368

Conclusions:

In conclusion, L(+)-lactic acid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation test (OECD 476, nowadays OECD 490) in the presence and absence of mammalian metabolic activation.

Executive summary:

In a mammalian cell gene mutation assay conducted in accordance to OECD guideline 476 (nowadays OECD 490), L5178Y mouse lymphoma cells cultured in vitro were exposed to L(+)-lactic acid (90% purity), solved in RPMI 1640 medium. In the first experiment, L(+)-lactic acid was tested up to concentrations of 901 µg/mL (0.01 M, the highest concentration recommended in the guidelines) in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, L(+)-lactic acid was again tested up to concentrations of 901 µg/mL in the absence S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9 mix. The induced mutation frequency with and without metabolic activation was not increased compared to control in all tested concentrations. The positive controls did induce the appropriate response. Based on the results, it can be concluded, that L(+)-lactic acid is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

This study is classified as acceptable. This study satisfies the requirement for OECD 476 (nowadays OECD 490 for in vitro mutagenicity (mammalian forward gene mutation) data.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

based on determination of the mitotic index.

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
- Effects of pH: At the highest concentration of the test substance (10 mM equal to 901 µg/ml) the pH was 7.1 compared to a pH of 7.8 in the solvent control.
- Effects of osmolality: At the highest concentration of the test substance (10 mM equal to 901 µg/mL) the osmolarity was 275 mOsm/kg compared to an osmolarity of 269 mOsm/kg in the solvent control.
- Water solubility: miscible
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test blood cultures were treated with 10, 33, 100, 333, 901 µg/mL L(+)-lactic acid/mL culture medium (equal to concentrations of 0.1, 0.4, 1.1, 3.7 and 10 mM) with and without S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

For individual results see box 'Any other information on results incl. tables'.

Mitotic Indices:

Table1: Mitotic index of human lymphocyte cultures treated with L(+)-lactic acid in the dose range finding test

L(+)-lactic acid concentration (µg/ml)	Number of metaphases:   Absolute	Number of metaphases:   Number of cells scored	Number of metaphases:   Percentage of control
Without metabolic activation (-S9-mix)
3 h exposure time, 24 h fixation time
Control a)	96	1004	100
10	99	1007	103
33	80	1045	83
100	60	1008	63
333	66	1009	69
901	63	1003	66
24 h exposure time, 24 h fixation time
Control a)	65	1007	100
10	62	1042	95
33	71	1041	109
100	66	1016	102
333	68	1048	105
901	36	1028	55
48h exposure time,48h fixation time
Control a)	68	1017	100
10	64	1026	94
33	51	1013	75
100	65	1010	96
333	57	1017	84
901	33	1033	49
With metabolic activation (+S9-mix)
3 h exposure time, 24 h fixation time
Control a)	85	1044	100
10	70	1013	82
33	71	1008	84
100	68	1006	80
333	66	1020	78
901	71	1007	84

a.) culture medium

Table 2: Mitotic index of human lymphocyte cultures treated with L(+)-lactic acid in the first cytogenetic assay

L(+)-lactic acid concentration (µg/ml)	Number of metaphases a)   Absolute	Number of metaphases a)   Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
3 h exposure time, 24 h fixation time
Control b)	3-36	1009-1028	100
10	3-35	1002-1012	103
100	3-28	1008-1033	90
901	1-9	1002-1040	34
MMC-C; 0.5 µg/ml	4-7	1026-1031	16
MMC-C; 0.75 µg/ml	7-5	1029-1004	17
With metabolic activation (+S9-mix)
3 h exposure time, 24 h fixation time
Control b)	3-48	1040-1028	100
10	3-27	1013-1007	79
100	4-35	1035-1001	101
901	4-32	1008-1007	93
CP; 10 µg/ml	21-14	1005-1025	43

a)     Duplicate cultures

b)     Culture medium

Table 3: Mitotic index of human lymphocyte cultures treated with L(+)-lactic acid in the second cytogenetic assay 

