Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471, WoE): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102 and TA 1538 and in E. coli WP2 uvrA pKM 101

Chromosome aberration (OECD 473/478, WoE): negative in Chinese hamster ovary cells and human lymphocytes with and without metabolic activation

Gene mutation in mammalian cells (OECD 476, WoE): negative in mouse lymphoma L5178Y cells with and without metabolic activation

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06 May - 29 Aug 1996
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
September 1995
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
September 1995
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium strains)
trp operon ( E. coli strains)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9-mix), prepared from livers of male rats treated with Aroclor 1254
Test concentrations with justification for top dose:
0, 10, 33, 100, 333 and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
- S9: sodium azide (1 μg/plate, TA1535 and TA100); 9-aminoacridine (75 µg/plate, TA 1537); 2-nitrofluorene (1 µg/plate, TA98 and TA 1538); methylmethanesulfonate (1000 µg/plate, WP2 uvrA); +S9: 2-aminoanthracene (1 μg/pate, all strains)
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h
- Expression time (cells in growth medium): 48 to 72 h

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn wit a dissecting microscope
Evaluation criteria:
Revertant colonies were counted and the mean and standard deviation were calculated and compared to the controls.
All Salmonella tester strains must demonstrate the presence of the deep rough mutation and the deletion of the uvrA gene. Cultures of the TA98 and TA100 strains must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion of the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls. Tester strain titers must be above 30.000.000 cells/ml. The mean of each positive control must be at least three-fold increased to the controlls. A minimum of three non-toxic dose levels are recquired to evaluate assay data.
Statistics:
Mean and standard deviation were calculated
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
other: Unacceptable vehicle control values for the tester strain TA1537 and TA 98 were repeated.
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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 specified
Positive controls validity:
valid
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
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: >100 µg/plate

RANGE-FINDING/SCREENING STUDIES: Yes

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Test results of experiment 1:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 1535

TA1537

TA98

TA100

 

TA 1538

WP2uvrA

-

Vehicle

12

4

17

111

5

21

 -

10

3

4

15

115

6

16

 -

33

5

5

19

113

6

11

-

100

9

4

19

98

7

12

-

333

7

5

11

116

4

10

-

1000

8

6

21

120

7

13

Positive

controls

- S9

Name

SA

9AA

2NF

SA

2NF

MMS

Concentrations

(μg/plate)

1.0

75

1.0

1.0

1.0

1000

Number of colonies/plate

241

40

105

377

179

143

+

Vehicle

14

5

18

133

7

11

 +

10

9

5

27

114

12

16

+

33

9

3

21

113

8

13

+

100

8

7

26

108

9

13

+

333

9

4

27

115

6

8

+

1000

10

5

17

111

9

13

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1.0

1.0

1.0

1.0

1.0

10

Number of colonies/plate

72

127

888

904

783

57

SA: sodium azide

9AA : 9-aminoacridine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

2NF: 2-nitrofluorene

 

 

Table 2: Test results of experiment 2/3:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 1535

TA1537

TA98

TA100

 

TA 1538

WP2uvrA

-

Vehicle

14

10

23

126

11

27

 -

10

7

13

16

117

7

27

 -

33

9

15

23

124

8

19

-

100

5

13

22

120

6

18

-

333

12

9

17

110

11

16

-

1000

8

14

17

125

5

21

Positive

controls

- S9

Name

SA

9AA

2NF

SA

2NF

MMS

Concentrations

(μg/plate)

1.0

75

1.0

1.0

1.0

1000

Number of colonies/plate

429

757

125

601

221

195

+

Vehicle

8

5

19

147

14

24

 +

10

9

7

17

142

16

30

+

33

10

5

19

136

18

25

+

100

10

5

20

132

13

31

+

333

10

4

17

138

12

19

+

1000

10

7

19

125

12

19

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1.0

1.0

1.0

1.0

1.0

10

Number of colonies/plate

85

97

530

647

1041

88

SA: sodium azide

9AA : 9 -aminoacridine

MMS: methylmethanesulfonate

2-AA: 2 -aminoanthracene

2NF: 2 -nitrofluorene

 

 

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
22 May - 28 Oct 1996
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adpoted in 1995
Deviations:
yes
Remarks:
Only basic data on test substance given
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy´s 5A medium supplemented with 10% FBS, 100 units penicillin and 100 µg streptomycin/ml and 2 mM L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9-mix (Spraque-Dawley)
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL
Chromosomal abberation assay: (157, 313), 625, 1250, 2500, 5000 µg/mL. Concentrations 157 and 313 µg/mL were not evaluated for CA
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, 0.08 and 0.15 µg/mL, -S9; cyclophosphamide 10 µg/mL, +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 20 h without S9 activation and 4 h with activation.

