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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

RA from source substance Ditridecyl adipate (CAS 16958 -92 -2) and Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9)

Gene mutation in mammalian cells (OECD 490, Thymidine kinase assay): negative in mouse lymphoma L5178 Tk +/- cells with and without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Feb - 10 Jun 2020
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
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Mainz, Germany
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
thymidine kinase locus (Tk1)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: ATCC (Wesel, Germany)
- Suitability of cells: The cell line is characterized by a high sensitivity to chemical mutagens, by a high proliferation rate, a high cloning efficiency and a stable spontaneous mutant frequency.
- Normal cell cycle time: 10 - 12 h

For cell lines:
- Absence of Mycoplasma contamination: Before freezing, each batch was screened for mycoplasma contamination.
- Methods for maintenance in cell culture: Cells were thawed 6 days prior treatment and cultivated in cell culture flasks.
- Doubling time: 10 - 12 h
- Modal number of chromosomes: 40 ± 2
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media:

Complete culture medium: RPMI 1640 medium supplemented with 10% heat inactivated horse serum, 1% penicillin/streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin) and 2% 100 mM sodium pyruvate.

Viability medium: RPMI 1640 medium supplemented with 15% heat inactivated horse serum, 1% penicillin/streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin) and 2% 100 mM sodium pyruvate.

Selection medium: RPMI 1640 medium supplemented with 15% heat inactivated horse serum, 1% penicillin/streptomycin (10,000 U/mL penicillin and 10 mg/mL streptomycin), 2% 100 mM sodium pyruvate and 5 µg/mL trifluorothymidine.

- Incubation (CO2 concentration, humidity level, temperature): The cells were cultured at 37.0 ± 1.0°C in a humidified atmosphere with 5.0 ± 0.5% CO2.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix)
- Source of S9 : Trinova Biochem GmbH, Gießen, Germany
- Method of preparation of S9 mix: S9 mix was prepared from the livers of male Sprague Dawley rats treated with an intraperitoneal injection of 500 mg/kg bw Aroclor 1254.
Test concentrations with justification for top dose:
Pre-test on toxicity
With and without S9 mix: 0.03, 0.06, 0.12, 0.24, 0.48, 0.95 and 1.907 mg/mL (4 h)

Experiment I
With and without S9 mix: 0.007, 0.015*, 0.030*, 0.060*, 0.119* and 0.238* mg/mL (4 h)

Experiment II
Without S9 mix: 0.003, 0.007, 0.015, 0.030*, 0.060*, 0.119* and 0.238* mg/mL (24 h)

* Concentrations evaluated for mutation rates
Vehicle / solvent:
- Vehicle used: acetone

- Justification for choice of vehicle: The solubility of the test item was determined in a non-GLP pre-test in culture medium (RPMI 1640) at a concentration of 19.07 mg/mL and in dimethyl sulfoxide (DMSO), ethanol as well as in acetone at a concentration of 381.4 mg/mL. The test item was completely insoluble in RPMI 1640, DMSO and ethanol, but soluable in acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: Three experiments were performed, one Experiment I and two Experiment II. In the first Experiment II various acceptability criteria were not fulfilled, therefore the experiment was considered invalid and repeated. Only the valid experiment (second Experiment II) was included in the study report.

METHOD OF TREATMENT:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 and 24 h
- Expression time (cells in growth medium between treatment and selection): 48 h
- Selection time: 10 - 12 days
- Method used: microtiter plates
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: After the expression period, 4000 cells/well from each experimental group were seeded into 2 microtiter plates with selective medium including trifluorothymidine (TFT). The viability (cloning efficiency 2) was determined by seeding 2 cells per well into 2 microtiter plates (same medium without TFT).
- Criteria for small (slow growing) and large (fast growing) colonies: Colonies were counted manually under a binocular magnifying glass. In accordance with their size, the colonies were classified into two groups:
Less than 25% of the well’s diameter = small colony
More than 25% of the well’s diameter = large colony

SELECTION AGENT: 5 μg/mL trifluorothymidine

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: Mutant frequency (MF)
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
• The induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
• The relative increase of the mutation frequency shows a dose relationship.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if:
• The induced mutation frequency does not exceed a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
• The relative increase of the mutation frequency does not show a dose relationship.
Statistics:
A linear regression (least squares) of the test item concentrations was performed to assess a possible dose dependent increase of mutant frequencies. With the assessment of this regression, it can be evaluated whether mutations increase with increasing dose of the test item. A p-value of 0.05 or lower (significance level 95%) is considered as critical.
The positive controls were tested at one concentration only, therefore, no dose-dependency could be evaluated. The chi-square test was used.
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
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of solutions that were used in the preliminary cytotoxicity test were determined to exclude a negative influence on the assay. There was no critical change in pH value, therefore a negative influence on the assay was excluded.
- Data on osmolality: The osmolality of solutions that were used in the preliminary cytotoxicity test were determined to exclude a negative influence on the assay. There was no critical change in osmolality, therefore a negative influence on the assay was excluded.
- Precipitation and time of the determination: Precipitation (oily drops) of the test item was visible in both experiments at the maximum concentration of the test item (0.238 mg/mL).

