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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro

Gene mutation in bacteria (Ames test, OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA

Cytogenicity in mammalian cells (Micronucleus test, OECD 487): negative in primary human lymphocytes with and without metabolic activation

Gene mutation in mammalian cells (Thymidine kinase assay, OECD 490): negative in mouse lymphoma L5178Y cells with and without metabolic activation

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Sep 2017- 15 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidin (Salmonella), tryptophan (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/P-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
The test item precipitated in the overlay agar in the test tubes at 5000 μg/plate . Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within 2 hours of preparation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);
- Cell density at seeding (if applicable):

Precultures of bacteria were grown in Erlenmeyer flasks and harvested at the late exponential or early stationary phase (10.0E+08 to 10.0E+09 cells/mL).

Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension,
2000 μL Overlay agar

Experiment II (Pre-Incubation)
In the pre-incubation assay 50 μL test solution (or solvent) or 100 μL reference mutagen solution (positive control), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After preincubation 2.0 mL overlay agar ( 45°C) was added to each tube.

The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL ( experiment I) or 100 μL ( experiment II) of the stock solution,
500 μl S9 mix/ S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

SELECTION AGENT (mutation assays): Plates with selective agar (without histidine/tryptophan) were used.

NUMBER OF REPLICATIONS: Triplicates

DETERMINATION OF CYTOTOXICITY
- Method: regular background growth

OTHER EXAMINATIONS:
Regular checking of the properties of the Salmonella typhimurium and Escherichia coli strains was done regarding the membrane permeability, ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontaneous mutation rates.

- OTHER:
Experimental period: 14 September 2017 - 23 October 2017
Rationale for test conditions:
Acceptability criteria:
- regular background growth in the negative and solvent control;
- the spontaneous reversion rates in the negative and solvent control in the range of laboratory's historical data;
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5;
Evaluation criteria:
- mutagen if biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice
(strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
- dose dependent increase considered biologically relevant if the threshold was exceeded at more than one concentration;
- increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment;
- dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
Whenever the colony counts remain within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All values were within the acceptable ranges of historicol control data of the laboratory.

Revertant Colony Counts (Mean of triplicates ±standard deviation)

Experiment I

without S9

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

Vehicle (ethanol)

10±4

8±2

33±6

158±8

31±8

0

13±5

7±2

33±5

162±13

43±3

3

8±3

9±4

37±11

158±6

33±2

10

11±3

6±1

30±5

151±28

33±9

33

12±4

10±2

29±6

142±23

27±5

100

11±3

6±1

31±6

152±10

29±9

333

10±6

12±4

28±4

158±23

33±7

1000

11±0

9±3

35±2

145±10

35±5

2500

8±3

10±3

34±4

145±9

34±7

5000

8±1P

8±4P

24±4P

167±5P

42±2P

sodium azide (10 µg)

1161±66

 

 

2439±49

 

4-nitro-o-phenylene-diamine (10 µg)

 

 

846±43

 

 

4-nitro-o-phenylene-diamine (50 µg)

 

77±9

 

 

 

methyl methane sulfonate (2 µL)

 

 

 

 

966±48

P = precipitation

 

Experiment I

with S9

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

Vehicle (ethanol)

15±1

17±3

48±8

172±17

42±5

0

12±2

18±2

43±10

159±22

40±9

3

14±3

19±6

44±3

132±23

52±3

10

10±2

18±3

42±6

152±6

42±10

33

14±4

15±6

42±12

146±9

46±7

100

11±4

17±6

44±8

142±11

47±6

333

10±3

16±6

36±4

135±9

40±7

1000

12±4

12±4

47±6

137±1

48±6

2500

12±3

16±2

42±1

137±15

55±9

5000

11±2P

9±3P

48±16P

120±10P

51±8P

2-aminoanthracene (2.5 µg)

388±32

176±21

5251±462

2924±370

 

2-aminoanthracene (10 µg)

 

 

 

 

427±16

P = precipitation

 

Experiment II

without S9

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

Vehicle (ethanol)

