Propanoic acid, 2-hydroxy-, ammonium salt (1:1), (2S)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ammonium-S-lactate dissociates into lactic acid/lactate and ammonium ions. Therefore, the genotoxic potential of ammonium-S-lactate is assessed in a weight-of-evidence approach based on several in vitro studies with the target substance itself, various dissociable ammonium compounds, and lactic acid.

In conclusion, these studies are considered negative with respect to genotoxicity, except for one cytogenicity assay on ammonium chloride. In combination with an in vivo micronucleus test on ammonium chloride, it is concluded that ammonium-S-lactate is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2005-07-15 to 2005-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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): Ammonium Lactate, PURASAL® NH
- Physical state: not reported
- Analytical purity: not reported
- Lot/batch No.: 0503002615
- Expiration date of the lot/batch: 2007-09-28
- Storage condition of test material: room temperature (20 ± 5 °C) in the dark
- Other: Reception date: 2005-04-01

Target gene:

S. typhimurium: Histidine locus
E. coli: Tryptophan locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary test: 100, 50, 25, 12.5, 6.25 and 3.125 µL/plate (with and without metabolic activation)
Main test: 50, 16.67, 5.56, 1.85, 0.62 and 0.21 µL/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
The solvent used for the test item was distilled water. No solubility tests were done because the sponsor defined the test item as completely soluble in water. The maximum concentration of 100 µL/plate, i.e. undiluted substance, was used for the performance of the preliminary test. The subsequent concentrations were prepared by 1/2 dilutions with sterile distilled water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, 1 µg/plate, all strains used

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, 1 µg/plate, TA 1535 and TA 100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, 50 µg/plate, TA 1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9, 1 µg/plate, E. coli WP2

Details on test system and experimental conditions:

PREINCUBATION METHOD:
The amount of 0.1 mL of the test item/test solvent was preincubated with 0.1 mL of the test strain culture and 0.5 mL of sterile buffer or metabolic activation system for 20 minutes at 37 ± 1 °C prior to mixing with 2.0 mL of the overlay agar and pouring onto the surface of a minimal agar plate. During pre-incubation the tubes were aired using a shaker.
This overlay melted at 45 ± 1 °C contained a sterile solution of 0.5 mM L-histidine/0.5 mM biotin in the proportion of 1:10 for Salmonella typhimurium and 0.25 mL of a sterile solution of tryptophan and 5 mL of nutrient broth for every 100 mL of top agar for Escherichia coli. The metabolising enzymes in the S-9 mixture are not stable at 45 °C and so the contents of the test tubes were mixed rapidly in a rotamixer and poured into plates containing a base layer of minimal agar. The added agar was allowed to settle down and the plates were incubated at 37 ± 1 °C for 48-72 hours. After this incubation period, the number of revertant colonies that had grown on each plate was counted. The experiment was repeated on another day using fresh bacterial cultures.

Evaluation criteria:

According to OECD 471

Statistics:

The comparisons between the results for the standard products and the control were made using Student's t-test. The statistical comparison of the different test-item concentrations was carried out, for all the bacterial strains, both with and without metabolic activation, using a one-way analysis of variance with a level of significance of p < 0.05.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: see box "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The maximum concentration of 100 µL/plate, i.e. undiluted substance, was used for the performance of the preliminary test. At the concentration of 100 µL/plate a slight decrease was observed in the number of revertants in strain TA-100 without S-9. In accordance with the OECD, which suggests 5 µL/plate as the high concentration for liquids, lower concentration were tested in the main test. 50 µL/plate was taken as the maximum concentration and 1/3 dilutions were prepared.

RESULTS (Experiment 1):
Positive controls: All the bacterial strains responded positively.
Negative controls: Normal values (within the range of historical data for negative controls) were obtained in the revertant colony counts on all the dishes treated only with the solvent.
Test item: The test item was toxic for TA-100 with and without S-9 and for TA-1535 with S-9 at the concentration of 50 µL/plate. No mutagenic response was observed in any of the tested strains, with or without S-9.

RESULTS (Experiment 2):
Positive controls: All the bacterial strains responded positively.
Negative controls: Normal values (within the range of historical data for negative controls) were obtained in the revertant colony counts on all the dishes treated only with the solvent. The mean of the control for the strain TA-1537 without S-9 fell slightly above the upper limit established by historical data although it fell within the range given in literature.
Test item: The test item was toxic for TA-100 without S-9 and for TA-1535 with S-9 at the concentration of 50 µL/plate.
No mutagenic response was observed in any of the tested strains, either with or without S-9.