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the concentration range applicable for Experiment I and II. Seven test item concentrations in the range of 0.03 - 1.907 mg/mL were tested for 4 hours in the presence and absence of metabolic activation. Cytotoxicity (relative cloning efficiency) was determined for treated cells in comparison to controls. Precipitation was noted at 0.24 mg/mL and above with and without S9 mix. There was no cytotoxicity at any concentration, neither with nor without S9 mix.

STUDY RESULTS : There was no statistically significant or reproducible dose-dependent increase in the number of mutant colonies observed in any experiment at any of the tested concentrations, neither in the presence, nor in the absence of metabolic activation. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. For details on gene mutation results and cytotoxicity in the main mutation experiments please refer to Table 2 under "Any other information on result incl. tables".

In total three experiments were performed (1x Experiment I and 2x Experiment II). The first Experiment II was invalid, since various acceptability criteria were not fulfilled. The invalid experiment was repeated and is reported as Experiment II in the study report. The invalid Experiment II is not included in the study report.

HISTORICAL CONTROL DATA: Please refer to Table 1 under "Any other information on materials and methods incl. tables".
- Positive historical control data: The mutation frequencies of both positive controls were within the range of the historical control data.
- Solvent historical control data: The mutation frequency of the solvent control acetone was outside the range of the historical control data. The historical control data for acetone only comprise two independent studies, therefore an untreated medium control was added to demonstrate that no deleterious or mutagenic effects were induced by acetone.
- Untreated negative historical control data: The mutation frequency of the untreated negative control was within the range of the historical control data.

Table 2: Experimental results

Content Relative Total Growth [%] Mutants per 106Cells
Exp I Exp II Exp I Exp II
4 h -S9 4 h +S9 24 h -S9 4 h -S9 4 h +S9 24 h -S9
Threshold - - - 207 190 250
Acetone - - - 81 64 124
Solvent pos. Control - - - 70 64 90
Positive control 60 35 33 365 491 575
0.003 mg/mL - - 105 - - n/e
0.007 mg/mL 103 83 91 n/e n/e n/e
0.015 mg/mL 111 83 90 76 83 n/e
0.030 mg/mL 102 89 103 86 82 103
0.060 mg/mL 108 86 100 87 71 106
0.119 mg/mL 105 96 100 83 62 113
0.238 mg/mL 101 66 108 71 77 101
Additional untreated control (medium control)
Solvent control (RPMI) - - - 85 64 90
Acetone 0.5% 90 109 98 81 64 124

n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations

Conclusions:
Under the conditions of the test the test item did not induce mutations in the Tk1 locus in the presence or absence of metabolic activation.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
/ Source CAS 16958-92-2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
/ precipitation was observed starting at 1500 μg/plate, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Remarks:
/ Source CAS 929696-90-9
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
/ precipitation was observed at 1600 and 5000 μg/plate, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
In two studies according to OECD 471, the source substances did not exhibit mutagenic properties in bacterial cells. As explained in the analogue justification, this result is considered to be valid also for the target substance.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 (incl. QA statement)
Remarks:
The Departement Of Health Of The Government Of The United Kingdom, UK
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon and trp operon
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4
Test concentrations with justification for top dose:
Preliminary toxicity test (in TA 100 and WP2 uvr A): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate with and without metabolic activation
Main Assay (Experiment I and II): 50, 150, 500, 1500 and 5000 μg/plate with and without metabolic activation

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL, but was fully soluble in dimethyl formamide at the same concentration and in acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL. Acetone was selected as the vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: ENNG (2-5 µg/mL for WP2 uvr A, TA 100, TA 1535); 9-AA (80 μg/plate for TA 1537); 4NQO (0.2 μg/plate for TA98); +S9: 2-AA (1-10 µg/plate for TA 100, TA 1535, TA 1537 and WP2 uvr A); BP (5 μg/plate for TA 98)
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine; 9-AA:9-Aminoacridine; 4-NQO: 4-nitroquinoline-1-oxide; 2-AA: 2-aminoanthracene; BP: benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (Preliminary toxicity test and Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 20 min (Experiment II)
- Exposure duration: 48 h (all experiments)

