(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

Genotoxicity studies of lactid-acid were used as read-across for L-lactide. Lactic acid was tested as not genotoxic in three in vitro OECD guideline tests (OECD 471, OECD 473 and OECD 476) and by supporting in vitro studies.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

2014-01-17 to 2014-05-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study in accordance with OECD guideline 473. Lactic acid used as read-across partner to L-lactide

Qualifier:

according to

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to

Guideline:

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

dose range finding assay: 0, 10, 33, 100, 333, 901 µg/ml (equal to concentrations of 0, 0.1, 0.4, 1.1, 3.7 and 10 mM)
1 experiment: 0, 10, 100, 901 µg/ml (equal to concentrations of 0, 0.1, 1.1 and 10 mM)
2 experiment: 0, 100, 333, 666, 901 µg/ml (equal to concentrations of 0, 1.1, 3.7, 7.4 and 10 mM)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

solvent: RPMI 1640

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

solvent for positive controls: Hank´s Balanced Salt Solution (HBSS)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 ± 2 hours
- Exposure duration: experiment 1: 3 hours (with and without metabolic activation); experiment 2: 24 and 48 hours without metabolic activation and 3 hours with metabolic activation
- Expression time (cells in growth medium): experiment 1: 20 to 22 hours; experiment 2: 44 to 46 hours
- Fixation time (start of exposure up to fixation or harvest of cells): experiment 1: 24 hours; experiment 2: 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µg/ml medium during the last 2.5 to 3 hours of the culture period
STAIN (for cytogenetic assays): 5 % (v/v) Giemsa for 10 to 30 minutes

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index from at least 1000 cells

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
None of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

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

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:

(N-1) (ad-bc)^2
X^2 = ------------------------
(a+b) (c+d) (a+c) (b+d)

Where:
a = the total number of aberrant cells in treated cultures to be compared with the control.
b = the total number of aberrant cells in the control cultures.
c = the total number of non aberrant cells in treated cultures to be compared with the control.
d = the total number of non aberrant cells in the control cultures.
N = sum of n0 and n1.
n0 = the total number of cells scored in the control cultures.
n1 = the total number of cells scored in the treated cultures.

(N-1) (ad-bc)^2
If P X^2 > ----------------------- (one-tailed) is small (p< 0.05) the hypothesis that the
(a+b) (c+d) (a+c) (b+d)

incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.

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.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

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:

Interpretation of results (migrated information):
negative

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 cytogenetics assay peripheral human lymphocyte cultures were exposed to L(+)-lactic acid (90%), solved 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 cofactors.

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 hour. 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

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 (gene mutation).

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

Justification for selection of genetic toxicity endpoint
An in vitro testing battery under GLP (OECD 471, OECD 476, OECD 473) has been conducted with a lactic acid, as degradation product of L-lactide.

Justification for classification or non-classification

Genotoxicity of the read-across partner L(+)-lactic acid was assessed in a standard in vitro testing battery under GLP. The substance was not genotoxic in any of the test systems. Therefore, based on the available data no classification of L-lactide is warranted.