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF CELLS EVALUATED: A minimum of 200 metaphase spreads (100 per duplicate flask) at each concentration used.
Evaluation criteria:
Criteria for cytotoxicity of the test substance: (1) cell growth inhibition relative to the solvent control
Criteria for chromosomal damage: (1) Number and types of aberrations found, the percentage of structurally and numerically damaged cells in the total population were counted.
Statistics:
The frequency of structural aberrations per cell was calculated. The statistical analysis of the percent aberrant cells was performed with Fisher´s Exact Test. It was used to compare pair wise the percent aberrant cells of each treatment group with that of the solvent control. In the event of positive control, the Cochran-Armitage test was used to measure dose- responsiveness.
The dose response was estimated by linear regression curves. Difference in the sensitivity of mutagenic compounds was established by comparing the slopes of corresponding dose-response regression curves. For a comparison of mean values, Student's t test was used.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
48% in the highest dose for treatment of 4 h without metabolic activation, 33% with metabolic activation and 3% for 20 h exposure without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 8 in the highest dose
- Effects of osmolality: 285 in the highest dose

RANGE-FINDING/SCREENING STUDIES: Yes, cell growth inhibition of 83% without and 3% with metabolic activation at the highest dose tested (5000 µg/mL).


COMPARISON WITH HISTORICAL CONTROL DATA: Yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Chromosomal aberration – Summary

 

Treatment

[µg/ml]

S9 activation

Treatment/

harvest time [h]

Mitotic index

Cells scored

Aberrations/cell (mean±SD)

Cells with aberrations [%]

numerical

structural

Vehicle (Ethanol)

-

4/20

6.9

200

0.005±0.071

2.5

0.5

625

-

4/20

5.9

200

0.000±0.000

2.0

0.0

1250

-

4/20

5.9

200

0.025±0.186

4.0

2.0

2500

-

4/20

6.5

200

0.025±0.186

2.5

2.0

5000

-

4/20

4.4

200

0.000±0.000

0.5

0.0

MMC (0.08)

-

4/20

6.1

200

0.150±0.788

3.5

9.0*

Vehicle (Ethanol)

+

4/20

7.8

200

0.025±0.157

2.0

2.5

625

+

4/20

7.0

200

0.020±0.140

1.0

2.0

1250

+

4/20

6.9

200

0.035±0.184

3.0

3.5

2500

+

4/20

7.4

200

0.030±0.222

2.0

2.0

5000

+

4/20

7.7

200

0.010±0.100

2.5

1.0

CP (10)

+

4/20

2.3

200

0.950±1.591

2.5

45.5*

Vehicle (Ethanol)

-

20/20

7.6

200

0.020±0.140

1.0

2.0

625

-

20/20

6.4

200

0.015±0.158

1.5

1.0

1250

-

20/20

5.5

200

0.025±0.186

2.0

2.0

2500

-

20/20

6.0

200

0.025±0.157

2.5

2.5

5000

-

20/20

6.2

200

0.020±0.172

2.0

1.5

MMC (0.08)

-

20/20

5.8

200

0.220±0.513

1.0

18.0*

MMC = Mitomycine C

CP = Cyclophosphamide

* = p≤0.01; Fisher´s exact test

Conclusions:
Interpretation of results (migrated information):
negative
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
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.

Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Prior to use cells were cleansed of pre-existing mutants.
Additional strain / cell type characteristics:
other: TK
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the results obtained in the preliminary toxicity trial, 2 independent assays for mutation to trifluorothymidine resistance were performed with this concentration: Assay n°1: with and without S9: 2500, 791, 250, 79.1, 25.0 g/mL Assay n°2 : without S9: 2500, 1250, 625, 313, 156, 78.1 g/mL Assay n°3: with S9: 2500, 1250, 625, 313, 156, 78.1g/mL
Vehicle / solvent:
Solvent/vehicle:
- acetone batch no. : 07L110525 obtained from BDH and 12L110525 obtained from VWR
- DMSO batch no.: 1473729V obtained from Fluka
Test item was evaluated in a preliminary trial using DMSO and acetone. This solvent were selected since they are compatible with the survival of the cells and the S9 metabolic activity. In addition, there are many historical control data demonstrating that no mutagenic effects are induced by these solvents. The test item is soluble in the acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Remarks:
Methylmethanesulphonate served as positive control in the absence of S9 and Benzo(a)pyrene (B(a)P) served as positive control in the presence of S9
Details on test system and experimental conditions:
1. Preparation of test cell cultures: A cell suspension (1X10^6 cells/mL) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.

2. Cytotoxicity assay: a preliminary cytotoxicity test was performed in order to select appopriated dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatment were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9. Wells containing viable clones were identified by eye using background illumination and then counted.

3. Mutation assay
3.1. Treatment of cell cultures: Preparation of test cultures was performed as described above. Duplicate cultures were prepared at each test point, with the exception of the positive controls, which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment was performed, where a longer treatment time (24 hours) was used in the absence of S9 metabolic activation.
After washing in PBS, cells were re suspended in fresh complete medium and cell densities were determined. The cultures were incubated at 37°C in 5% CO2 atmosphere to allow for expression of the mutant phenotype.

3.2. Expression period: During the expression period, the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determine and adjusted.

3.3. Plating for 5-trifluorothymidine resistance: after dilution, the cell suspensions in complete medium were suplemented with trifluorothymidine and an estimated 2X 10^3 cells were plated in each well of four 96-well plates. Plate were incubated at 37°C in a 5% CO2 atmosphere for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containg large colonies as well as the number of those containing small colonies were scored.

3.4. Plating for viability: After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of 2 96-wells plates. These plates were incubated at 37°C in a 5% CO2 atmosphere for 14 days and wells containing clones were identified as above and counted.
Evaluation criteria:
1. Acceptance criteria: The assay was considered valid if the following criteria were met: - The cloning efficiencies at Day 2 in the solvent/vehicle control cultures in the absence of S9 metabolic activation fell within the range od 65-120% - The solvent/vehicle control growth factor in the absence of S9 metabolic activation over 2 days fell within the range of 8-32 - The mutant frequencies in the solvent/vehicle control cultures fell within the range of 50-200X10^6 viable cells. - The positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). 2. Criteria for OUTCOME of assay: For a test item to be considered mutagenic in this assay, it is required that: - The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126X10^-6) at one or more doses. - There is a significant dose-relationship as indicated by the linear trend analysis. Results which only partially satisfy the above criteria are dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that Trimethylolpropane Tripelargonate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

The test item Trimethylolpropane Tripelargonate was examined for mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistance mutants in mouse lymphoma L5178 cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. This trial was performed in according to OECD 476 guidelines and GLP.

A preliminary solubility trial indicated that the maximum feasible concentration of the test item in the final treatment medium was 1250 µg/mL using acetone as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the presence and absence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77, and 4.88 µg/mL.

No relevant toxicity was observed at any concentration tested, in any treatment series. Slight opacity was observed at the end of treatment period at the five highest concentrations. No precipitation was noted at any concentration tested.

Based on the results obtained in the preliminary toxicity trial, two independent assays for mutation to trifluorothymidine resistance were performed using the following dose levels:

Assay n°1 with and without S9, treatment of 3 hours doses: 2500, 791, 250, 79.1, 25 µg/mL

Assay n°2: without S9, treatment of 24 hours doses: 2500, 1250, 625, 313, 156, 78.1 µg/mL

Assay n°3 with S9, treatment of 3 hours doses: 2500, 1250, 625, 313, 156, 78.1 µg/mL

No relevant toxicity was observed at any concentration tested in any treatment series. in both experiments, at the end of the treatment time, test item particles in suspension were noted at the highest or two highest concentrations. No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolim.