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of the number of revertant colonies and inspection of the bacterial background lawn
Evaluation criteria:
There were several criteria for determining a positive result. Any, one, or all of the following were used to determine the overall result of the study:
- a dose-related increase in mutant frequency over the dose range tested.
- a reproducible increase at one or more concentrations.
- biological relevance against historical control ranges.
- statistical analysis of data as determined by UKEMS.
- fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item was considered non-mutagenic (negative) in the test system if the above criteria were not met.
Statistics:
Mean values and standard deviations of the mean number of revertants were calculated. Statistical analysis of data was performed according to guidelines from UKEMS.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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 was observed starting at 1500 μg/plate, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (globular in appearance) was noted by eye at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: In the Preliminary toxicity test, the test item was non-toxic to strains TA 100 and WP2 uvrA up to the maximum concentration of 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean number of revertants of vehicle and positive controls was within the normal range of the respective historical control data.

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

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT I (plate incorporation)

S9-Mix

Without

 

Concentration (per plate)

TA 100

TA1535

WP2 uvrA

TA98

TA 1537

SC

108 ± 14

14 ± 2

19 ± 2

21 ± 4

10 ± 1

Test material

 

 

 

 

 

50 µg

91 ± 12

13 ± 2

18 ± 6

19 ± 2

7 ± 4

150 µg

95 ± 21

15 ± 2

22 ± 2

18 ± 5

8 ± 4

500 µg

91 ± 7

14 ± 4

21 ± 5

16 ± 1

8 ± 2

1500 µg

94 ± 6

13 ± 1

18 ± 10

22 ± 8

6 ± 2

5000 µg

93 ± 3

10 ± 2

20 ± 2

16 ± 3

8 ± 3

PC

 

 

 

 

 

ENNG

614 ± 28

442 ± 54

937 ± 42

-

-

4-NQO

-

-

-

239 ± 30

-

9-AA

-

-

-

-

912 ± 96

S9-Mix

 

With

Concentration (per plate)

TA 100

TA 1535

WP2 uvr A

TA98

TA 1537

NC

91 ± 2

11 ± 3

26 ± 8

27 ± 3

11 ± 0

Test material

 

 

 

 

 

50 µg

101 ± 12

11 ± 2

25 ± 4

24 ± 4

9 ± 3

150 µg

96 ± 11

11 ± 1

28 ± 6

31 ± 2

10 ± 1

500 µg

104 ± 11

10 ± 1

29 ± 4

23 ± 8

10 ± 4

1500 µg

89 ± 3

11 ± 1

22 ± 7

23 ± 10

12 ± 5

5000 µg

99 ± 10

12 ± 1

24 ± 8

24 ± 6

10 ± 2

PC

 

 

 

 

 

2-AA

1238 ± 270

231 ± 11

286 ± 14

-

237 ± 12

BP

-

-

-

199 ± 11

-

SC = Solvent control; PC = Positive control substances; SD = standard deviation;

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine; 9-AA: 9-Aminoacridine; 4-NQO: 4-nitroquinoline-1-oxide; 2-AA: 2-aminoanthracene; BP: benzo(a)pyrene

Table 2. Test results of experiment II (pre-incubation)

Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD)

EXPERIMENT II (pre-incubation)

S9-Mix

Without

 

Concentration (per plate)

TA 100

TA1535

WP2 uvrA

TA98

TA 1537

SC

79 ± 6

15 ± 5

26 ± 3

15 ± 3

10 ± 4

Test material

 

 

 

 

 

50 µg

72 ± 2

12 ± 2

24 ± 5

26 ± 12

9 ± 1

150 µg

82 ± 13

12 ± 2

27 ± 5

16 ± 3

11 ± 3

500 µg

66 ± 1

8 ± 2

18 ± 1

22 ± 5

11 ± 1

1500 µg

74 ± 3

13 ± 4

27 ± 5

16 ± 1

5 ± 1

5000 µg

79 ± 1

11 ± 4

23 ± 6

21 ± 7

11 ± 4

PC

 

 

 

 

 

ENNG

728 ± 131

892 ± 86

946 ± 35

-

-

4-NQO

-

-

-

118 ± 8

-

9-AA

-

-

-

-

534 ± 206

S9-Mix

 

With

Concentration (per plate)

TA 100

TA1535

WP2 uvrA

TA98

TA 1537

NC

75 ± 5

14 ± 6

34 ± 9

24 ± 4

9 ± 2

Test material

 

 

 

 

 