9±3

8±3

31±6

183±11

42±1

0

13±5

9±2

25±8

196±13

45±8

33

10±1

8±3

38±6

181±21

50±3

100

12±3

10±3

29±4

168±21

48±3

333

10±4

8±3

29±8

187±23

45±4

1000

11±3

7±3

26±6

186±13

47±2

2500

12±2

8±3

42±5

193±15

43±5

5000

7±2P

9±2P

41±3P

193±14P

51±7P

sodium azide (10 µg)

1321±129

 

 

2200±65

 

4-nitro-o-phenylene-diamine (10 µg)

 

 

330±22

 

 

4-nitro-o-phenylene-diamine (50 µg)

 

157±3

 

 

 

methyl methane sulfonate (2 µL)

 

 

 

 

843±42

P = precipitation

 

Experiment II

with S9

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

Vehicle (ethanol)

18±6

13±2

48±9

172±8

61±7

0

12±3

9±2

43±15

203±23

54±3

33

15±6

12±2

45±4

160±11

62±10

100

17±4

13±5

46±5

152±6

52±4

333

16±4

9±2

39±12

171±18

53±10

1000

16±6

10±4

39±5

165±11

57±2

2500

13±3

13±5

35±12

169±13

45±7

5000

16±6P

11±2P

40±12P

163±12P

59±5P

2-aminoanthracene (2.5 µg)

499±25

138±29

4428±281

4575±143

 

2-aminoanthracene (10 µg)

 

 

 

 

524±19

P = precipitation

 

 

Conclusions:
No biologically relevant increase in revertant colony numbers of any of the five tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Nov 2017 - 02 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy non-smoking male donor (34 years old) for Experiment I and healthy non-smoking female donor (35 years old) for Experiment II
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohemagglutinine (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Cell cycle length, doubling time or proliferation index: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time).
- Whether whole blood or separated lymphocytes were used if applicable: Blood cultures were established by preparing an 11% mixture of whole blood in medium within 30 h after blood collection.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- Properly maintained: yes, all incubations were done at 37 °C with 5.5% CO2 in humidified air.
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Experiment I: 0, 7.4, 13.0, and 22.7 µg/mL
Experiment II: 0, 18.7, 32.7, 57.1 µg/mL
Phase separation occurred at the end of treatment at the top dose of each experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (0.5% in culture medium)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
All formulations were prepared freshly before treatment and used within two hours of preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine (continous treatment)
Details on test system and experimental conditions:
The induction of cytogenetic damage in human lymphocytes was assessed in two independent experiments with one preparation interval (40 hours).
Treatments started after a 48 hour stimulation period with phytohemeagglutinine (PHA) when cells were actively proliferating and the cells were prepared at approx. 2 – 2.5 fold of the normal cell cycle time.

A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI (cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay. The pre-test was performed with 11 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. This preliminary test was designated Experiment I, since the cultures fulfilled the acceptability criteria and appropriate concentrations could be selected for cytogenetic evaluation.

Experiment I
- Exposure duration: 4 hours (with and without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours after start of exposure
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were washed. The washing procedure was repeated once. The cells were resuspended in complete culture medium with 10% FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Experiment II
- Exposure duration: 20 hours (without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 hours after start of exposure
About 48 hours after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10% FBS) containing the test item. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed. The washing procedure was repeated once. After washing the cells were re-suspended in complete culture medium containing 10% FBS (v/v). Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

NUMBER OF REPLICATIONS: Two parallel cultures were analyzed.

Preparation of cells
The cultures were harvested by centrifugation 40 h after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

DETERMINATION OF CYTOTOXICITY
Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the cytokinesis block proliferation index (CBPI) was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

- OTHER:
Experimental period: 04 Oct 2017 - 21 Nov 2017
Rationale for test conditions:
Acceptability criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realized as 95% confidence interval)
− The concurrent positive controls should produce a statistically significant increase in the micronucleus frequency and should be within the laboratory historical positive control data range.
− Cell proliferation criteria in the solvent control are considered to be acceptable
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations
Evaluation criteria:
A test item was considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. narrow concentration spacing, other metabolic activation conditions, i.e. S9 concentration or S9 origin) could be useful.
However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.
Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Key result
Species / strain:
lymphocytes: human, primary
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: The cytokinesis block proliferation index (CBPI) was calculated in 500 cells per culture as following:
CBPI = ((Mononucleate cells x 1) + (Binucleated cells x 2) + (multinucleate cells x 3)) / total number of cells