Negative and positive control treatments were included in each mutation experiment, both in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is conclude that Trimethylolpropane Tripelargonate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: OECD 487 (in vitro micronucleus test in human lymphocytes)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation
Species / strain / cell type:
other: peripheral blood for lymphocytes cultures
Details on mammalian cell type (if applicable):
For each experiment, whole blood was collected from healthy volunteer donors. The volunteers were non-smoker and were not receiving any medication or radiation exposure prior to the time of sampling.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First experiement: Dose levels 625, 357, 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 µg/mL
Second experiment: Dose levels 204, 117, 66.6, 38.1, 21.8, 12.4, 7.10 and 4.06 µg/mL
Vehicle / solvent:
DMSO and acetone. These solvents were selected since they are compatible with the survival of the cells and the S9 metabilic activity
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine
Remarks:
Mitomycin C and colchicine in absence of S9 metabolic activation and Cyclophosphamide in presence of S9 metabolic activation
Details on test system and experimental conditions:

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolims, respectively.

The haverest time of 32 hours corresponding to approximately 2.0 cell cycles was used.
As a negative results were obtained, a second experiment was performed in the absence of S9 metabolism usung approximately the same haverst time. A continous treatment until harvest at 31 hours was used.

48 hours after initiation of lymphocytes cultures are initiated, they are centrifuged at 1000 rpm for 10 min and the culture medium is decanted and replaced with treatment medium.
After exposure period the lymphocytes cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.

A few drops of the cell suspension obtained in this way were dropped onto clean, wet grease-free glass slides.
Three slides were prepared for each test point and each was labelled with the identity of the culture.

The slides were allowed to air dry kept at room temperature prior to staining with a solution of Acridine Orange in PBS.
The slides will be randomly assigned code numbers by a person not subsequently involved in slide evaluation and any other identification marks will be concealed. For the three selected doses, for the solvent and for the negative and positive controls, at least 1000 binucleated cells per cell cultures will be scored to assess the frequency of micronucleated cells, but with the presence of cytokinesis block, a greater magnitude of response may be observed in mononucleated cells in compare with binucleated cells, it is why at least 1000 mononucleated cells per culture may be scored instead of binucleated cells.
Evaluation criteria:
The criteria for identifying micronuclei are as follows: (i) the micronucleus diameter must be less than 1/3 of the diameter of the nucleus, (ii) the micronucleus diameter must be greater than 1/16 of the diameter of the nucleus, (iii) no overlapping with the nucleus must be osberved, (iv) the aspect must be the same as the chromatin. In this assay, the test item is considered as clearly positive if the following criteria are met: - Significant increases in the proportion of micronucleus cells over the concurrent controls occur at one or more concentrations. - The proportion of micronucleus cells at such data points exceeds the normal range. If the increases fall within the range of values normally observed in the negative control cultures, the test item can not be classified as positive. Any significant increases over the concurrent negative controls are therefore compared with historical control values derived from recent studies. - There is a significant dose-relationship
Statistics:
For the statistical analysis, a modified chi-squared test was used tocompare the number of binucleated cells with micronuclei in control and treated cultures.
Species / strain:
lymphocytes: 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
Remarks on result:
other: strain/cell type: lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

On the basis of this results, it is concluded that TMP perlargonate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.
Executive summary:

The test item TMP pelargonate was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. This study was performed in accordance with OECD guideline 487 and GLP without significant deviation.

Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively.

The harvest time of 32 hours corresponding to approximately 2.0 cells cycles was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 31 hours was used. Solutions of the test item were prepared in acetone.

For the main experiment, the maximum dose level for treatment was selected in agreement with the sutdy protocol and on the basis of the solubility of the test item. Dose levels of 625, 357, 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 g/mL were used for the first main experiment. Based on the results obtained, dose levels of 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 g/mL were used for the second main experiment.

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.

The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micronuclei taking into account the cytotoxicity of the test item treatments, calculated by the cytokinesis-block proliferation index (CBPI), and the observed test item precipitation by the end of treatment.

1000 binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrend vehicule control value was observed at any dose level in any treatment series.

Statistically increase in the incidence of micronuclei cells were observed following treatments with the positive controls Cyclosphosphamide, Motomycin-C and Colchicine indicating the correct functioning of the test system.