50 µg

75 ± 4

12 ± 3

25 ± 0

21 ± 1

7 ± 3

150 µg

83 ± 22

10 ± 2

22 ± 9

26 ± 2

12 ± 4

500 µg

70 ± 18

10 ± 7

29 ± 4

21 ± 2

8 ± 2

1500 µg

76 ± 12

11 ± 6

24 ± 2

21 ± 3

11 ± 2

5000 µg

76 ± 22

10 ± 2

29 ± 5

26 ± 3

10 ± 3

PC

 

 

 

 

 

2-AA

1506 ± 102

201 ± 30

246 ± 22

-

316 ± 32

BP

-

-

-

130 ± 7

-

SC = Solvent control; PC = Positive control substances; SD = standard deviation;

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine; 9-AA: 9-Aminoacridine; 4-NQO: 4-nitroquinoline-1-oxide; 2-AA: 2-aminoanthracene; BP: benzo(a)pyrene

Conclusions:
Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28 Aug - 26 Sep 2016
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 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon; trp operon
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:
Cofactor supplemented post-mitochondrial factor (S9 mix), prepared from the livers of male rats treated with Aroclor.
Test concentrations with justification for top dose:
Dose range finding test / Experiment I (TA 100 and WP2 uvr A): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate with and without metabolic activation
Experiment I (TA 1537, TA 1535 and TA 98): 52, 164, 512, 1600 and 5000 µg/plate with and without metabolic activation
Experiment II (all strains): 17, 52, 164, 512 and 1600 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item was observed to be insoluble in water and dimethyl sulfoxide. In ethanol, the test item was fully soluble at 50 mg/mL. Based on these solubility findings, ethanol was selected as vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: -S9: acridine mutagen ICR-191 (2.5 µg/plate) for TA 1537 (Exp. I); +S9: 2-amino-anthracene (1, 2.5 or 5 µg/plate) for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, Experiment I) and preincubation (Experiment II)

DURATION
- Preincubation period: 30 min (Experiment II)
- Exposure duration: 48 ± 4 h (Experiment I and II)

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the mean number of revertants, reduction in the background lawn and the increase in the size of the microcolonies
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 or WP2 uvr A is not greater than twice the concurrent control, and the total number of revertants in tester strains TA 1535, TA 1537 or TA 98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 or WP2 uvr A is greater than twice the concurrent control, or the total number of revertants in tester strains TA 1535, TA 1537 or TA 98 is greater than three times the concurrent control at one or more concentrations with or without metabolic activation and/or a concentration-related increase over the range tested.
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 follow-up experiment.
Statistics:
Mean values and standard deviations were calculated.
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 was observed at 1600 and 5000 µg/plate, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 was observed at 1600 and 5000 µg/plate, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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 was observed at 1600 and 5000 µg/plate, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 was observed at 1600 and 5000 µg/plate, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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 was observed at 1600 and 5000 µg/plate, with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the dose range finding study precipitation of the test material on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 and 5000 µg/plate. In Experiment I the test substance precipitated on the plates at the start of the incubation period at 5000 µg/plate and at 1600 and 5000 µg/plate at the end of the incubation period. Precipitation at 5000 µg/plate was observed as oily droplets at the start and the end of the incubation period. In Experiment II the test substance precipitated at the start of the incubation period at concentrations of 512 and 1600 µg/plate in TA 98 without metabolic activation only. At the end of the incubation period precipitation of the test substance was observed at 1600 µg/plate in all tester strains with and without metabolic activation. Slight precipitate did not influence automated counting of the plate.

RANGE-FINDING/SCREENING STUDIES: The test substance was initially tested in TA100 and WP2 uvr A as a dose range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed in the dose range finding test. The results of the range-finding test were included in Experiment I.

HISTORICAL CONTROL DATA (ranges)
- Positive historical control data:
TA 1535 (-S9: 78-1381; +S9: 78-1058)
TA 1537 (-S9: 55-1565; +S9: 55-1112)
TA 98 (-S9: 410-2057; + S9: 263-1907)
TA 100 (-S9: 549-1848; +S9: 620-2651)
WP2 uvr A (-S9: 127-1951; +S9: 85-1390)
The positive controls were within the range of the historical control data.

- Negative (solvent/vehicle) historical control data:
TA 1535 (-S9: 4-36; +S9: 3-34)
TA 1537 (-S9: 3-25; +S9: 3-28)
TA 98 (-S9: 9-50; + S9: 9-57)
TA 100 (-S9: 63-153; +S9: 60-456)
WP2 uvr A (-S9: 12-68; +S9: 12-70)
The solvent control was within the range of the historical control data.