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes, given in attachment
- Negative (solvent/vehicle) historical control data: yes, given in attachment

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, which showed precipitation.
In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.
In Experiment II, in the absence of S9 mix, the value of the solvent control, as well as the value after continuous treatment with 57.1 μg/mL (1.05% micronucleated cells, each), slightly exceeded the range of the 95% control limit (0.12 – 1.03% micronucleated cells), but were clearly within the min-max range (0.05 – 1.35% micronucleated cells) of the historical control data and therefore acceptable. No statistical significance was observed.
Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

The number of micronucleated cells in % was determined in a sample of 2000 bi-nucleated cells.

Table 1: Experiment I: 4 hours exposure without S9 mix, preparation interval 40 hours

 

Proliferation index CBPI

Cytostasis in %

Micronucleated cells in %

Solvent control, 0.5% ethanol

1.95

 

1.10

Mitomycin C, 0.8 µg/mL

1.71

24.9

11.55, S

Test item 7.4 µg/mL

1.93

2.1

0.55

Test item 13.0 µg/mL

1.97

nc

1.00

Tes item 22.7 µg/mL, PS

19.4

0.3

0.90

PS = Phase separation occurred at the end of treatment

nc = Not calculated as the CBPI is equal or higher than the solvent control value

S = The number of micronucleated cells is statistically significantly higher than corresponding control values

 

Table 2: Experiment II: 20 hours exposure without S9 mix, preparation interval 40 hours

 

Proliferation index CBPI

Cytostasis in %

Micronucleated cells in %

Solvent control, 0.5% ethanol

1.83

 

1.05

Demecolcine, 100 ng/mL

1.51

38.5

7.05, S

Test item 18.7 µg/mL

1.88

nc

1.00

Test item 32.7 µg/mL

1.86

nc

0.75

Tes item 57.1 µg/mL, PS

1.93

nc

1.05

PS = Phase separation occurred at the end of treatment

nc = Not calculated as the CBPI is equal or higher than the solvent control value

S = The number of micronucleated cells is statistically significantly higher than corresponding control values

Table 3: Experiment I: 4 hours exposure with S9 mix, preparation interval 40 hours

 

Proliferation index CBPI

Cytostasis in %

Micronucleated cells in %

Solvent control, 0.5% ethanol

2.01

 

1.00

Cyclophosphamide, 17.5 µg/mL

1.58

42.4

6.90, S

Test item 7.4 µg/mL

2.05

nc

1.05

Test item 13.0 µg/mL

2.09

nc

0.55

Tes item 22.7 µg/mL, PS

2.02

nc

0.65

PS = Phase separation occurred at the end of treatment

nc = Not calculated as the CBPI is equal or higher than the solvent control value

S = The number of micronucleated cells is statistically significantly higher than corresponding control values

Conclusions:
The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, when tested up to precipitating concentrations.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Sep 2017 - 01 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
doubling time 10 - 12 h in stock cultures
Details on mammalian cell type (if applicable):
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit in Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time. Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of Envigo allowing the repeated use of the same cell culture batch in experiments. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Metabolic activation:
with and without
Metabolic activation system:
Liver microsome preparations (S9 mix) from rats induced with Phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
1st Experiment: 4 hours without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125 µg/mL
1st Experiment: 4 hours with metabolic activation: 7.8, 15.6, 31.3, 62.5, 125 µg/mL
2nd Experiment: 24 hours without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125 µg/mL
The concentration range of the main experiments was limited by precipitation of the test item. Phase separation occurred at 125.0 μg/mL and above in the presence and absence of metabolic activation following 4 and 24 hours treatment. Relevant cytotoxic effects indicated by a relative total growth of less than 50% of survival in both parallel cultures was observed in the 2nd experiment at 125.0 μg/mL without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol. On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol. The final concentration of ethanol in the culture medium was 0.5% (v/v).
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol (99.99% purity)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, microwells.

Thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured two times prior to treatment. The cell cultures were incubated at 37 ± 1.5 °C in a humidified atmosphere with 4.5% carbon dioxide and 95.5% ambient air.

Experimental Performance
In the mutation experiment 1×10e7 (3×10e6 during 24 h exposure) cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3% horse serum (15% during 24 h exposure) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. Positive and solvent controls were performed in parallel. After 4 h (24 h in the second experiment without metabolic activation) the test item was removed by centrifugation and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of totally 48 h.
The cell density was determined each day and adjusted to 3×10e+5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector.
After the expression period the cultures were seeded into microtiter plates. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4×10e+3 cells in selective medium (see below) with trifluorothymidine (TFT). The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37°± 1.5 °C in 4.5% carbon dioxide/95.5% humidified air for 10 - 15 days. Then the plates were evaluated. The relative total growth (RTG) was calculated by the RSG multiplied by the viability.

The cell density was determined using Casy® or Beckman Coulter cell counters.

Size Distribution of the Colonies:
Colonies were counted manually. In accordance with their size the colonies were classified into two groups. The colony size distribution was determined in the controls and at all concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 25% of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity:
Pre-Test on Toxicity: 1×10e+7 cells (3×10e+6 during 24 h exposure) in 10 mL RPMI medium were exposed to each concentration of the test item in the presence (4 hours) and absence of metabolic activation (4 and 24 hours). During the 4 hours treatment period the serum concentration was reduced from 15% to 3%. Following treatment the cells were washed twice. Subsequently the cells were resuspended in 30 mL complete culture medium for a 2-day growth period. The cell density was determined each day and adjusted to 3×10e+5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector.
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 15.6 μg/mL and 2000 μg/mL were used with respect to the OECD guideline 490. No relevant toxic effects indicated by a relative suspension growth (RSG) below 50% were observed up to the highest concentration with and without metabolic activation after 4 h and 24 h treatment.

- OTHER:
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed.
Rationale for test conditions:
Acceptability of the Assay
A mutation assay is considered acceptable if it meets the following criteria:
All plates, from either the cloning efficiency or the TFT resistance-testing portion of the experiment are analysable.
The absolute cloning efficiency at the time of mutant selection (CE) of the solvent controls is 65 – 120%.
The total suspension growth of the solvent control calculated by the day 1 fold-increase in cell number multiplied by the day 2-fold-increase in cell number is 8 – 32. Following 24 h treatment the total suspension growth is 32 – 180.
The range of the solvent control mutant frequency is in the range of 50 – 170 per 10e+6 cells.
The positive controls (MMS and CPA) should yield an absolute increase in total MF (Mutation Frequency, number of mutant colonies per 106 cells), that is an increase above spontaneous background MF (an induced MF [IMF]) of at least 300 per 10e+6 cells. At least 40% of the induced mutation frequency (IMF) should be reflected in the small colony MF. Alternatively, the positive controls should induce at least 150 small colonies.
The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures (i.e. the relative total growth – RTG – should be greater than 10% of the concurrent selective control group).
The highest concentration of the test item should be 2000 μg/mL, unless limited by toxicity or solubility of the test item. If toxicity occurred, the highest concentration should lower the relative total growth to approximately 10 to 20% of survival. If precipitation is noted, the highest analyzed concentration should be the lowest concentration where precipitation is observed by the naked eye.
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10e+6 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10% of the vehicle control.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10e+6 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.
Statistics:
A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend was judged as significant whenever the p-value (probability value) was below 0.05. However, both, biological relevance and statistical significance were considered together.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
1st experiment, 4 hours
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
1st experiment, 4 hours
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
2nd experiment, 24 hours
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 125 µg/mL (less than 50% survival in both parallel cultures)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments.
No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

No substantial and reproducible increase of the mutation frequency was noted in the main experiments with and without metabolic activation.

The mutation frequency exceeded the threshold of 126 above the corresponding solvent control at 31.3 and 125.0 μg/mL in culture II of the second experiment without metabolic activation. However, these increases were judged as biologically irrelevant as they were not reproduced in the parallel culture under identical experimental conditions. Furthermore the mean values (31.3 μg/mL: 144; 125,0 μg/mL: 179) are below the threshold.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of p<0.05 was determined in the second culture of experiment II without metabolic activation. Since the effect was not reproduced in the parallel culture, it is judged as biologically irrelevant.

