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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Jun - 28 Jun 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
GLP - Guideline study with acceptable restrictions. No S. typhimurium TA102 or E. coli strain was included.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983 (2)
Deviations:
yes
Remarks:
no S. typhimurium TA102 or E. coli strain was included
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted March 2, 1983
Deviations:
yes
Remarks:
no S. typhimurium TA102 or E. coli strain was included
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
First and second experiment: 8, 40, 200, 1000 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Suspension medium Tween 80/bidest. water
- Justification for choice of solvent/vehicle: According to the author, the suspension medium was chosen based on the solubility properties preliminary tested before start of the study.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tween 80/bidest. water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (2 µg/plate, ±S9, TA1535 and TA100); 9-Aminoacridine (80 µg/plate,±S9, TA1537); 4-Nitro-o-phenylendiamine (40 µg/plate, ±S9,TA98 and TA1538); 2 Amino-anthracene (2.5 or 5 µg/plate, ±S9, TA1535 and TA1357; TA100, TA1538 and TA98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments
Evaluation criteria:
The test material may be considered positive in this test system if a combination of the following criteria are met:
- the plate background of non-reverted bacteria does not show any growth reduction versus the respective negative controls.
- the spontaneous mutation rates of each tester strain per plate are within the characteristic spontaneous mutation range.
- the positive controls show mutation rates exceeding the control values of TA100 at least two fold and those of the other strains at least by the factor 3.
- at more than one dose tested, at least a two-fold (or more) increase in comparison with the negative controls in the tester strain TA100. For the other strains, an increase in the mutation rate of more than 3 above the corresponding negative controls.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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
- Other confounding effects: Contamination of 1 plate of strain TA98 in the second experiment (1000 µg/plate), therefore only 2 individual values were used for calculation of the mean number of revertant colonies at this concentration.

COMPARISON WITH HISTORICAL CONTROL DATA:
- see Table 1 and Table 2 under "Any other information on results incl. tables".
Remarks on result:
other: all strains/cell types tested

COMPARISON WITH HISTORICAL DATA

Table 1. Characteristic spontaneous mutation range of the test batches without S9-mix.

Tester strains

TA1535

TA100

TA1537

TA1538

TA98

Historical laboratory values (Mean values)

Extreme values

 

6

1-25

 

87

35-201

 

7

1-24

 

15

6-27

 

20

5-39

Ames et al.*

(Mean values)

Extreme values

 

20

5-50

 

160

60-220

 

7

3-25

 

25

5-40

 

40

15-75

 *Ames, BN, McCann, J, Yamasaki, E 1977, ´Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test.`in Kilbey et al. Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 1 -17.

Table 2. Characteristic spontaneous mutation range of the test batches containing S9-mix.

Tester strains

TA1535

TA100

TA1537

TA1538

TA98

Historical laboratory values (Mean values)

Extreme values

 

8

1-25

 

105

54-252

 

6

1-23

 

18

3-48

 

27

7-76

STUDY RESULTS

Table 3. Test results of experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

TA1537

TA1538

TA98

Negative control

9.7 ± 3.1

97.7 ± 2.1

6.3 ± 3.2

6.7 ± 1.2

46.7 ± 3.2

Solvent control

7.7 ± 3.5

94.3 ± 17.4

8.0 ± 1.7

7.3 ± 2.3

46.3 ± 6.0

8

8.3 ± 2.1

70.0 ± 7.8

9.0 ± 1.0

6.7 ± 1.2

33.7 ± 0.6

40

11.0 ± 2.7

74.3 ± 3.2

7.3 ± 2.1

5.3 ± 0.6

40.3 ± 4.9

200

12.0 ± 4.6

67.3 ± 11.2

6.3 ± 1.2

5.7 ± 1.2

37.3 ± 3.2

1000

10.0 ± 4.0

62.7 ± 10.8

10.3 ± 1.2

4.3 ± 1.5

42.7 ± 3.1

5000

9.0 ± 1.7

83.0 ± 6.9

5.7 ± 1.5

4.3 ± 0.6

48.3 ± 3.5

Positive

controls,

–S9

Name

SA

SA

9AA

4NPD

4NPD

Concentrations

(μg/plate)