Tabe 1: Summary of the dose range finding test / Experiment I

  TA 100   WP2 uvr A  
  -S9 mix + S9 mix -S9 mix + S9 mix
Ethanol 86 ± 6 93 ± 5 22 ± 3 21 ± 5
Test substance
[µg/plate]
       
1.7 72 ± 18 88 ± 23 24 ± 5 16 ± 2
5.4 68 ± 10 71 ± 3 32 ± 4 16 ± 0
17 87 ± 16 72 ± 6 22 ± 5 19 ± 4
52 65 ± 8 85 ± 15 25 ± 2 22 ± 2
164 73 ± 10 79 ± 6 21 ± 5 13 ± 3
512 73 ± 20 73 ± 5 20 ± 2 19 ± 7
1600 68 ± 8 SP 71 ± 3 SP 28 ± 5 SP 18 ± 3SP
5000 82 ± 19 SP 87 ± 20 SP 21 ± 2 SP 21 ± 4SP
Positive controls methylmethane sulfonate 2-aminoanthracene 4-nitroquinoline N-oxide 2-aminoanthracene
  1102 ± 106 749 ± 105 433 ± 60 1230 ± 94

SP: Slight precipitation

Table 2: Summary of Experiment I

  TA 1535   TA 1537   TA 98  
  -S9 mix + S9 mix -S9 mix + S9 mix -S9 mix + S9 mix
Solvent control 6 ± 2 10 ± 5 5 ± 2 11 ± 4 15 ± 4 15 ± 4
Test substance
[µg/plate]
           
52 8 ± 5 7 ± 3 7 ± 3 12 ± 2 10 ± 6 15 ± 4
164 9 ± 2 10 ± 6 4 ± 4 10 ± 4 9 ± 6 17 ± 3
512 6 ± 3 10 ± 2 7 ± 3 5 ± 2 14 ± 3 18 ± 2
1600 4 ± 3 SP 7 ± 2 SP 4 ± 1 SP 6 ± 1 SP 16 ± 5 SP 20 ± 5 SP
5000  11 ± 3 SP 8 ± 3 SP 11 ± 8 SP 6 ± 4 SP 13 ± 3 SP 15 ± 9 SP
Positive control sodium azide 2-aminoanthracene ICR-191 2-aminoanthracene 2-nitrofluorene 2-aminoanthracene
  753 ± 67 170 ± 20 932 ± 72 230 ± 54 1283 ± 54 982 ± 46

SP: Slight precipitation

Table 3: Summary of Experiment II

  TA 1535   TA 1537   TA 98   TA 100   WP2 uvr A  
  -S9 mix + S9 mix -S9 mix + S9 mix -S9 mix + S9 mix -S9 mix + S9 mix -S9 mix + S9 mix
Solvent control 9 ± 5 8 ± 2 20 ± 21 23 ± 29 19 ± 5 27 ± 6 126 ± 4 114 ± 20 28 ± 9 29 ± 9
Test substance
[µg/plate]
                   
17 13 ± 7 6 ± 2 5 ± 2 10 ± 2 21 ± 2 25 ± 8 110 ± 15 112 ± 8 37 ± 11 23 ± 4
52 8 ± 1 13 ± 1 14 ± 11 5 ± 2 19 ± 7 43 ± 35 124 ± 23 102 ± 3 32 ± 6 25 ± 9
164 5 ± 3 9 ± 6 5 ± 2 9 ± 8 21 ± 1 20 ± 0 118 ± 6 106 ± 18 28 ± 10 25 ± 9
512 7 ± 4 12 ± 2 5 ± 2 9 ±6 17 ± 3 39 ± 32 98 ± 11 106 ± 8 35 ± 11 33 ± 9
1600 8 ± 4 SP 11 ± 4 SP 5 ± 2 SP 14 ± 11 SP 19 ± 5 SP 28 ± 5 SP 9 ± 8 SP 112 ± 6 SP 26 ± 2 SP 33 ± 7 SP
Positive control sodium azide 2-aminoanthracene ICR-191 2-aminoanthracene 2-nitrofluorene 2-aminoanthracene methylmethane sulfonate 2-aminoanthracene 4-nitroquinoline N-oxide 2-aminoanthracene
  875 ± 31 161 ± 8 100 ± 15 221 ± 47 1312 ± 52 671 ± 56 735 ± 35 1390 ± 112 225 ± 159 520 ± 26

SP: Slight precipitation

Conclusions:
Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Gene mutation in somatic cells in vivo (similar to OECD 474, Micronucleus test): negative for induction of micronuclei in the bone marrow of male and female Sprague-Dawley rats