Table 2: Summary of the results obtained with the test item (experiment 1)
		Mean revertants per plate (µL/plate)
Test strain		% S-9	Control	0.21	0.62	1.85	5.56	16.67	50
TA 1535		0.0	21.7	20.3	21.7	20.7	20.3	22.3	21.0
TA 1537		0.0	19.7	19.0	19.0	19.0	18.7	20.7	19.3
TA 98		0.0	31.0	31.0	31.0	30.3	30.7	31.7	32.0
TA 100		0.0	133.3	132.3	132.7	131.0	133.0	132.7	-
E. coli WP2		0.0	143.3	143.0	142.3	141.3	142.3	141.0	141.7
TA 1535		10.0	21.0	20.0	20.7	19.0	20.7	20.7	-
TA 1537		10.0	20.3	19.0	19.3	18.7	20.7	19.7	19.7
TA 98		10.0	35.7	36.3	36.7	35.3	35.7	36.0	37.3
TA 100		10.0	120.7	121.3	119.3	120.7	119.7	120.0	-
E. coli WP2		10.0	143.0	141.7	142.3	142.0	142.0	141.3	142.0

Table 3: Summary of the results obtained with the test item (experiment 2)
		Mean revertants per plate (µL/plate)
Test strain		% S-9	Control	0.21	0.62	1.85	5.56	16.67	50
TA 1535		0	25.0	24.3	22.3	25.3	23.3	22.0	23.3
TA 1537		0	24.0	22.3	25.0	22.3	25.0	23.3	22.7
TA 98		0	29.7	30.0	32.0	29.0	28.7	28.7	28.7
TA 100		0	140.3	140.7	140.3	139.0	139.7	141.0	-
E. coli WP2		0	140.0	139.7	140.3	140.0	141.0	141.0	140.0
TA 1535		10	22.7	22.7	21.0	21.3	20.7	22.0	-
TA 1537		10	25.7	25.7	25.3	26.0	26.7	26.3	25.3
TA 98		10	32.3	34.3	33.7	34.3	33.7	32.7	33.7
TA 100		10	141.0	142.0	140.3	140.3	140.3	141.3	141.3
E. coli WP2		10	141.7	141.3	140.7	140.3	141.7	140.7	141.3

Conclusions:

The test item showed no evidence of mutagenic activity in a bacterial reverse mutation assay (OECD 471).

Executive summary:

In a bacterial reverse mutation assay (according to OECD 471) Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one strain of Escherichia coli (WP2 uvrA) were exposed to the test item at concentrations of 0, 0.21, 0.62, 1.85, 5.56, 16.67 and 50 µL per plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies above background.

This study is classified as acceptable. This study satisfies the requirement of OECD test guideline 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

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

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

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 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.

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 5:

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.

Conclusions:

In conclusion, L(+)-lactic acid is considered not clastogenic in the in vitro mammalian chromosomal aberration test, with and without metabolic activation.

Executive summary:

In a mammalian cell cytogeneticity assay conducted according to OECD guideline 473, peripheral human lymphocyte cultures were exposed to L(+)-lactic acid (90% purity), dissolved in RPMI 1640 cell culture medium. In the first and second experiment the doses were 0, 10, 100, 901 µg/mL for 3 hours with and without metabolic activation. In the second experiment additional treatment to doses of 0, 100, 333, 666 and 901 µg/mL was carried out for 24 and 48 hours exposure time. S9 was derived from phenobarbital plus ß-naphtoflavone treated rats and supplemented with co-factors.

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 59%. 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 response. 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 justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reference 3

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 report attached in section 13 of IUCLID.

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

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 8.0E+06 cells (1.0E+06 cells/ml for 3 hours treatment) or 5 x 106 cells (1.25E+05 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 1.25E+05 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.

Conclusions:

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

Executive summary:

In a mammalian cell gene mutation assay conducted in accordance with OECD guidline 476 (nowadays OECD 490), L5178Y mouse lymphoma cells cultured in vitro were exposed to L(+)-lactic acid (90% purity), dissolved in RPMI 1640 medium at concentrations of 0.54, 1.7, 5.4, 17, 52, 164, 512 and 901 µg/mL in the presence and absence of mammalian metabolic activation. Metabolic activation was only performed in experiment 1 with a treatment duration of 3 hours, but not in experiment 2 with a treatment duration of 24 hours.