It is concluded that TMP perlagonate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013-09-24 - 2013-09-25
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial gene mutation assay
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
liver S9 fraction from rat
Test concentrations with justification for top dose:
Toxicity test:

50, 158, 500, 1580 and 5000 µg/plate

Main Assay I:
5000, 2500, 1250, 625 and 313 µg/plate (-S9)
1600, 800, 400, 200, 100 µg/plate (+S9)

In Main Assay II, the test item was assayed at the same dose levels used in Main Assay I.
Vehicle / solvent:
The test item was used as a solution in acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone, DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work PRELIMINARY TOXICITY TEST A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. MAIN EXPERIMENTS Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures. The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The second experiment was performed using a pre-incubation method. The components were added in turn to an empty test-tube. The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify. INCUBATION AND SCORING The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were scored by counting the number of revertant colonies on each plate.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Species / strain:
E. coli WP2 uvr A
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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY TEST
The test item TMP pelargonate was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration in the absence of metabolic activation and at the two highest concentrations in the presence of metabolic activation. No toxicity was observed with any tester strain at any dose level, both in the absence or presence of S9 metabolism. ASSAY FOR REVERSE MUTATION
Two experiments were performed. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate (-S9) and 1600, 800, 400, 200, 100 μg/plate (+S9). No toxicity was observed with any tester strain at any dose level both in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations, both in the absence and presence of metabolic activation. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentrations in the presence of S9 metabolism only. No relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce two-fold increases in the number of revertant
colonies in the plate incorporation or pre-incubation assay, at any dose level,
in any tester strain, in the absence or presence of S9 metabolism.
It is concluded that the test item TMP pelargonate does not induce reverse
mutation in Salmonella typhimurium or Escherichia coli under the reported
experimental conditions.
Executive summary:

The test item TMP pelargonate was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in acetone.

No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate.

As no relevant increase in revertant numbers was observed at any concentration tested in Main Assay I, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item TMP pelargonate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.

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

Genetic toxicity in vivo

Description of key information

Micronucleus test (OECD 474, WoE): negative in mouse bone marrow cells with and without metabolic activation

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
13 May - 08 July 1992
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted in 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK Limited), Margate, Kent, UK
- Age at study initiation: 5-9 weeks for phase I (determination of the maximum tolerated dose) and 7-9 weeks for phase II (Micronucleus test) of the study
- Assigned to test groups randomly: Yes
- Housing: 5 per cage in mobile mouse cage racks, housed per sex
- Diet: Porton Combined Diet, ad libitum
- Water: filtered tap water, ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
The study consisted in two phases: in phase I the maximum tolerated dose (MTD) was determined, on the basis of lethalities or severe toxicity observed over a four-day observation period following a single intraperitoneal injection.
In phase II, male and female animals were weighed and given a single intraperitoneal injection of corn oil (vehicle control), cyclophosphamide (positive control) or test substance prepared in corn oil.

Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 h and 48 h
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: 65 mg/kg bw in physiological saline
Tissues and cell types examined:
Monochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No deaths or severe adverse effects occurred in Phase I of the study with doses up to 5000 mg/kg bw. This dose was selected as MTD.

TREATMENT AND SAMPLING TIMES: 24 h and 48 h after dosing


DETAILS OF SLIDE PREPARATION: Bone Marrow smears were stained with polychrome methylene blue and eosin


METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were evaluated for micronuclei per slide. In addition, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.

Evaluation criteria:
Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point.
Percentage of polychromatic erythrocytes.
Statistics:
The incidence of micronucleated polychromatic erythrocytes and percentage of polychromatic erythrocytes in the erythrocyte sample were considered by analysis of variance regarding each combination of sampling time, dose level and sex as a separate group. Results were examined to determine wether any differences between vehicle control and test substance treated groups were consistent between sexes and across sampling times.
Each group mean was compared with the vehicle control group mean at the corresponding sampling time using a one-sided Student´s t-test based on the error mean square in the analysis.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times.
Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be statistically significant compared to the concurrent control values.
The positive control induced stastistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Mean incidence of micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

 

Table 1: Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 mL/kg

0.8 ± 0.8

1.0 ± 1.2

12

Cyclophosphamide

65 mg/kg

24.4 ± 6.0**

 

13

Test substance

5000 mg/kg

0.6 ± 0.6

0.4 ± 0.6

 

Table 2: Females

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

0.2 ± 0.5

1.4 ± 1.1

12

Cyclophosphamide

65 mg/kg

18.4 ± 7.3**

 

13

 Test substance

5000 mg/kg

0.4 ± 0.9

0.4 ± 0.9

 

 

Mean percentage of polychromatic erythrocytes ± Standard Deviation at two sampling times. n=5

 