RA from source substance Ditridecyl adipate (CAS 16958 -92 -2)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: source: CAS 16958-92-2
Conclusions:
In a study simmilar to OECD 474, the source substance did not induce clastogenicity in male and female Sprague Dawley rats in the micronucleus assay. As explained in the analogue justification, this result is considered to be valid also for the target substance.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study report, comparable to guideline study with acceptable restrictions (no data on analytical purity, non-GLP, no positive control)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
no positive control
Principles of method if other than guideline:
The present study (Study ID 40553) was part of a multiple endpoint study (Study ID 40552). Details on methods for dosing, animal care etc. were derived from the 13-weeks-repeated dose dermal study.
GLP compliance:
no
Remarks:
The study was conducted according to GLP regulations, but the report was not reviewed by the quality assurance unit.
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Details on the strain: Rat/crl COBS CD[SD] BR/Charles River, Lakeview, New Jersey
- Source: Charles River, Lakeview, New Jersey
- Age at study initiation: 49 days
- Weight at study initiation: males: 161.9 - 166.8 g; females: 149.2 - 150.5 g
- Housing: individually, in suspended, stainless steel cages, with wire mesh bottoms and fronts
- Diet: Purina Certified Lab Chow #5002 in pellet form; ad libitum
- Water: tap water, delivered by an automatic watering system; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: none
Details on exposure:
TEST SITE
- Area of exposure: on the backs of the animals, beginning at the scapula and continuing laterally and posteriorly
- Type of wrap if used: no wrap. Animals were fitted with cardboard "Elisabethan" collars to minimize ingestion of test material.
- Time intervals for shavings or clipplings: approx. 24 h before the start of dosing, hair was then reclipped as necessary, but at least once per week

REMOVAL OF TEST SUBSTANCE
- Washing: approx. 24 h after the last dose each week, as much residual test substance as practical was wiped off with gauze pads.

TEST MATERIAL
- Constant volume or concentration used: yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
24 h, 5 days/week
Post exposure period:
none
Dose / conc.:
800 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Positive control(s):
None.
Since the study was part of a 13-weeks repeated dose dermal study, no positive control was run in parallel.
Tissues and cell types examined:
Peripheral blood smears and bone marrow were taken from the femurs of 5 animals/sex/dose.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The doses were the same as chosen for the 13-weeks repeated dermal dose study.

TREATMENT AND SAMPLING TIMES:
To ascertain if the bone marrow and peripheral blood were equally sensitive for micronuclei detection, each target tissue was scored as an independent phase of the study and the results were compared. Three peripheral blood slides and four bone marrow slides were made for each animal.

DETAILS OF SLIDE PREPARATION:
Slides were air dried and fixed in absolute methanol for 15 min. After the slides had dried, they were placed in an acridine orange (0.125 mg/mL in Giordano's buffer, pH 5.4-6.5) solution for 7 to 10 min, rinsed in Giordano's buffer and washed in fresh Giordano's buffer for an additional 5-10 min.

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes and 1000 normochromatic erythrocytes were scored in each target tissue to determine the incidence of micronuclei.
Statistics:
Several statistical methods, including ANOVA (analysis of variance) and SAS GLM (general linear model) models, were applied to the data. The ANOVA F test was used to determine significant differences between mean values. The Tukey's Studentized Range Test and the Scheffe's Test were performed to determine which means were different. SAS GLM is a studentized linear regression analysis that can be used to determine dose responsiveness.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable

Table 1: Micronucleus assay of bone marrow from rats treated dermally with the substance for 13 weeks (averaged data)

 

Dose [mg/kg bw/day]

Sex

No. of animals

PCE/NCE

% PCE with MN

% NCE with MN

0

F

5

0.38 (0.19)

0.12 (0.11)

0.04 (0.05)

0

M

5

0.44 (0.17)

0.04 (0.05)

0.02 (0.04)

0

M+F

10

0.41 (0.17)

0.08 (0.09)

0.03 (0.05)

800

F

5

0.40 (0.12)

0.14 (0.11)

0.04 (0.05)

800

M

5

0.40 (0.08)

0.08 (0.13)

0.00 (0.00)

800

M+F

10

0.40 (0.10)

0.11 (0.12)

0.02 (0.04)

2000

F

5

0.46 (0.16)

0.06 (0.05)

0.02 (0.04)

2000

M

5

0.41 (0.09)

0.02 (0.04)

0.08 (0.08)

2000

M+F

10

0.44 (0.12)

0.04 (0.05)

0.05 (0.07)

Averaged data: means (standard deviation)

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

MN: micronuclei

 

 

Table 2: Micronucleus assay of peripheral red blood cells from rats treated dermally with the substance for 13 weeks (averaged data)

 

Dose [mg/kg bw/day]

Sex

No. of animals

PCE/NCE

% PCE with MN

% NCE with MN

0

F

5

0.02 (0.00)

0.02 (0.04)

0.02 (0.04)

0

M

5

0.02 (0.00)

0.14 (0.11)