L(+)-lactic acid was tested to the maximum concentration of 0.01 M, equivalent to 901 µg/mL. The induced mutation frequency with and without metabolic activation was not increasedcompared to control in all tested concentrations. The positive controls did induce the appropriate response. The spontaneous mutation frequencies in the solvent-treated controls were within the historical control data ranges.

This study is classified as acceptable. This study satisfies the requirements of the OECD test guideline 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 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

Description of key information

No in vivo genotoxicity study is available for the target substance. As the target substance dissociates into lactic acid and the ammonium ion it is therefore acceptable to use available data from the read-across partner ammonium chloride. Ammonium chloride was tested negative in an in vivo micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

see Table 1 (box "any other information on results incl. tables")

Table 1: Summary of the results

Compound

Vehicle

Route

Number of doses

Time between doses (hr)

Sampling time (hr)

Dose level (mg/kg bw)

MNPCE (%)

PCE (%)

Mortality

Single dosing

Ammonium chloride

Saline

ip

1

24

0

0.18 ± 0.18

56.8 ± 4.7

0/6

62.5

0.12 ± 0.12

60.9 ± 4.2

0/6

125

0.15 ± 0.14

61.7 ± 3.8

0/6

250

0.13 ± 0.05

64.3 ± 2.5

0/6

500

0.12 ± 0.08

56.9 ± 6.1

0/6

Mitomycin C

ip

1

24

2.0

4.18 ± 1.30*

52.3 ± 4.6

0/6

Repeated dosing

Ammonium chloride

Saline

ip

4

24

24

0

0.20 ± 0.09

59.9 ± 8.3

0/6

31.3

0.25 ± 0.19

67.2 ± 13.5

0/6

62.5

0.17 ± 0.10

63.7 ± 4.5

0/6

125

0.20 ± 0.18

64.0 ± 9.2

0/6

250

0.17 ± 0.08

61.6 ± 6.9

0/6

Mitomycin C

ip

1

24

2.0

7.15 ± 3.92*

32.2 ± 11.0

0/6

*= statistically significant (p< 0.01)

Conclusions:

Under the reported experimental conditions Ammonium chloride did not induce structural and/or numerical chromosomal damage in the bone marrow cells of the mouse.

Executive summary:

In a ddY mouse bone marrow micronucleus test conducted similar to OECD guideline 474, six male mice per dose were treated intraperitoneally with Ammonium chloride (99.7% purity) at doses of 0, 62.5, 125, 250 and 500 mg/kg bw (experiment 1) and at doses of 0, 31.3, 62.5, 125 and 250 mg/kg bw (experiment 2). The animals were injected intraperitoneally with the test substance once (experiment 1) or four times at 24 h intervals (experiment 2) with an additional 24 hours period after the last dosing. The vehicle was physiological saline. Ammonium chloride did not increase the level of micronuclei in comparison to the concurrent vehicle control. Thus, Ammonium chloride is considered to be non-mutagenic according to the results of the in vivo micronucleus test reported.

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)

Additional information

Ammonium-S-lactate dissociates into ammonium and lactate in aqueous media. Thus, the assessment of the genotoxic potential can be based on available data on dissociable ammonium compounds and lactic acid.

Further details on the read-across rationale are provided in IUCLID section 13. The target substance itself was tested negative in a bacterial reverse mutation test (OECD 471). The read-across partner lactic acid was tested negative in a L5178Y mouse lymphoma mutagenicity assay (OECD 490) and in an in vitro chromosome aberration assay (OECD 473). The read-across partner ammonium sulfate was tested negative in two in vitro chromosome aberration assays (Obe, 1986; Tuschy, 1988). Ammonium chloride induced chromosome aberrations in vitro (Ishidate, 1984), but was tested negative in an in vivo micronucleus test (Hayashi, 1988).

Based on available data from the target substance itself and from its suitable read-across partners, the target substance ammonium-S-lactate is considered negative for mutagenicity based on a weight-of-evidence approach.

Justification for classification or non-classification

Based on available data from the target substance itself and also from its read-across partners, the target substance ammonium-S-lactate is considered negative and no classification in accordance with CLP Regulation 1272/2008 is warranted for mutagenicity.