Table 3: Males

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

48.0 ± 5.6

44.3 ± 7.5

12

Cyclophosphamide

65 mg/kg

41.4 ± 4.4*

 

13

 Test substance

5000 mg/kg

42.2 ± 7.0*

43.3 ± 1.9

 

Table 4: Female

Group

Compound

Dose

Mean Incidence

24 h

48 h

11

Vehicle control

(corn oil)

10 ml/kg

41.9 ± 4.8

41.9 ± 1.7

12

Cyclophosphamide

65 mg/kg

45.9 ± 3.49

 

13

 Test substance

5000 mg/kg

46.5 ± 5.8

48.0 ± 5.2

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

Additional information

Justification for analogue read-across

Data on the in vitro genetic toxicity in bacterial and mammalian cells of Pentaerythritol, mixed esters with linear and branched fatty acids are not available. The genetic toxicity assessment was therefore based on studies conducted with analogue substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. 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 substance are the basis of read-across. A detailed justification for analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 67762-53-2

The in vitro mutagenic potential of Fatty acids, C5-9, tetraesters with pentaerythritol was assessed in a reverse mutation assay according to OECD Guideline 471 and under GLP conditions (Mecchi, 1999). The plate incorporation method was applied, using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvrA. The tester strains were incubated with test substance dissolved in ethanol at concentrations of 33.3 to 5000 µg/plate, with and without the addition of a metabolic activation system (Aroclor 1254 induced rat liver S9-mix). Vehicle and positive controls were included and shown to be valid. No cytotoxicity was observed. Slight precipitation was observed from 100 µg/plate without metabolic activation, and from 1000 µg/plate with metabolic activation. The test substance did not induce an increase in reversions in the S. typhimurium strains, with or without metabolic activation and is considered to be not mutagenic under the conditions of this study.

 

CAS 126-57-8

A bacterial reverse mutation assay (Ames test) was performed according to a protocol similar to OECD guideline 471 and under GLP conditions. The test item TMP pelargonate was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in acetone. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate. As no relevant increase in revertant numbers was observed at any concentration tested in Main Assay I, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that the test item TMP pelargonate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.

 

CAS 11138-60-6

The potential mutagenicity of Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol was assessed in a bacterial reverse mutation assay (Ames test) according to OECD guideline 471 and under GLP conditions (Bailey, 1996). The test substance was diluted in ethanol and test substance concentrations of 0, 10, 33, 100, 333 and 1000 µg/plate were tested in triplicate on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E.coli WP2 uvr A, with and without metabolic activation. No cytotoxicity was observed, although precipitation was noted at concentrations from 100 µg/plate. No increase in the mean number of revertants per plate was observed when compared to controls. The positive and negative controls included for each tester strain were shown to be valid. Based on the study results, the test substance was considered non-mutagenic in the selected strains of S. typhimurium in the presence and absence of metabolic activation.

 

Genetic toxicity (cytogenicity) in mammalian cells in vitro

CAS 11138-60-6

An in vitro mammalian chromosome aberration test was performed with Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol in Chinese hamster ovary cells (CHO) according to OECD guideline 473 and under GLP conditions (Gudi, 1996). Duplicate cultures of CHO cells were evaluated for chromosome aberrations in the presence and absence of metabolic activation (Arochlor 1254-induced rat liver S9-mix). Cells were exposed to test substance concentrations of 625, 1250, 2500 and 5000 µg/mL for 4 and 20 hours without and for 4 hours with metabolic activation. The fixation time was 20 hours for all exposure times, with and without metabolic activation. Cytotoxicity was observed at the highest concentration tested with and without metabolic activation. The vehicle (solvent) controls induced aberration frequencies within the expected range. The positive controls were shown to be valid. Evaluation of 200 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations at any dose level tested in comparison to the negative controls. The test material was therefore considered to be non-clastogenic to CHO cells in vitro.