2

2

80

40

40

Mean No. of colonies/plate

(average of 3 ± SD)

744.7 ± 49.3

894.0 ± 62.0

939.7 ± 181.4

1541.0 ± 96.0

1410.7 ± 154.4

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

5

2.5

5

5

Mean No. of colonies/plate

(average of 3 ± SD)

9.0 ± 1.7

88.7 ± 17.7

7.0 ± 2.7

10.3 ± 1.5

46.0 ± 8.7

+

Negative control

5.7 ± 0.6

80.3 ± 15.3

8.7 ± 2.9

11.3 ± 2.3

53.3 ± 3.2

+

Solvent control

9.7 ± 4.6

80.3 ± 14.4

9.3 ± 1.2

13.7 ± 2.3

44.3 ± 6.7

+

8

12.3 ± 3.1

82.3 ± 12.1

7.7 ± 3.5

14.7 ± 2.9

49.7 ± 4.2

+

40

11.3 ± 4.6

92.0 ± 11.8

6.3 ± 0.6

13.0 ± 4.2

47.7 ± 4.0

+

200

8.0 ± 2.0

84.3 ± 4.9

8.0 ± 1.0

16.3 ± 1.5

53.3 ± 4.0

+

1000

10.0 ± 2.0

70.7 ± 9.7

9.3 ± 3.8

13.0 ± 1.0

50.5 ± 3.5C

+

5000

10.0 ± 3.6

74.7 ± 11.0

7.0 ± 1.7

13.0 ± 3.0

54.0 ± 7.0

Positive controls, +S9

Name

SA

SA

9AA

4NPD

4NPD

Concentrations

(μg/plate)

2

2

80

40

40

Mean No. of colonies/plate

(average of 3 ± SD)

213.3 ± 19.8

230.0 ± 4.6

283.3 ± 27.8

1434.0 ± 68.9

1307.6 ± 68.3

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

5

2.5

5

5

Mean No. of colonies/plate

(average of 3 ± SD)

186.3 ± 19.4

1328.3 ± 166.5

52.3 ± 1.5

1711.3 ± 25.0

1587.7 ± 237.7

SA = sodium azide

4NPD = 4-nitro-o-phenylendiamine

9AA = 9-aminoacridine

2AA = 2-Amino-anthracene

C = Contamination, only 2 individual values

Table 4. Test results of experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA1535

TA100

TA1537

TA1538

TA98

Negative control

13.3 ± 1.5

117.7 ± 10.0

7.7 ± 2.9

9.0 ± 4.4

33.0 ± 1.0

Solvent control

10.3 ± 5.8

111.7 ± 5.0

7.3 ± 0.6

9.3 ± 1.5

30.0 ± 6.3

8

11.3 ± 2.3

118.3 ± 12.5

7.3 ± 1.5

13.0 ± 2.0

32.0 ± 12.5

40

14.0 ± 2.7

105.0 ± 17.4

9.3 ± 0.6

9.3 ± 0.6

25.0 ± 6.2

200

13.0 ± 1.0

97.7 ± 2.9

8.0 ± 1.7

7.7 ± 2.1

32.0 ± 6.6

1000

13.3 ± 2.1

111.7 ± 9.0

9.7 ± 4.0M

8.7 ± 4.0

41.3 ± 4.2

5000

12.0 ± 1.0

125.3 ± 4.9

7.3 ± 2.1

11.0 ± 4.6

30.7 ± 2.5

Positive

controls,

–S9

Name

SA

SA

9AA

4NPD

4NPD

Concentrations

(μg/plate)

2

2

80

40

40

Mean No. of colonies/plate

(average of 3 ± SD)

807.3 ±10.6

945.3 ± 37.5

1156.0 ± 216.5

2159.3 ± 106.6

226.7 ± 24.7

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

5

2.5

5

5

Mean No. of colonies/plate

(average of 3 ± SD)