0.02 (0.04)

0

M+F

10

0.02 (0.00)

0.08 (0.10)

0.02 (0.04)

800

F

5

0.02 (0.00)

0.10 (0.12)

0.04 (0.05)

800

M

5

0.03 (0.01)

0.08 (0.18)

0.00 (0.00)

800

M+F

10

0.02 (0.01)

0.09 (0.14)

0.02 (0.04)

2000

F

5

0.02 (0.01)

0.08 (0.08)

0.02 (0.04)

2000

M

5

0.03 (0.01)

0.06 (0.09)

0.00 (0.00)

2000

M+F

10

0.02 (0.00)

0.07 (0.08)

0.01 (0.03)

Averaged data: means (standard deviation)

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

MN: micronuclei

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

Additional information

Justification for read-across

There are only limited substance-specific data on genetic toxicity available for Fatty acids, C18 -unsaturated, 1,6 Hexanediol Diester(EC 947-912-3). The assessment of genetic toxicity was therefore based on experimental data with the registered substance itself and on studies conducted with analogue substances as part of a read-across approach, in order to fulfil the standard requirements set out in Annex VII - VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No. 1907/2006.

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”.

For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substances are the basis of read-across. For gene mutation in bacteria, read-across was conducted with the structurally related analogue substances Ditridecyl adipate (CAS 16958-92-2) and Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9). For gene mutation in mammalian cells, experimental data with the registered substance Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester is available. Data on cytogenicity are covered with ani n vivo micronucleus assay conducted with the analogue substance Ditridecyl adipate (CAS 16958-92-2). A detailed justification for the analogue read- across approach is provided in the technical dossier (see IUCLID Section 13).

 

Gene mutation in bacteria

 As no reliable experimental data are available on genetic toxicity in bacterial cells for Fatty acids, C18 -unsaturated, 1,6 Hexanediol Diester(EC 947-912-3), read-across to reliable data on the analogue substances Ditridecyl adipate (CAS 16958-92-2) and Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9) was conducted.

 

CAS 16958-92-2

The in vitro genetic toxicity of the test substance was assessed in a bacterial reverse mutation assay (Ames test) according to OECD 471 and in compliance with GLP (Chemtura, 2013, WoE). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains including TA 98, 100, 1535 and 1537 and in E. coli WP2 uvr A bacterial cells at concentrations up to 5000 µg/plate in 2 independent experiments, including plate incorporation and preincubation method. Precipitates were observed starting at a concentration of 1500 µg/plate (all tester strains). The test substance did not exhibit mutagenic properties in the absence or presence of metabolic activation. Cytotoxicity was not observed. The vehicle and positive controls were valid and lay within the range of historical control data, demonstrating the validity of the assay. Based on the results of the conducted study, the test substance is not considered to exhibit mutagenic properties in bacterial cells.

 

CAS 92969-90-9

The in vitro genetic toxicity of the test substance was assessed in a bacterial reverse mutation assay (Ames test) according to OECD 471 and in compliance with GLP (Zschimmer and Schwarz, 2017, WoE). The mutagenic potential of the test substance was assessed in S. typhimurium tester strains including TA 98, 100, 1535 and 1537 and in E. coli WP2 uvr A bacterial cells at concentrations up to 5000 µg/plate in the plate incorporation test and up to 1600 µg/plate in the preincubation test. Precipitates were observed in both tests starting at a concentration of 1600 µg/plate (all tester strains). The test substance did not exhibit mutagenic properties in the absence or presence of metabolic activation. Cytotoxicity was not observed. The vehicle and positive controls were valid and lay within the range of historical control data, demonstrating the validity of the assay. Based on the results of the conducted study, the test substance is not considered to exhibit mutagenic properties in bacterial cells.

 

Gene mutation in mammalian cells in vitro

 

Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester (EC 947-912-3) was investigated for its potential to induce gene mutation in mammalian cells in vitro by using the Thymidine kinase assay (Dako AG, 2020, key). The test was conducted in L5178Y mouse lymphoma cells in compliance with GLP and according to OECD guideline 490. Based on a preliminary solubility test, the test item was dissolved in acetone. A preliminary cytotoxicity test with a 4 h incubation time in the presence and absence of S9 mix (Aroclor 1254-induced rat S9 liver fraction) revealed no cytotoxicity up to 1.907 mg/mL, but precipitation of the test material in culture medium at 0.24 mg/mL Thus, concentrations for the main mutagenicity test were selected based on solubility issues.

A total of three independent experiments were performed (Experiment I and twice Experiment II) whereby the first Experiment II was invalid since various acceptability criteria were not fulfilled. Therefore only the valid Experiment II was reported.