L(+)-lactic acid concentration (µg/ml)	Number of metaphasesa   Absolute	Number of metaphasesa   Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
24 h exposure time, 24 h fixation time
Control b)	90-85	1000-1000	100
100	75-83	1000-1003	90
333	67-65	1008-1000	75
666	73-66	1001-1000	79
901	39-42	1002-1000	46
MMC-C; 0.2 µg/ml	24-34	1000-1003	33
MMC-C; 0.3 µg/ml	21-33	1003-1000	31
48 h exposure time, 48 h fixation time
Control b)	93-88	1005-1000	100
100	71-87	1001-1000	87
333	66-51	1000-1000	65
666	34-37	1000-1002	39
901	22-24	1003-1000	25
MMC-C; 0.1 µg/ml	18-20	1002-1003	21
MMC-C; 0.15 µg/ml	17-19	1000-1004	20
With metabolic activation (+S9-mix)
3 h exposure time, 48 h fixation  time
Control b)	88-87	1000-1000	100
10	66-75	1000-1045	81
100	62-64	1003-1005	72
901	71-63	1000-1000	77
CP; 10 µg/ml	22-18	1005-1000	-c.)

a) Duplicate cultures

b) Culture medium

c) CP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

Chromosome aberrations in human lymphocyte cultures treated with L(+)-lactic acid

1. Cytogenetic Assay:

Conc	Exposure Time [h]	Fixation Time [h]	Metabolic Activation	Culture	Mitotic Index [%]	Cells  scored	Cells  + gaps	Cells - gaps
Culture medium	3	24	without	A+B	100	200	0	0
10 µg/ml	3	24	without	A+B	103	200	5	5
100 µg/ml	3	24	without	A+B	90	200	0	0
901 µg/ml	3	24	without	A+B	34	200	4	4
MMC-C 0.5 µg/ml	3	24	without	A+B	16	150	76***	76***
Culture medium	3	24	with	A+B	100	200	2	2
10 µg/ml	3	24	with	A+B	79	200	3	3
100 µg/ml	3	24	with	A+B	101	200	2	2
901 µg/ml	3	24	with	A+B	93	200	2	2
CP 10 µg/ml	3	24	with	A+B	43	200	59***	59***

*) Significantly different from control group (Chi-square test), p < 0.001

2. Cytogenetic Assay:

Conc	ExposureTime [h]	FixationTime [h]	Metaboic Activation	Culture	Mitotic Index  [%]	Cells  scored	Cells  + gaps	Cells - gaps
Culture medium	3	48	with	A+B	100	200	2	1
10 µg/ml	3	48	with	A+B	81	200	1	1
100 µg/ml	3	48	with	A+B	72	200	0	0
10 µg/ml	3	48	with	A+B	77	200	3	3
CP 10 µg/ml	3	48	with	A+B	n.d.^b	100	53***	53***
Culture medium	24	24	without	A+B	100	200	1	1
100 µg/ml	24	24	without	A+B	90	200	1	1
666 µg/ml	24	24	without	A+B	79	200	0	0
901 µg/ml	24	24	without	A+B	46	200	3	3
MMC- 0.1 µg/ml	24	24	without	A+B	33	100	53***	53***
Culture medium	48	48	without	A+B	100	200	1	1
100 µg/ml	48	48	without	A+B	87	200	3	3
333 µg/ml	48	48	without	A+B	65	200	2	2
666 µg/ml	48	48	without	A+B	39	200	2	2
MMC- 0.1 µg/ml	48	48	without	A+B	21	100	53***	53***

^bCP was fixed after 24 hours. Therefore, the mitotic index could not be calculated as percentage of control.

*) Significantly different from control group (Chi-square test), p < 0.001

Conclusions:

L(+)-lactic acid is considered to be not clastogenic in the in vitro mammalian chromosomal aberration test conducted according to the OECD TG 473 using human lymphocytes in the presence or absence of metabolic activation.

Executive summary:

In an in vitro cytogenicity assay conducted according to OECD TG 473, peripheral human lymphocyte cultures were exposed to L(+)-lactic acid (90% purity), solved in RPMI 1640 cell culture medium. In the first experiment, the doses were 0, 10, 100, 901 µg/mL with and without metabolic activation. In the second experiment doses were 0, 100, 333, 666, 901 µg/mL without metabolic activation and 0, 10, 100, 901 µg/mL with metabolic activation (rat liver S9-mix).