 

CAS 126-57-8

The test item TMP pelargonate was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. This study was performed in accordance with OECD guideline 487 and GLP without significant deviation. Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 32 hours corresponding to approximately 2.0 cells cycles was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 31 hours was used. Solutions of the test item were prepared in acetone. For the main experiment, the maximum dose level for treatment was selected in agreement with the sutdy protocol and on the basis of the solubility of the test item. Dose levels of 625, 357, 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 g/mL were used for the first main experiment. Based on the results obtained, dose levels of 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 g/mL were used for the second main experiment. Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. Dose levels were selected for the scoring of micronuclei taking into account the cytotoxicity of the test item treatments, calculated by the cytokinesis-block proliferation index (CBPI), and the observed test item precipitation by the end of treatment. 1000 binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrend vehicule control value was observed at any dose level in any treatment series. Statistically increase in the incidence of micronuclei cells were observed following treatments with the positive controls Cyclosphosphamide, Motomycin-C and Colchicine indicating the correct functioning of the test system. It is concluded that TMP perlagonate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 126-57-8

The test item Trimethylolpropane Tripelargonate was examined for mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistance mutants in mouse lymphoma L5178 cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. This trial was performed in according to OECD 476 guidelines and GLP. A preliminary solubility trial indicated that the maximum feasible concentration of the test item in the final treatment medium was 1250 µg/mL using acetone as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the presence and absence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77, and 4.88 µg/mL. No relevant toxicity was observed at any concentration tested, in any treatment series. Slight opacity was observed at the end of treatment period at the five highest concentrations. No precipitation was noted at any concentration tested. Based on the results obtained in the preliminary toxicity trial, two independent assays for mutation to trifluorothymidine resistance were performed using the following dose levels: Assay n°1 with and without S9, treatment of 3 hours doses: 2500, 791, 250, 79.1, 25 µg/mL Assay n°2: without S9, treatment of 24 hours doses: 2500, 1250, 625, 313, 156, 78.1 µg/mL Assay n°3 with S9, treatment of 3 hours doses: 2500, 1250, 625, 313, 156, 78.1 µg/mL No relevant toxicity was observed at any concentration tested in any treatment series. in both experiments, at the end of the treatment time, test item particles in suspension were noted at the highest or two highest concentrations. No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment, both in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is conclude that Trimethylolpropane Tripelargonate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

 

Genetic toxicity in mammalian cells in vivo

CAS 68424-31-7

An in vivo mammalian erythrocyte micronucleus test was performed according to OECD guideline 474 and under GLP conditions, using Fatty acids, C5-10, esters with pentraerythritol (Griffiths and Mackay, 1992). 5 mice/sex/dose were administered 5000 mg/kg bw of the test substance via intraperitoneal injection and sacrificed after 24 and 48 hours respectively. Bone marrow cells from the femur were extracted, and slides with bone marrow smears were prepared and stained with polychrome methylene blue and eosin. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes compared with the control values were seen in either sex at either of the sampling times. No increase in the percentage of polychromatic erythrocytes was observed in the females, compared with the control group. A small, but significant decrease was noted in treated male mice. This small decrease is considered not to be biologically significant compared with the concurrent control values. No toxicity was observed at the limit dose of 5000 mg/kg bw. The positive control substance (cyclophosphamide) induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes and a reduction in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls, showing the positive control was valid.

 

Overall conclusion for genetic toxicity

There are no studies available on the in vitro and in vivo genetic toxicity of the target substance Pentaerythritol, mixed esters with linear and branched fatty acids. Therefore analogue read-across from source substances was applied from in vitro studies on bacterial and mammalian cells, and from in vivo studies, using five source substances. The results of the available in vitro and in vivo studies on source substances were negative. Based on the available data, and following the analogue approach Pentaerythritol, mixed esters with linear and branched fatty acids is not expected to be mutagenic and clastogenic in vitro and in vivo.

 

Justification for selection of genetic toxicity endpoint

Hazard assessment is conducted by means of read-across from structural analogues. All available in vitro genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

 

Short description of key information:

Genetic toxicity in vitro:

Ames test (OECD 471, WoE): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102 and TA 1538 and in E. coli WP2 uvrA pKM 101

Chromosome aberration (OECD 473/478, WoE): negative in Chinese hamster ovary cells and human lymphocytes with and without metabolic activation

Gene mutation in mammalian cells (OECD 476, WoE): negative in mouse lymphoma L5178Y cells with and without metabolic activation

 

Genetic toxicity in vivo:

Micronucleus test (OECD 474, WoE): negative in mouse bone marrow cells with and without metabolic activation

 

Endpoint Conclusion: No adverse effect observed (negative)

 

 

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Pentaerythritol, mixed esters with linear and branched fatty acids, data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

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


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