12.3 ± 5.5

110.3 ± 13.1

9.3 ± 3.8

22.0 ± 6.1

29.3 ± 3.8

+

Negative control

12.3 ± 5.8

121.7 ± 10.3

9.3 ± 2.3

14.0 ± 7.2

39.7 ± 3.1

+

Solvent control

12.3 ± 3.1

120.3 ± 17.0

8.7 ± 1.2

11.3 ± 1.5

45.0 ± 1.0

+

8

15.0 ± 3.5

122.3 ± 12.7

6.7 ± 2.5

9.7 ± 1.2

40.1 ± 2.3

+

40

13.3 ± 2.3

123.0 ± 7.6

8.7 ± 5.0

13.7 ± 6.7

41.3 ± 8.3

+

200

16.0 ± 3.0

122.3 ± 13.3

9.0 ± 3.6

9.7 ± 6.4

45.0 ± 10.2

+

1000

16.0 ± 4.6

104.0 ± 19.3

8.3 ± 1.2

12.3 ± 3.2

41.7 ± 6.4

+

5000

16.0 ± 1.7

95.0 ± 8.5

11.7 ± 3.5

12.0 ± 5.6

39.0 ± 13.1

Positive controls, +S9

Name

SA

SA

9AA

4NPD

4NPD

Concentrations

(μg/plate)

2

2

80

40

40

Mean No. of colonies/plate

(average of 3 ± SD)

232.0 ± 15.1

286.0 ± 29.2

748.0 ± 139.2

455.3 ± 14.2

531.3 ± 22.7

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

2.5

5

2.5

5

5

Mean No. of colonies/plate

(average of 3 ± SD)

131.0 ± 5.2

1735.0 ± 84.5

81.3 ± 8.4

585.3 ± 32.1

594.3 ± 56.6

SA = sodium azide

4NPD = 4-nitro-o-phenylendiamine

9AA = 9-aminoacridine

2AA = 2-Amino-anthracene

M = Manual evaluation

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Comparable to guideline study with acceptable restrictions. No discussion of results and no historical control data.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no discussion of results and no historical control data.
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no discussion of results and no historical control data.
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
MEM medium supplemented with 2 mM L-Glutamine and
- 4% (v/v) fetal calf serum (MEM4) or
- 0% (v/v) fetal calf serum (MEM0)
- 100 IU/mL penicillin/streptomycin
During exposure to the test substancewith S9 mix, MEM0 medium was used and replaced by MEM4 after 3 h after test substance administration.
Additional strain / cell type characteristics:
other: modal chromosome number of 22 and a cell cycle length of approx. 16.5 h
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
18h treatment: 10, 40 and 80 µg/mL (without metabolic activation)
18h treatment: 10, 60, 80 and 100 µg/mL (with metabolic activation)
28h treatment: 80 µg/mL (without metabolic activation)
28h treatment: 100 µg/mL (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
MEM4 medium (resp. MEM0 medium in the test with S9 mix) containing 1% (v/v) ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, 3 and 4 µg/mL in MEM0 medium, +S9; mitomycin C, 0.03 and 0.04 µg/mL in MEM4 medium, -S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18 and 28 h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h treatment: 18 h; 28 h treatment: 28 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 replications each in one (28 h treatment) or two (18 h treatment) independent experiment

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells per slide

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreduplicated cells: yes
Evaluation criteria:
The test chemical is to be considered clastogenic in this assay if:
- it induces chromosomal aberrations (excl. gaps) in a statistically significant manner in one or more concentrations
- the induced proportion of aberrant cells at such test substance concentrations exceeds the normal range of the test system
- positive results can be verified in an independent experiment.
Statistics:
The number of aberrant cells in each replica was used to establish acceptable homogeneity between replicates by means of a binomial dispersion test ( Richardson, C., Williams, D. A., Allen, J. A., Amphlett, G., Chanter, D. O. and Phillips, B. Analysis of Data from In Vitro Cytogenetic Assays, in: Statistical Evaluation of Mutagenicity Test Data, Kirkland, D. J., (ed) Cambridge University Press, Cambridge, pp. 41-64., 1990 ). The proportion of cells that was treated with the test substance and harboured structural aberrations (excl. gaps) was compared with the corresponding proportion of the negative controls in the Chi-square test. Probability values of p < 0.05 were accepted as statistically significant.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
systematic influence of the test compound which led to a reduction in the mitotic index from 10 µg/mL.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was soluble in ethanol, in MEM4 medium containing 1% ethanol the solubility limit of the test substance was determined to be 100 µg/mL (homogeneous emulsion).

