Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not considered to be genotoxic, based on the overall negative results from an Ames test andin vitrochromosomal aberration assay conducted with the test substance as well as mouse lymphoma assays available with the read across substances.

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:
From November 20, 2011 to December 14, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test substance was dissolved in dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany). The stock solution was treated with ultrasonic waves until the test substance had completely dissolved. Test substance concentrations were used within approximately 1½ hours after preparation.
Target gene:
- Histidine
- Tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
The strains TA1537 and TA98 are capable of detecting frameshift mutagens, strains TA1535, TA100 and WP2uvrA are capable of detecting base-pair substitution mutagens.
Metabolic activation:
with and without
Metabolic activation system:
metabolizing system (S9-mix)
Test concentrations with justification for top dose:
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.
Vehicle / solvent:
dimethyl sulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle alone: dimethyl sulfoxide
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoantracene
Details on test system and experimental conditions:
Salmonella typhimurium bacteria and Escherichia coli bacteria.
Source:
- Salmonella typhimurium strains: Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames): TA100, TA98, TA1537, TA1535
- Escherichia coli strain: Trinova Biochem GmbH, Germany (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) WP2uvrA
The Salmonella typhimurium strains were regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).
Rationale for test conditions:
Recommended test system in international guidelines (e.g. OECD and EC).
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
Statistics:
No formal hypothesis testing was done.
- A test substance is considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three times the concurrent vehicle control. b) The negative response should be reproducible in at least one independently repeated experiment.
- A test substance is considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three times the concurrent vehicle control. b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Experiment 1:
Based on the results of the dose range finding test, the test substance was tested up to the dose level of 3330 µg/plate in the absence and presence of 5% (v/v) S9-mix with the Salmonella typhimurium strains, TA1535, TA1537 and TA98. Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at the highest dose level of 3330 µg/plate. With the exception of tester strain TA98 in the absence of S9-mix (where a moderate reduction in the number of revertant colonies was observed at the two highest tested concentrations), there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all three tester strains in the absence and presence of S9-mix. Finally, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.
- Experiment 2:
To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 3330 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Precipitation of the test substance on the plates was observed at the start of the incubation period at a concentration of 3330 µg/plate. At the end of the incubation period precipitation was observed at 1000 and 3330 µg/plate in all tester strains, with the exception of tester strain WP2uvrA. In this tester strain precipitation on the plates was already observed at a dose level of 333 µg/plate. With the exception of tester strain TA100 in the presence of S9-mix (where an extreme reduction in the number of revertant colonies was observed at the highest tested concentration), there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. Finally, in the second mutation experiment, no biological relevant increase in the number of revertants was observed upon treatment with the test substance under all conditions tested. Although an increase above the historical control data range was observed at a dose of 333 µg/plate in tester strain TA1537 in the presence of S9-mix, this increase was not dose dependent and caused by one outlier. Therefore this increase was considered not biologically relevant.
Conclusions:
Under the study conditions, it was concluded that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according OECD Guideline 471 and EU Method B.13/14. The substance was tested up to the dose level of 3330 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA with (5 or 10% (v/v)) and without metabolic activation (S9-mix). Precipitates, toxicity and mutagenicity were evaluated. All bacterial strains showed negative responses over the entire dose range, i.e. there was no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays (Verbaan, 2011).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosomal aberration assay
Specific details on test material used for the study:
Batch HTIF11021 of the test substance was a clear green to brown highly viscous liquid and it was dissolved in dimethyl sulfoxide. Test substance concentrations were used within 2 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v).
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
HBSS without calcium and magnesium
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Cultured peripheral human lymphocytes (from healthy adult, non-smoking, male volunteers).
Cell culture:
- Blood samples were collected by venapuncture and immediately after blood collection lymphocyte cultures were started.
- Culture medium Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
- Lymphocyte cultures: whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added.
- Environmental conditions: all incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 - 98%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.6 - 37.5°C).
Rationale for test conditions:
Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD, EC)
Evaluation criteria:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, onesided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.
Statistics:
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.
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.
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
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:
- Dose range finding test: test blood cultures were treated with the test substance at concentrations of 10, 33, 100, 333 and 1000 µg/ml in culture medium with and without S9-mix. At a concentration of 333 µg/ml, the test substance precipitated in the culture medium. Based on the results of the dose range finding test, different dose levels were selected for the cytogenetic assay and the scoring of chromosome aberrations, with and without metabolic activation.
- Both in the absence and presence of S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Moreover, both in the absence and presence of S9-mix, the test substance did not show a biologically relevant increase in the number of polyploid cells and cells with endoreduplicated chromosomes.

It can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Conclusions:
Under the study conditions, it was concluded that this test was valid and that the test substance was not clastogenic in human lymphocyte.
Executive summary:

A study was conducted to determine the clastogenecity potential of the test substance according to OECD Guideline 473 and EU Method B.10. In the first assay, the substance was tested up to 40 and 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels. In the second assay, the substance was tested up to 40 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 45 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix the substance was tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance was considered not clastogenic in human lymphocyte (Verbaan, 2011).