L(+)-lactic acid was tested up to 901 µg/mL, which was cytotoxic based on determination of the mitotic index after an exposure time of 24 and 48 hours. The percentage of the mitotic index after 24 hours of 901 µg/mL was 55%, that after 48 hours of 901 µg/mL 49%. Concentrations lower than 901 µg/mL did not cause a dose-dependent decrease in the percentage of the mitotic index after 24 and 48 hours of exposure. The mitotic index after 3 hours of exposure was lower compared to control (66% in experiment 1, 84% in experiment 2) but did not reach the threshold value of 45 ± 5% according to OECD guideline 473 for cytotoxicity. Positive controls induced the appropriate responses. There was no evidence for a concentration related positive response of chromosome aberration induced over background.

This study is classified as acceptable and satisfies the requirement for the in vitro mammalian chromosomal aberration test according to OECD 473.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

All results are presented in Tables 1-8 in the attached background material (NOTOX Project 494586).

Conclusions:

Ethyl (S)-lactate is considered to be not clastogenic in the in vitro mammalian chromosomal aberration test using human lymphocytes, with and without metabolic activation.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration) according to OECD Guideline 473, primary peripheral human lymphocytes cultures were exposed to Ethyl (S)-lactate (purity 99.83%) at concentrations of 0, 100, 333 and 1180 µg/mL with and without metabolic activation.

The test item was tested up to the limit concentration of 1180 µg/mL (0.01 M). Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background.

In conclusion, Ethyl (S)-lactate is not clastogenic in human lymphocytes.

This study is classified as acceptable and satisfies the requirement for the in vitro mammalian chromosomal aberration test according to OECD 473.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003-04-09 to 2003-06-16

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 21st July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): n-propyl (S) lactate
- Analytical purity: 99.5%
- Lot/batch No.: 0210001541
- Appearance: colourless liquid
- Storage conditions: at room temperature in the dark
- Expiry date: 2004-08-30

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
The test substance was dissolved in dimethyl sulfoxide of spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.

Target gene:

Salmonella typhimurium: Histidine
E. coli: Tryptophane

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Salmonella typhimurium: Dr. Bruce Ames, University of California at Berkeley, USA
Escherichia coli: Prof. Dr. B.A. Bridges, University of Sussex, Brighton, UK
- Suitability of cells: recommended tester strains


For cell lines:
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampililcin resitance (TA98,100), UV-sensitivity and the number of spontaneous revertants.
The used Escherichia coli strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

MEDIA USED
- Type and composition of media:
Agar plates: Agar plates contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid, code L28) in Vogel-Bonner medium E, 20 g glucose. The agar plates for the test with the S. typhimurium strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the E. coli strain contained 15 µg/plate tryptophan.
Top agar: Top agar medium containing 0.6% agar and 0.5% NaCl was heated to dissolve the agar.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- S9 fractions isolated from the liver of Wistar rats after induction with Aroclor-1254.

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA.
Main test:
Strains TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Strains TA1535, TA1537 and TA98: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (0.1 mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, with S9 (TA1535/TA98/TA100: 1 µg/plate; TA1537: 2.5 µg/plate; WP2uvrA: 5 µg/plate); Daunomycin, without S9 (TA98: 4 µg/plate)

Remarks:

With S9: 2-aminothracene; without S9: SA (TA1535), 9AC(TA1537), DM (TA98), 4-NQO (WP2uvrA)

Details on test system and experimental conditions:

Dose Range Finding Test:
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate.

Mutation Assay:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplcate in each strain. The test substance was tested both in the absence and presence of S9 mix in each strain, in two independent experiments.
Top agar in top agar tubes was melted by heating to 45 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test substance in DMSO and either 0.5 mL S9-mix or 0.5 mL 0.1 M phosphate buffer. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 °C +/- 1.0 °C for 48 hours. After this period revertant colonies were counted.

NUMBER OF REPLICATIONS: 3

Colony counting:
The revertant colonies were counted automatically with a Protos model 5000 or manually, if less than 40 colonies per plate were present.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined..