- Other confounding effects: In the first test without metabolic activation the negative controls exhibited only a mitotic index of 2.0%. Therefore, test 1 without metabolic activation was completely repeated with the same doses and named test #1a.

Remarks on result:
other: all strains/cell types tested

Table 1. Summary of data obtained in experiment #1a.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in % mean

with gaps

without gaps

Exposure period 18h, without S9 mix

Control, Ethanol

1.0% (v/v)

10.7

2.0

0.0

MMC

0.03

2.1

24.6

18.5*

MMC

0.04

2.9

40.0

31.9*

Test substance

10

8.0

3.5

1.5

40

6.7

3.0

0.5

80

5.3

5.0

0.0

Exposure period 18h, with S9 mix

Control, Ethanol

1.0% (v/v)

7.0

6.5

4.0

CP

3.0

2.7

45.0

34.0*

Test substance

10

6.7

3.5

1.5

60

6.1

4.5

1.0

100

5.7

4.0

2.0

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls) 

*: significant, no statistical evaluation

Table 2. Summary of data obtained in experiment #2.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in % mean

with gaps

without gaps

Exposure period 18h, without S9 mix

Control, Ethanol

1.0% (v/v)

7.9

6.0

3.5

MMC

0.03

3.4

23.5

14.0*

Test substance

10

7.5

4.0

0.5

40

8.0

9.5

3.0

80

5.8

5.5

0.5

Exposure period 18h, with S9 mix

Control, Ethanol

1.0% (v/v)

6.3

8.0

2.0

CP

3.0

1.6

42.0

33.3*

Test substance

10

6.5

5.5

0.0

60

5.7

4.5

1.5

100

6.7

6.5

1.5

Exposure period 28h, without S9 mix

Control, Ethanol

1.0% (v/v)

7.9

3.5

0.0

MMC

0.03

4.4

43.5

32.5*

Test substance

80

5.8

4.5

0.5

Exposure period 28h, with S9 mix

Control, Ethanol

1.0% (v/v)

9.2

6.5

1.5

CP

3.0

6.0

36.5

27.5*

Test substance

100

8.4

3.5

1.0

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

 *: significant, no statistical evaluation

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Jun 2010 - 17 Aug 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
GLP-Guideline study, tested with the source substance Fatty acids, C16-18, esters with ethylene glycol(CAS 91031-31-1). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin, sodium pyruvate and L-glutamin amd 10% (v/v) heat-inactivated horse serum (24 h exposure); for 3 h exposure only 5% (v/v) heat-inactivated horse serum were included. Selective medium consisted of the basic medium and 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphtoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1:
With and without S9-mix: 0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 100.0 and 333.0 µg/mL (3 h)

Experiment 2:
In the absence of S9-mix: 3.0, 10.0, 33.0, 100.0, 125.0, 140.0 and 175.0 µg/mL for 24h
In the presence of 12% (v/v) S9-mix: 0, 0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 100.0 and 333.0 µg/mL for 3h
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, 7.5 µg/mL, with S9; methylmethanesulfonate, 15 µg/mL and 5 µg/mL without S9, for 3 h and 24 h treatment, respectively
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 h
- Expression time (cells in growth medium): 48 h

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine
- Selection time (if incubation with a selection agent): 11-12 days

NUMBER OF REPLICATIONS: 2 replications each in 5 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth, relative survival, suspension growth, relative suspension growth, growth rate

Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120%. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50E-06 and ≤ 170E-06.
c) The growth rate (R) over the 2-day expression period for the negative controls should be between 8 and 32 (3 h treatment) and between 32 and 180 (24 h treatment).
d) The mutation frequency of MMS should not be below 500E-06, and fro CP not below 700E-06.

