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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.