Duplicate cultures per condition were exposed for 4 h with and without S9 mix to test item concentrations in the range of 0.007 – 0.238 mg/mL (first experiment) and for 24 h in the absence of S9 mix to concentrations of 0.003 – 0.238 mg/mL (second experiment).Corresponding negative (culture medium), solvent (acetone) and positive controls (methylmethane sulfonate (MMS), 19.5 µg/mL forExperiment Iand 12.5 µg/mL forExperiment II, -S9and cyclophosphamide (CPA), 5.5 µg/mL, +S9) were included.Following exposure, the cells were incubated for 48 hto allow expression of the mutant phenotype. The expression period was followed by a selection period, where cells were incubated in selection medium containing trifluorothymidine (5 µg/mL) for 10 - 12 days.

Precipitation of the test item was noted in both experiments at the highest concentration of 0.238 mg/mL. There was no cytotoxicity observed in any experiment at any concentration, neither with nor without S9 mix.

There was no statistically significant or reproducible dose-dependent increase in the number of mutant colonies observed in any experiment at any of the tested concentrations, neither in the presence, nor in the absence of metabolic activation. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Mutant frequencies obtained with the untreated medium were within the range of the laboratories historical data as well as in the range of the spontaneous mutant frequency of the L5178Y cells. The mutation frequency of the solvent control acetone was outside the range of the historical control data. The historical control data for acetone only comprise two independent studies, therefore an untreated medium control was added to demonstrate that no deleterious or mutagenic effects were induced by acetone. The positive controls MMS and CPAshowed a distinct increase in induced total mutant colonies, demonstrating the sensitivity of the test and the functionality of the S9 mix. All acceptance criteria were fulfilled.

Under the conditions of the test,Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester (EC 947-912-3) did not induce forward mutations in Tk1 locus in the absence or presence of metabolic activation. Thus, the test item is considered not mutagenic in mammalian cells in vitro.

 

Mammalian Erythrocyte Micronucleus Test

As no reliable experimental data are available on the clastogenic potential ofFatty acids, C18-unsaturated, 1,6 Hexanediol Diester,read-across to reliable data on the analogue substance Ditridecyl adipate (CAS 16958-92-2) was conducted.

 

CAS 16958-92-2

The clastogenic potential of Ditridecyl adipate (CAS 16958-92-2) was investigated in an in vivo Mammalian Erythrocyte Micronucleus Test in Sprague-Dawley rats similar to OECD Guideline 474 (Exxon, 1985, key). The test substance was applied to the clipped dorsal skin of 10 animals per sex and group at dose levels of 800 and 2000 mg/kg bw/day. Treatment of the animals was performed daily for 5 days/week over a period of 13 weeks. A similar constituted control group remained untreated and served as controls. At the end of exposure, the femoral bone marrow and peripheral blood smears of 5 animals per sex and dose were prepared and the incidence of micronucleated cells per 1000 polychromatic or normochromatic erythrocytes were scored in the respective tissues. In addition, the number of polychromatic and normochromatic erythrocytes was determined and a ratio of both was calculated in order to determine cytotoxic effects of the test substance. The obtained ratios of poly- to normochromatic cells did not indicate cytotoxicity in bone barrow and peripheral blood of treated animals. The frequency of micronuclei in polychromatic and normochromatic erythrocytes of the femoral bone marrow and peripheral blood of treated animals was not increased at any dose level compared to controls after 13-week dermal exposure.

Under the conditions of this experiment, Ditridecyl adipatedid not induce clastogenicity in male and female Sprague Dawley rats in the micronucleus assay.

 

Overall conclusion for genetic toxicity

There are only limited information available on the genetic toxicity of Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester(EC 947-912-3). Analogue read-across from source substances was applied for gene mutation in bacteria and for clastogenicity. The available measured data on gene mutation in bacteria for the read-across analogue substances Ditridecyl adipate (CAS 16958-92-2) and Hexanedioic acid, di-C16-18 (even numbered)-alkyl esters (CAS 92969-90-9) and the available data on genotoxicityin vivofor the read-across analogue substance Ditridecyl adipate (CAS 16958-92-2) reveal no genetic toxicity. A test for gene mutation in mammalian cellsin vitrowith the registered substance Fatty acids, C18 -unsaturated, 1,6 Hexanediol Diester (EC 947-912-3) demonstrated that the test item has no mutagenic potential. In conclusion, based on the experimental data available, no potential forgenetic toxicity is expected for Fatty acids, C18-unsaturated, 1,6 Hexanediol Diester.

Justification for classification or non-classification

Based on the analogue read-across approach and on the available experimental data with the test item itself, there is no indication that the substance induces genetic toxicity. The available data do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.