The global evaluation factor (GEF) has been identified by the IWTG as the mean of the negative/solvent mutation frequeny (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than the MF of the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations reaches a mutation frequency of the MF of the controls + 126. b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility and precipitation: The test substance precipitated in the exposure medium at concentrations of 100 µg/mL and above. The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range ending test was 333 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, the relative suspension growth was 66% at the test substance concentration of 333 µg/mL compared to the relative suspension growth of the solvent control. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent treated control cultures were within the ranges of the historical controls.
Remarks on result:
other: strain/cell type:

Table 1: Cytotoxic and mutagenic responses

Treatment

Concentration [µg/mL]

Cloning efficiency [%]

Relative total growth [%]

Mutation frequency x 10-6

 

total

small colonies

large colonies

3 h treatment without S9-mix

Solvent control (mean)

--

86

100

61

42

19

Test substance

0.1

101

114

56

29

25

0.3

101

108

52

28

23

1.0

118

140

66

48

16

3.0

91

109

63

46

15

10.0

95

108

66

32

33

33.0

97

104

76

46

28

100.0*

91

97

81

32

46

333.0*

102

77

64

48

14

MMS

15

60

45

685

521

118

3 h treatment with 8% (v/v) S9-mix

Solvent control (mean)

--

91

100

67

36

29

Test substance

0.1

108

111

74

47

25

0.3

89

94

71

45

24

1.0

108

113

64

42

20

3.0

98

107

78

53

23

10.0

95

100

87

54

29

33.0

108

96

55

32

22

100.0*

98

89

83

60

21

333.0*

94

92

63

23

39

CP

7.5

60

32

1074

829

144

24 h treatment without S9-mix

Solvent control (mean)

--

115

100

51

26

23

Test substance

3

135

92

58

42

14

10

137

109

51

39

11

33

133

85

71

32

35

100*

110

30

100

35

60

125*

118

31

70

31

36

140*

118

20

103

51

47

175*

102

9

134

53

72

MMS

5

101

73

865

463

233

3 h treatment with 12% (v/v) S9-mix

Solvent control (mean)

--

109

100

72

47

23

Test substance

0.1

110

100

73

49

21

0.3

123

117

72

47

23

1.0

104

105

82

52

27

3.0

97

104

94

62

29

10.0

115

126

87

57

26

33.0

107

108

85

59

23

100.0*

131

123

80

51

26

333.0*

97

83

92

64

24

CP

7.5

70

59

979

621

221

*precipitation of test substance in the exposure medium

MMS = methylmethanesulfonate

CP = cyclophosphamide

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for read-across

There are no data for genetic toxicity available for Propyleneglycol dioleate. In accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 read-across from appropriate substances is conducted to fulfill the standard information requirements set out in Regulation (EC) No 1907/2006, Annex VIII, 8.4.

According to Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met”. In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”. 

Propyleneglycol dioleate represents an UVCB substance comprised of diesters of 1,2-propyleneglycol chemically linked to mainly oleic acid (C18:1) and/or palmitic and/or stearic acid (C16, C16:1).Gylcol esters are in general known to bestepwise hydrolysed by gastrointestinal enzymes into the free fatty acid component and the respective alcohol (Long, 1958; Lehninger, 1970; Mattson and Volpenhein, 1972).

Based on thecommon metabolic fate of glycol esters, the read-across approach is based on the presence ofcommon functional groups, common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals,common functional groups, structural similarities and similar physico-chemical, toxicological and toxicokinetic behaviour. For further details on the read-across approach, please refer to the analogue justification in section 13 of the technical dossier.

 

As no data are available on mutagenic properties of Propyleneglycol dioleate, read-across to reliable data on the analogue substances Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol (CAS 84988-75-0), C8-C10-1,3-Butandiolester (CAS 853947-59-8) and Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1) was conducted.

