(3S-cis)-3,6-dimethyl-1,4-dioxane-2,5-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For the assessment of the potential to induce genetic toxicity no studies are available for the target substance, L-lactide. Therefore, data from suitable read-across partners, L(+)-lactic acid and lactic acid was used to assess genetic toxicity of the target substance. For details and justification of read-across please refer to the report attached in section 13 of IUCLID.

Data from an in vitro genetox testing battery conducted in accordance with OECD guidelines with the suitable read-across partner L(+)-lactic acid was used in a weight of evidence approach. L(+)-lactic acid was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471, in an in vitro chromosome aberration assay conducted according to OECD 473 and in a mammalian cell gene mutation assay conducted according to OECD 476 (nowadays OECD 490). In supporting studies, the second source substance lactic acid showed no potential to induce any mutagenic or clastogenic effect. Based on results, L-lactide is considered to not induce genetic toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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

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: No

RANGE-FINDING/SCREENING STUDIES: Dose range finding test performed with TA100 and WP2uvrA. Doses tested: 0, 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate. Based on the results, the following doses were selected for the main experiment with TA1535, TA1537 and TA98 in the presence and absence of S9-mix: 100, 333, 1000, 330 and 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative control values were within the laboratory historical control data ranges, except for TA100 in the absence of S9-mix, second experiment. Evaluation: The mean plate count (146) was just outside the limit of the range (144) and clear negative results are observed in all experiments. Therefore, this deviation in the mean plate count of the solvent control had no effect on the results of the study. The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1535 in the absence of S9-mix, first experiment. Evaluation: The value (257) was just below the limit of the range (262). The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

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

MUTAGENICITY:
Experiment 1: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
Experiment 2: Based on the results from the first experiment, the test item was tested up to 5000 µg/plate. No increase in the number of revertants was observed.

Conclusions:

In conclusion, L(+)-lactic acid 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 conducting according to OECD guideline 471, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2uvrA were exposed to L(+)-lactic acid (90% purity) at concentrations of 0, 100, 333, 1000, 3330 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

L(+)-lactic acid 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).

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:

Table 1: 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
48 h exposure time, 48 h 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 cytegenetic assay

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)	34-36	1009-1028	100
10	37-35	1002-1012	103
100	35-28	1008-1033	90
901	15-9	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)	33-48	1040-1028	100
10	37-27	1013-1007	79
100	47-35	1035-1001	101
901	43-32	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 metaphases a)   Absolute	Number of metaphases a)   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 using human lymphocytes, with and without metabolic activation.

Executive summary:

In a mammalian cell cytogenetic assay conducted according to OECD guideline 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 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 details and 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 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 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 hydrolysis of lactide into lactoyl lactic acid and subsequently lactic acid, under acid and neutral conditions, the toxicology of lactides can be understood in terms of the toxicology of lactic acid. As no data was available for L-lactide, genotoxicity studies of lactic acid were used for read-across. L(+)-lactic acid was tested in three independent test system as not genotoxic, which were performed in accordance to the OECD guidelines 471 (Ames test), 473 (chromosome aberration) and 476 (nowadays OECD 490, gene mutation).

Based on the available data of the read-across partner lactic acid, L-lactide is considered to be non-genotoxic.

Justification for classification or non-classification

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