In this study the range of the solvent controls was from 77 up to 149 mutant colonies per 10e+6 cells; the range of the groups treated with the test item was from 52 up to 226 mutant colonies per 10e+6 cells.

MMS (19.5 μg/mL in experiment I and 13.0 μg/mL in experiment II) and CPA (3.0 μg/mL and 4.5 μg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the positive controls. The positive controls remained within the range of the historical positive control data throughout the study.

The viability (CE) exceeded the higher limit of 120% in the first culture of the first experiment without metabolic activation (127%). The mean value of both parallel cultures however, (127% and 73% equal to a mean of 100%) was fully acceptable. In experiment II the viability was exceeded in the second culture (153%). The mean value of both parallel cultures was fully acceptable (111% and 153% equal to a mean of 132%).

Precipitation occurred at 125.0 μg/mL and above in the presence and absence of metabolic activation following 4 and 24 hours treatment.

Table 1: Summary of results / Culture I

 

µg/mL

 

 

 

 

Experiment 1, 4h treatment

 

S9 mix

relative total growth

mutant colonies per 10e+6 cells

treshold

Solvent control with ethanol

-

--

100

149

275

Methyl Methane Sulfonate

19.5

--

33.3

237

275

Test item

7.8

--

109.3

66

275

Test item

15.6

--

77.2

104

275

Test item

31.3

--

66.6

98

275

Test item

62.5

--

69.7

80

275

Test item

125

--

72.2

162

275

 

 

 

 

 

 

Solvent control with ethanol

-

+

100

128

254

Cyclophosphamide

3.0

+

70.3

252

254

Test item

4.5

+

35.5

461

254

Test item

7.8

+

140.1

92

254

Test item

15.6

+

147.2

121

254

Test item

31.3

+

132.1

115

254

Test item

62.5

+

121.6

106

254

Test item

125

+

114.2

90

254

 

 

 

 

 

 

Experiment 2, 24h treatment

 

 

 

 

 

Solvent control with ethanol

-

--

100

125

251

Methyl Methane Sulfonate

13.0

--

8.7

611

251

Test item

7.8

--

167.8

52

251

Test item

15.6

--

63.4

141

251

Test item

31.3

--

97.3

84

251

Test item

62.5

--

73.9

101

251

Test item

125

--

44.3

132

251

Table 2: Summary of results / Culture II

 

µg/mL

 

 

 

 

Experiment 1, 4h treatment

 

S9 mix

relative total growth

mutant colonies per 10e+6 cells

treshold

Solvent control with ethanol

-

--

100

146

272

Methyl Methane Sulfonate

19.5

--

18.9

548

272

Test item

7.8

--

158.4

108

272

Test item

15.6

--

111.7

139

272

Test item

31.3

--

96.9

127

272

Test item

62.5

--

101

167

272

Test item

125

--

128

108

272

 

 

 

 

 

 

Solvent control with ethanol

-

+

100

105

231

Cyclophosphamide

3.0

+

40.6

325

231

 

4.5

+

19.6

620

231

 

7.8

+

66.4

105

231

 

15.6

+

61.6

166

231

 

31.3

+

83.5

106

231

 

62.5

+

62.3

129

231

 

125

+

76.5

109

231

 

 

 

 

 

 

Experiment 2, 24h treatment

 

 

 

 

 

Solvent control with ethanol

-

--

100

77

203

Methyl Methane Sulfonate

13.0

--

8.1

345

203

Test item

7.8

--

109.7

55

203

Test item

15.6

--

57.5

109

203

Test item

31.3

--

26.9

204

203

Test item

62.5

--

37.5

168

203

Test item

125

--

33.1

226

203

 

Cultures were not continued at 250 and 500 µg/mL to avoid analysis of too many precipitating concentrations.