 

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 84988-75-0

A study investigating the genetic toxicity in vitro of Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol is available. The study was conducted according to OECD guideline 471 under GLP conditions (Banduhn, 1991). In two independent experiments, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were tested according to the plate incorporation procedure. Concentrations from 8 to 5000 µg/plate were investigated with and without a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results and were therefore considered as valid. Thus, under the conditions of this study, the test substance did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

 

CAS 853947-59-8

An in vitro mammalian chromosome aberration test was conducted with C8-C10-1,3-Butandiolester in accordance with OECD guideline 473 under GLP conditions (Dechert, 1997). The induction of structural chromosome aberrations was evaluated in vitro in Chinese hamster lung fibroblasts (V79 cells), incubated for 18 and 28 h with and without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 10-100 µg/mL (18 h incubation) and 80 and 100 µg/mL (28 h incubation) of the test substance in the vehicle ethanol were applied. The solubility limit of the test substance in the vehicle ethanol in the culture medium was determined to be 100 µg/mL. In the first experiment without metabolic activation, the negative controls exhibited a mitotic index of 2.0% only and the experiment was therefore repeated. Thereafter, the negative as well as the positive controls showed the expected results and were within the range of historical control data. The frequency of polyploidy cells with and without metabolic activation was within the expected range (< 10%). In the experiments both with and without metabolic activation, a systematic influence of the test substance was observed, which led to a reduction in the mitotic index. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed.

Therefore, under the conditions of the study, C8-C10-1,3-Butandiolester did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in Chinese hamster lung fibroblasts in vitro.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

 

CAS 91031-31-1

Mutagenic properties of Fatty acids, C16-18, esters with ethylene glycol were characterized in an in vitro mammalian cell gene mutation study according to OECD guideline 476 under GLP conditions (Verspeek-Rip, 2010). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/β-naphtoflavone-induced rat liver S9). In the first experiment, cells were exposed for 3 h to the test substance at concentrations of 0.1-333 µg/mL (in DMSO) with and without metabolic activation. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 3-175 µg/mL and with metabolic activation (3 h; 12% S9-mix) from 0.1-333 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data. No cytotoxicity was observed up to the precipitating concentration of 100 µg/mL and up to 333 µg/mL, respectively. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, Fatty acids, C16-18, esters with ethylene glycol did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

 

Conclusion on genetic toxicity

The available data on read-across analogue substances do not provide evidence that Fatty acids, C14-18 and C16-18-unsatd., esters with propylene glycol (CAS 84988-75-0), C8-C10-1,3-Butandiolester (CAS 853947-59-8) or Fatty acids, C16-18, esters with ethylene glycol (CAS 91031-31-1) exhibit mutagenic or clastogenic properties in either bacteria or mammalian cells. Therefore, no properties for genetic toxicity are expected for Propyleneglycol dioleate.

 

References

Agency for Toxic Substances and Disease Registry (ATSDR) (1997). Toxicological Profile for Propylene Glycol. US Department of Health and Human Services. Atlanta, US.

 

Agency for Toxic Substances and Disease Registry (ATSDR) (2010). Toxicological Profile for Ethylene Glycol. US Department of Health and Human Services. Atlanta, US.

Lehninger, A.L. (1970). Biochemistry. Worth Publishers, Inc.Long, C.L. et al. (1958). Studies on absorption and metabolism of propylene glycol distearate. Arch Biochem Biophys, 77(2):428-439.

Mattson, F.H. and Volpenhein, R.A. (1972). Hydrolysis of fully esterified alcohols containing

from one to eight hydroxyl groups by the lipolytic enzymes of the rat pancreatic juice. Journal

of Lipid Research 13: 325-328

 

Miller, O.N., Bazzano, G. (1965).Propanediol metabolism and its relation to lactic acid -metabolism. Annals of the New York Academy of Sciences 119:957-973.

 

Ritchie, A.D. (1927). Lactic acid in fish and crustacean muscle. Journal of Experimental Biology 4:327-332.


Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from structural analogues. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details). No study was selected, since all available in vitro genetic toxicity studies were negative.

Short description of key information:
Genetic toxicity in vitro:
Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Chromosome aberration (OECD 473): negative with and without metabolic activation in Chinese hamster lung fibroblasts (V79) cells
Gene mutation in mammalian cells (OECD 476): CAS 91031-31-1: negative with and without metabolic activation in L5178Y mouse lymphoma cells

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the analogue read-across approach, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.