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with and without S9.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in any tester strain is greater than two times the solvent control, either with or without S9. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Not available

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation of the test material was not observed in any plate of the tester strains.


RANGE-FINDING/SCREENING STUDIES:
- Precipitation: precipitation of the test material was not observed in any plate of the tester strains.
- Toxicity: no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Precipitate:

The test substance did not precipitate in the top agar. Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation period in all tester strains.

Toxicity:

The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Number of revertants:

All bacterial strains showed negative responses over the entire dose range. The negative and positive controls were within the historical control data ranges indicating that the test conditions were adequate.

Conclusions:

Propyl (S)-lactate is not genotoxic in the bacterial reverse gene mutation assay (OECD 471) in the presence and absence of mammalian metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria conducted according to OECD guideline 471, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2uvrA were exposed to Propyl (S)-lactate (99.5% purity) at concentrations of 0, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test item was tested up to the limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. Based on the results, the test item can be considered to be non-mutagenic.

 

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation assay).

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

All results are presented in Tables 1–10 (see attachments).
Dose range finding test:
At a concentration of 1000 µg/mL the test substance precipitated in the culture medium at the 3 h exposure time experiment. Based on the results of the DRF for the 3h exposure time, 24 h fixation time experiment the following dose levels were selected: 100, 125, 150, 175, 200, 235, 270 and 300 µg/mL culture medium.
First experiment:
Table 2 shows the mitotic index of treated cultures. In the presence of S9-mix no appropriate dose level could be selected for scoring of chromosome aberrations since at 270 µg/mL no cytotoxicity was observed (9%), whereas the next higher concentrations of 300 µg/mL was too toxic for scoring (79%). This part of the experiment was repeated.
Repeated first experiment (1a):
With S9 -mix: 150, 200, 250, 270, 275, 280, 285, 290, 295 and 300 µg/mL culture medium
Table 3 shows the mitotic index of the treated cultures and appropriate dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9 -mix, the test substance did not induce a statistically or biologically relevant increase in the number of cells with chromosome aberrations (Table 4, 5).
Second experiment:
A second experiment was performed in which the cultures were exposed to the test substance in the absence of S9 -mix for 24 or 48 hours. In the presence of S9 mix, cells were fixed after 48 hours following a 3 hour exposure. Table 6 shows the mitotic index of the treated cultures. In the absence of S9-mix at the 48 h continuous exposure time, no appropriate dose levels could be selected for scoring since no concentration was available with cytotoxicity of greater 50%. This part of the experiment was repeated.
Repeated second experiment (2a):
Without S9 mix: 150, 200, 250, 300, 310, 320, 330, 340 and 350 µg/mL culture medium (48 h exposure time, 48 fixation time)
Table 7 shows the mitotic index of the treated cultures and appropriate dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix, the test substance did not induce a statistically or biologically relevant increase in the number of cells with chromosome aberrations (Table 8,9, 10).
Both in absence and presence of S9-mix, the test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Controls:

the number of cells with chromosome aberrations, number of polyploid cells and cells with endoreduplicated chromosomes found in the solvent control cultures was within the laboratory historical control data range. Both positive control chemicals produced statistically significant increases in the frequency of aberrant cells. It can be therefore concluded, that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:

2-Ethylhexyl (S)-lactate is considered to be not clastogenic in the in vitro mammalian chromosomal aberration test using human lymphocytes in the presence or absence of metabolic activation.

Executive summary:

In a mammalian chromosomal aberration test conducted according to OECD TG 473, primary peripheral human lymphocytes cultures were exposed to 2-Ethylhexyl (S)-lactate (purity 99%) at concentrations of up to 350 µg/mL without metabolic activation and up to 330 µg/mL with metabolic activation.

The test substance was tested up to cytotoxic concentrations and appropriate concentration were selected for scoring of chromosome aberrations. There was no evidence of chromosome aberration induced over background. Positive and negative controls induced the appropriate response and were within the laboratory historical control data range. In conclusion, 2-Ethylhexyl (S)-lactate is not clastogenic in human lymphocytes under the experimental conditions described.