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
This endpoint is read across from 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid; Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: No data; solubility limit in the culture medium: 340.9-681.8 μg/mL
- Precipitation: Yes; in the range-finding study, precipitation was observed at concentrations of 93.75-1500 μg/mL tested in the absence and presence of S-9

RANGE-FINDING/SCREENING STUDIES: Six concentrations were tested with and without S-9 ranging from 46.88-1500 μg/mL (limited by solubility in culture medium).
- Precipitation was observed at concentrations of 93.75 to 1500 μg/mL tested with and without S-9. Lowest precipitating concentration was retained and the higher concentration was discarded.
- Without S-9: complete toxicity was observed at all concentrations; with S-9: highest concentration providing > 10% RS was 46.88 μg/mL
- See table 1 for more details


COMPARISON WITH HISTORICAL CONTROL DATA: Yes; historical mean mutant frequencies for the vehicle controls were 4.34 without S-9 and 4.22 with S-9

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the range-finding study, complete toxicity was observed at all concentrations in the absence of S-9. In the presence of S-9, highest concentration providing > 10% RS was 46.88 μg/mL which gave 21% RS.

Table 1: RS values - range-finder experiment

Treatment (µg/mL)

-S-9 %RS

+S-9 %RS

0

100

100

46.88

0

21

93.75 P

0

0

187.5 P

0

0

375 P

0

0

750 P, PP

0

0

% RS: Percentage Relative Survival

P: Precipitation observed at time of treatment

PP: Precipitation observed following treatment incubation period

Table 2: Summary of mutation data

Experiment 1: (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)

-S-9

Treatment (µg/mL)

+S-9

%RS

MF§

%RS

MF§

0

100

2.21

 

0

100

1.34

 

0.0977

91

4.25

NS

3.75

103

NE

 

0.1953

84

2.3

NS

7.5

113

NE

 

0.3906

105

NE

 

15

107

NE

 

0.7813

90

NE

 

30

98

3.53

NS

1.563

84

NE

 

35

92

1.72

NS

3.125

79

3.81

NS

40

77

NE

 

6.25

75

2.24

NS

45

81

3.45

NS

12.5

58

6.66

*

50

83

NE

 

25

4

2.97

NS

60

61

9.53

*

50

0

NE

 

75

45

5.23!

NS

Linear trend

 

NS

Linear trend

**

NQO

 

 

 

B[a]P

 

 

 

0.1

61

45.16

 

2

30

97.71

 

0.15

47

49.83

 

3

11

190.6

 

Experiment 2: (3 hour treatment in the absence and presence of S-9)

Treatment (µg/mL)

-S-9 %

Treatment (µg/mL)

+S-9

%RS

MF§

%RS

MF§

0

100

2.89

 

0

100

3.56

 

3

101

4.05

NS

15

86

5.71

NS

6

71

4.69

NS

30

63

3.61

NS

9

76

7.85

*

40

29

4.24

NS

12

57

3.38

NS

50

21

6.69

NS

15

59

3.15

NS

60

6

4.67

NS

18

27

3.66

NS

70

4

NE

 

21

18

6.87

NS

80

3

NE

 

24

5

NE

 

90

1

NE

 

27

2

NE

 

100

0

NE

 

30

0

NE

 

125

0

NE

 

Linear trend

 

NS

Linear trend

NS

NQO

 

 

 

B[a]P

 

 

 

 10 

 34 

 29.68 

 

 2 

 70 

 85.43 

 

 15 

 40 

 52.00 

 

 3 

 25 

 61.29 

 

§: 6TG resistant mutants/106 viable cells 7 days after treatment

% RS: Percent relative survival adjusted by post treatment cell counts

* Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

NS: Not significant

*, **, *** Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively

NE: Not evaluated for mutant frequency due to excessive toxicity. Presented for information only

! Based on one replicate only

Conclusions:
Under the test conditions, the read across substance (BADGEDA) is not considered as mutagenic in L5178Y cells. This indicates absence of mutagenic potential of the SoI.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD guideline 476, in compliance with GLP. Under the test conditions, BADGEDA is not considered as mutagenic in L5178Y cells indicating absence of mutagenic potential of the SoI.  As explained in the read across justification, the differences in the structure of the components between the SoI and the read across substance are unlikely to lead to differences in toxicities in the mouse lymphoma assay that are higher than typical experimental error of the test method.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Mouse lymphoma assay
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
This endpoint is read across from nonanoic acid, which is a potential hydroytic product of one of the major components; Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Details on test system and experimental conditions:
4 hours exposure
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
- Nonanoic acid was not considered to be mutagenic under the conditions of this study without exogenous metabolic activation.
- Nonanoic acid, in the presence of S9 metabolic activation, induced a weak mutagenic response. Increases in the numbers of mutants per plate were seen at all test substance concentrations, and doubled at ≥300 µg/mL in trial 1, and at doses ≥100 µg/mL in trial 2. This occurred only in the presence of increasing moderate to severe cytotoxicity and small colony development and may indicate damage to the chromosome carrying the TK locus, rather than actual mutagenicity within the TK gene locus.
Remarks on result:
other:
Remarks:
Increases in the numbers of mutants per plate doubled at ≥300 µg/mL in trial 1, and at doses ≥100 µg/mL in trial 2. This occurred only in the presence of increasing moderate to severe cytotoxicity and small colony development and may indicate damage to the chromosome carrying the TK locus, rather than actual mutagenicity within the TK gene locus.
Conclusions:
Overall, based on the available weight of evidence from in vitro and in vivo genotoxicity assays with the hydrolytic product/metabolite, the SoI can be expected not to have gentoxic potential.
Executive summary:

A mouse lymphoma forward mutation assay was available as additional background information in the RAC opinion on nonanoic acid. Overall, based on the available weight of evidence from in vitro and in vivo genotoxicity assays with the hydrolytic product/metabolite, the SoI can be expected not to have gentoxic potential. As explained in the read across justification, the differences in the structure of the components between the SoI and the read across substance are unlikely to lead to differences in toxicities in the genotoxicity assays that are higher than typical experimental error of the test methods.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A study was conducted to determine the mutagenic potential of the test substance according OECD Guideline 471 and EU Method B.13/14. The substance was tested up to the dose level of 3330 µg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA with (5 or 10% (v/v)) and without metabolic activation (S9-mix). Precipitates, toxicity and mutagenicity were evaluated. All bacterial strains showed negative responses over the entire dose range, i.e. there was no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assays (Verbaan, 2011).

A study was conducted to determine the clastogenecity potential of the test substance according to OECD Guideline 473 and EU Method B.10. In the first assay, the substance was tested up to 40 and 100 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels. In the second assay, the substance was tested up to 40 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 45 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix the substance was tested up to 100 µg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Under the study conditions, the test substance was considered not clastogenic in human lymphocyte (Verbaan, 2011).

 

A study was conducted to determine the mutagenicity potential of a read across substance, BADGEDA, according to OECD Guideline 476 and compliance with GLP. In this study, mouse lymphoma L5178Y TK+/-(3.7.2C) cells were exposed to the read across substance BADGEDA in DMSO at concentrations of 0, 46.88, 93.75, 187.5, 375 or 750 μg/mL in RPMI 1640 medium with and without 2% S-9 metabolic activation for a preliminary cytotoxicity test. In the main test, two experiments were performed at the following concentrations:

- Experiment 1 (3 hour exposure): Without S-9: 0, 0.0977, 0.1953, 0.3906, 0.7813, 1.563, 3.125, 6.25, 12.5, 25 or 50 μg/mL; with S-9: 0, 3.75, 7.5, 15, 30, 35, 40, 45, 50, 60 or 75 μg/mL

- Experiment 2 (3 hour exposure): Without S-9: 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 or 30 μg/mL; with S-9: 0, 15, 30, 40, 50, 60, 70, 80, 90, 100 or 125 μg/mL 

Mutant frequencies in negative control cultures fell within normal ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). In Experiment 1, statistically significant increases in mutant frequency were observed at 12.5 (without S-9) and 60 µg/mL (with S-9). A significant linear trend was observed in the presence of S-9 only. In Experiment 2, a statistically significant increase in mutant frequency was observed at 9 µg/mL in the absence of S-9 only. However, no significant increases were observed following treatment with S-9 and there were no significant linear trends in either the absence or presence of S-9. Further, in both experiments, the increases in mutant frequency at the significant concentrations were all within three times the historical mean value and the increases were not considered concentration related or biologically relevant. Under the test conditions, BADGEDA is not considered as mutagenic in L5178Y cells indicating absence of mutagenic potential of the test substance. 

A mouse lymphoma forward mutation assay was available on the metabolite, nonanoic acid. In this study, L5178TK+/- cells were exposed for 4 h to non-activated doses ranging from 150 to 1600 µg Nonanoic acid per mL and to S9-activated concentrations ranging from 37.5 to 600 µg a.i./mL. Nonanoic acid was not considered to be mutagenic under the conditions of this study without exogenous metabolic activation. Nonanoic acid, in the presence of S9 metabolic activation, induced a weak mutagenic response. Increases in the numbers of mutants per plate were seen at all test substance concentrations, and doubled at ≥300 µg/mL in trial 1, and at doses ≥100 µg/mL in trial 2. This occurred only in the presence of increasing moderate to severe cytotoxicity and small colony development and may indicate damage to the chromosome carrying the TK locus, rather than actual mutagenicity within the TK gene locus. Therefore, it was concluded that the results do not reflect intrinsic mutagenicity. However the study was considered to be of limited value since the purity of the substance tested was not available in the study summary and there was no access to the fully study. Further, nonanoic acid was not found to be genotoxic in two otherin vitroassays i.e., in an bacterial reverse mutation assay conducted with Salmonella and E.coli strains as well as in an chromosomal aberration assay conducted with peripheral human lymphocytes. It also did not show evidence of clastogenicity or aneunogenicity in anin vivomicronucleus assay conducted in mice (ECHA RAC, 2013).

Justification for classification or non-classification

Based on the overall negative genotoxicity results from an Ames test and in vitro chromosomal aberration assay conducted with the test substance as well as mouse lymphoma assays available with the read across substances, no classification is required for genotoxicity according to CLP (1272/2008) criteria.