Conclusions:
The test item did not induce gene mutations at the mouse lymphoma thymidine kinase locus using the mammalian cell line L5178Y.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

Genetic toxicity (mutagenicity) in bacteria in vitro

The mutagenic potential of Propylene dinonanoate (CAS 41395-83-9) was tested in a reverse mutation assay according to OECD guideline 471 under GLP conditions (key study, 2018). Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA were used in two experiments. Tester strains were incubated with test material at concentrations of 0, 3, 10, 33, 100, 333, 1000, 2500, and 5000 μg/plate with and without the addition of a metabolic activation system (phenobarbitone and β-naphthoflavone induced rat liver S9 mix). Precipitation occurred at doses ≥ 5000 µg/plate. Vehicle and appropriate positive controls were included into the study design. Positive control materials induced statistically significant increases in the frequency of revertant colonies indicating the satisfactory performance of the test and the activity of the metabolizing system. No increase in the frequency of revertant colonies compared to concurrent vehicle and negative controls was observed in all strains treated with the test material, neither in the presence nor in the absence of metabolic activation. Thus, Propylene dinonanoate (CAS 41395-83-9) did not induce point mutations by base-pair changes or frame-shifts in the genome of the bacterial strains tested.

Genetic toxicity (cytogenicity)/ micronucleus study

Propylene dinonanoate (CAS 41395-83-9) was tested in an in vitro mammalian cell micronucleus test according to OECD Guideline 487 under GLP conditions (key study, 2018). Human primary lymphocytes were obtained from a healthy non-smoking male donor (34 years old) for Experiment I and healthy non-smoking female donor (35 years old) for Experiment II. Treatments started after a 48 hour stimulation period with phytohemeagglutinine (PHA) when cells were actively proliferating. In the first experiment, cells were exposed to the test substance with and without metabolic activation for 4 h, followed by 16 h of recovery. After this period Cytochalasin B was added and the cells were cultured another approximately 20 hours until preparation (harvest of cells).The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). In the second experiment cells were treated as in first experiment, except that they were exposed for 20 hours without S9 mix. Test concentrations were 0, 7.4, 13.0, and 22.7 μg/mL for the first experiment, and 0, 18.7, 32.7, and 57.1 μg/mL for the second experiment. Precipitation occurred at the end of treatment at the top dose of each experiment. The number of micronucleated cells in % was determined in a sample of 2000 bi-nucleated cells. Vehicle (solvent) controls induced frequencies within the range expected for normal human lymphocytes. Cyclophosphamide (with S9 for 4 hours), mitomycin C (without S9 for 4 hours) and Demecolcine (without S9 for 24 hours) were used as positive control materials inducing statistically significant increases in percentage of micronucleated cells indicating the satisfactory performance of the test and of the activity of the metabolizing system. No increase in the frequency of micronucleated cells at any dose level tested in comparison to the negative controls occurred. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, when tested up to precipitating concentrations.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

The mutagenic potential of Propylene dinonanoate (CAS 41395-83-9) was tested in a in vitro gene mutation study according to OECD guideline 490 under GLP conditions (key study, 2017). Duplicate cultures of mouse lymphoma L5178Y cells were evaluated. In the first experiment, the cells were treated for 4 h with 0, 7.8, 15.6, 31.3, 62.5, 125 µg/mL in the presence or absence of metabolic activation (phenobarbitone and β-naphthoflavone induced rat liver S9 mix). In the second experiment, concentrations of 0, 7.8, 15.6, 31.3, 62.5, 125 µg/mL were applied without metabolic activation for 24 h. Precipitation occurred at 125.0 μg/mL and above in the presence and absence of metabolic activation following 4 and 24 hours treatment. Relevant cytotoxic effects indicated by a relative total growth of less than 50% of survival in both parallel cultures was observed in experiment II at 125.0 μg/mL without metabolic activation. Cyclophosphamide and methylmethanesulfonate were used as positive controls with and without S9 mix, respectively. Positive and negative controls were valid and in the range of historical control data. No significant increase in the mutation frequency at the thymidine kinase locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. It was concluded that the test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

Overall conclusion for genetic toxicity

The results of the available in vitro mutagenicity and in vitro cytogenicity studies were consistently negative. Based on the available data, no hazard regarding genotoxicity is identified for Propylene dinonanoate (CAS 41395-83-9).

Justification for classification or non-classification

The available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.