This study is classified as acceptable and satisfies the requirement for the in vitro mammalian chromosomal aberration test according to OECD 473.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 6

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Table 1: Results of the in vitro micronucleus test

Chemical		Dose		Number of MN/1000 cells (mean +/- SE)
Medium		50 µL/mL		4.00 +/- 0.71
Propanol-1		50 µl/mL		3.50 +/- 0.65
MMS		100 µg/mL		16.5 +/- 0.50
EMS		100 µg/mL		7.0 +/- 0.69
MMNG		0.08 µg/mL		8.2 +/- 0.83

 

Conclusions:

In conclusion, it can be stated that propanol-1 did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, the test substance is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro mammalian cell micronucleus test.

Executive summary:

In an in vitro mammalian cell micronucleus test conducted similar to OECD test guideline 487, V79 Chinese hamster cells cultured in vitro, were exposed for 1 hour to propanol-1 at a concentration of 50 µL/mL without metabolic activation. No increase of the micronucleus frequency was noted after treatment with the test item. The positive controls did induce distinct and biologically relevant increases of the micronucleus frequency. Therefore, it can be stated that propanol is considered to be non-mutagenic under the described test conditions.

This information is used in a read-across approach in the assessment of the target substance. For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no toxicity in experiment 1, no severe toxicity in experiment 2.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
PURASOLV® EL did not precipitate in the exposure medium up to and including the concentration of 1180 μg/ml (= 10 mM). Since testing up to 0.01 M is recommended in the guidelines, this concentration was used as the highest test substance concentration in the dose range finding test.
The pH and osmolarity of a concentration of 1180 μg/ml were 7.4 and 0.302 Osm/kg respectively (compared to 7.5 and 0.286 Osm/kg in the solvent control).

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 10 to 1180 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
Table 1 (Tables see attached background information) shows the cell counts of the cultures after 3 hours of treatment with various concentrations of PURASOLV® EL and after 24 and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 1180 μg/mL compared to the suspension growth of the solvent controls.
Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of PURASOLV® EL and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth.
In the absence of S9-mix, the relative suspension growth was 38% at the test substance concentration of 1180 µg/ml compared to the relative suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Tables are given in the attached background information.

Conclusions:

In conclusion, Ethyl (S)-lactate is considered to be non-mutagenic in the in vitro mammalian cell gene mutation test (OECD 476, nowadays OECD 490) in the presence and absence of mammalian metabolic activation.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD Guideline 476 (nowadays OECD 490), L5178Y mouse lymphoma cells cultured in vitro were exposed to Ethyl (S)-lactate (purity 99.83%) in RPMI 1640 medium at concentrations of 0, 0.3, 1, 3, 10, 33, 100, 333 and 1180 µg/mL in the presence and absence of mammalian metabolic activation. The test item was tested up to the highest concentration recommended in the guideline (0.01 M = 1180 µg/mL). Positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background. Based on the results, it can be concluded, that Ethyl (S)-lactate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

This study is classified as acceptable. This study satifies the requirement for Test Guideline OECD 476 (nowadays OECD 490) for in vitro mutagenicity (mammalian forward gene mutation) data.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- The pH and osmolarity of a concentration of 500 μg/ml were 7.3 and 0.410 Osm/kg respectively (compared to 7.5 and 0.414 Osm/kg in the solvent control).
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 10 to 500 µg/mL in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
Table 1 (Tables see attached background information) shows the cell counts of the cultures after 3 hours of treatment with various concentrations of the test substance and after 24 and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.
In the absence of S9-mix, the relative suspension growth was 78% at the test substance concentrations of 100 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test substance concentrations of 300 μg/mL and above.
In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 300 μg/mL compared to the solvent control. Hardly any cell survival was observed at the test substance concentration of 500 μg/mL.
Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of the test substance and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth.
In the absence of S9-mix, the relative suspension growth was 5% at the test substance concentrations of 300 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test substance concentration of 500 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Experiment 1 & 2: For individual results see Tables in box 'Attachments'.

1. Mutation experiment

The results of the two mutation experiments were within the validity criteria. Table 3 and 4 show the percentages of cell survival and the mutation frequencies for various concentrations of the test substance. Individual colony counts of cloning and selective plates and cell counts during subculturing are listed in Tables 5-11 of Appendix I.

1.1. First mutagenicity test

Evaluation of toxicity

In the absence of S9-mix, no dose level with a cell survival below 40% was reached; therefore this part of the experiment was rejected. The following dose range was selected for mutation experiment 1A: 10, 33, 100, 150, 175, 200, 210, 220, 230, 240, 250 and 260 µg/mL. The dose levels of 100 to 210 μg/mL and 220 to 240 µg/mL showed similar cytotoxicity. Therefore, the dose levels of 175 and 230 µg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 250 and 260 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 400 to 425 μg/mL showed similar cytotoxicity. Therefore, the dose level of 425 µg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 450 to 500 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:

Without S9-mix: 10, 33, 100, 150, 200, 210, 220 and 240 μg/mL exposure medium.

With S9-mix: 10, 30, 100, 300, 325, 350, 375 and 400 μg/mL exposure medium.

In the absence of S9-mix (Table 3), the relative total growth of the highest test substance concentration of 240 µg/mL was reduced by 65% and the dose level of 220 µg/mL was reduced by 74 % compared to the total growth of the solvent controls.In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 90% compared to the total growth of the solvent controls.

Evaluation of the mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

1.2. Second mutagenicity test

To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period and in the presence of 12 % (v/v) S9-mix with a 3 hour treatment period.

Evaluation of toxicity

In the absence of S9-mix, the dose levels of 1 to 75 μg/mL showed no cytotoxicity. Therefore, the dose levels of 1 and 10 µg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 265 to 335 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

In the presence of S9-mix, the dose levels of 300 to 365 μg/mL showed similar cell growth delay. Therefore, the dose level of 365 µg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 425 to 475 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were:

Without S9-mix: 3, 30, 75, 100, 135, 165, 200 and 235 µg/mL exposure medium.

With S9-mix: 10, 30, 100, 200, 300, 350, 385 and 400 μg/mL exposure medium.

In the absence of S9-mix (Table 4), the relative total growth of the highest test substance was reduced by 92 % compared to the total growth of the solvent controls.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 70 % compared to the total growth of the solvent controls (see protocol deviation 3).

Evaluation of mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Conclusions:

In conclusion, the test substance is considered to be non-mutagenic in the in vitro mammalian cell gene mutation test (OECD 476, nowadays OECD 490) in the presence and absence of mammalian metabolic activation.

Executive summary:

In a mammalian cell gene mutation assay (thymidine kinase (TK) locus), L5178Y mouse lymphoma cells cultured in vitro were exposed to 2-ethylhexyl-S-lactate (purity 99%) at concentrations up to 500 and 335 µg/mL in the presence and absence of mammalian metabolic activation.

The test substance was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background. Based on the results, it can be concluded

that 2-ethylhexyl (S)-lactate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

This study is classified as acceptable. This study satifies the requirement for test guideline OECD 476 (nowadays OECD 490) for in vitro mutagenicity (mammalian forward gene mutation).

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Due to the rapid, enzymatically catalyzed, hydrolysis of Propyl (S)-lactate into propanol and L-lactic acid, the toxicology of Propyl (S)-lactate can be understood in terms of the toxicology of the metabolites. Lactic acid is a ubiquitous and integral part of mammalian metabolism and therefore of minor toxicological relevance in comparison to propanol which is, as an alcohol, more important for the toxicological assessment. No data is available for the target substance, except for a bacterial reverse mutation test (OECD 471), in which Propyl (S)-lactate was tested negative. Thus, data from the suitable read-across partners lactic acid, propanol and from the structurally related ethyl (S)-lactate and ethylhexyl (S)-lactate were used in a weight-of-evidence approach to assess the genotoxic potential of the target substance. All read-across partners were tested as not genotoxic. L-lactic acid, ethyl (S)-lactate and ethylhexyl (S)-lactate were tested negative each in two in vitro genotoxicity tests conducted according to OECD 473 and OECD 476 (nowadays OECD 490). Propanol was tested negative in an in vitro mammalian cell micronucleus test.

Based on the available data from suitable read-across partners, the target substance can be considered to be non-genotoxic.

Justification for classification or non-classification

Based on the available results, the target substance Propyl (S)-lactate is not considered to be genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.