Octanoic acid, mixed tetraesters with 2-ethylhexanoic acid, heptanoic acid and pentaerythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

RA study

Justification for type of information:

Refer to the section 13 of IUCLID dataset for details on the read across justification. The bacterial reverse mutation assay with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium TA98; hisD 3052_GC rfa uvrB pKM101cS. typhimurium TA100; hisG 46_GC rfa uvrB pKM101S. typhimurium TA1535; hisG 46_GC rfa uvrBS. typhimurium TA1537; hisC 3076_GC rfa uvrBE. coli WP2uvrA; trpE65_AT rfa uvrA

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose-range finding: 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000 µg/plate
Main test: 0, 78.1, 156, 313, 625, 1250, 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: insoluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min.
- Exposure duration: 48 hrs.

Evaluation criteria:

If the number of revertant colonies on the test plates increased significantly in comparison to that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the results for the test substance was to be judged positive.

Statistics:

No statistical analysis has been done.

Key result

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

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium 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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose range: During test substance treatment, an oil-drop precipitate was observed on the plates with a dose of 2000 μg/plate or more for both treatment methods (±S9). After the completion of pre-incubation, the oil-drop precipitate had disappeared from the plates at 2000 μg/plate or more without S9 treatment (−S9 mix). During measurement of the colony count, an oil-drop and oil-film precipitate was observed on the plates with a dose of 2000 μg/plate or more for both treatment methods.
Main study: During test substance treatment, an oil-drop precipitate was observed on the plates at 1250 μg/plate or more for both treatment methods (±S9). After the completion of pre-incubation, the oil-drop precipitate had disappeared from the plates with a dose of 1250 μg/plate or more without S9 treatment (−S9 mix). During measurement of the colony count, an oil-drop and oil-film precipitate was observed on the plates with a dose of 1250 μg/plate or more for both treatment methods.

RANGE-FINDING/SCREENING STUDIES:

Dose range: The revertant colony count during pentaerythritol tetraiso-octylate treatment showed no clear tendency to increase at any of the doses for any of the study strains, excluding TA1535 (−S9 mix). Bacterial contamination was observed in some of the plates for TA1535 (−S9 mix), so no measurements were made of the colony count. No growth inhibition effects were observed in any of the treatment groups of the study strains.
Main study: In two independent experiments, in the case of the test substance treatment group, no clear trend for an increase in the revertant colony count was observed in any of the strains either with or without metabolic activation. No growth inhibition effects was observed in relation to any of the study strains in any of the treatment groups.

Conclusions:

Under the study conditions, the substance was considered to be non-mutagenic in bacterial reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the read-across substance CAS 7299 -99 -2 according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TAl00 and Escherichia coli strain WP2uvrA' were treated with solutions of the test substance using the Ames protocol at six dose levels with and without metabolic activation (10% liver S9 in standard co-factors). Acetone was used as the negative control and sodium azide, furylfuramide, 9 -aminoacridine and 2 -aminoanthracene were used as the positive controls for different test strains. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at any of the tested doses in the presence and absence of metabolic activation. The negative and positive controls produced expected results and were considered valid. Under the study conditions, the substance was considered to be non-mutagenic in bacterial reverse mutation assay (MHLW, 2005).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

RA study

Justification for type of information:

Refer to the section 13 of IUCLID dataset for details on the read across justification. The in vitro genetic toxicity study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell chromosomal aberration assay

Species / strain / cell type:

other: CHL/IU (Chinese hamster lung fibroblasts)

Details on mammalian cell type (if applicable):

- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Short-term treatment with metabolic activation: 1250, 2500 and 5000 μg/mL
Short-term treatment without metabolic activation: 1250, 2500 and 5000 μg/mL
Continuous treatment: 1250, 2500 and 5000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: For solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION- Exposure duration: 6 hrs. and 24 hrs.
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY- Method: cell growth
OTHER EXAMINATIONS:- Determination of polyploidy: yes

Evaluation criteria:

The frequency (%) of structural aberrations excluding gaps and numerical aberrations was determined. If significant differences were noted in the results between the frequency of test group and that of negative control group and dose-response and reproducibility were also observed, the results were judged to be positive. However final judgment has been done with considering with biological relevance of a result under the test condition.

Statistics:

The statistical analysis was done using Fisher's exact test (Level of Significance: 2.5% at one-side test) for frequency and Cochran-Armitage trend test (Level of Significance: 2.5% at one-side test)for dose-dependency.

Key result

Species / strain:

other: CHL/IU (Chinese hamster lung fibroblasts)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the study conditions, the substance was considered to be non-clastogenic.

Executive summary:

A study was conducted to determine the clastogenic potential of the read-across substance CAS 7299 -99 -2 according to OECD Guideline 473, in compliance with GLP. A chromosome aberration assay using Chinese hamster lung cells (CHL/IU) was performed with the test substance in the presence and absence of metabolic activation at dose levels of 1250, 2500 and 5000 μg/mL. The cells were exposed to the test substance for a duration of 6 h in the presence and absence of metabolic activation and for a duration of 24 h in the absence of metabolic activation. No treatment-related increase in the frequency of chromosome aberrations were observed for both structural and numerical aberrations at any of the doses at either of the exposure durations. The negative and positive controls were considered valid in the assay. Under the study conditions, the substance was considered to be non-clastogenic (MHLW, 2005).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 2nd April 2004 to 14th April 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

two strains were used

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

furylfuramide

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the study conditions, the substance was considered to be non-mutagenic in bacterial reverse mutation assay.

Executive summary:

A study was conducted to determine the mutagenic potential of the substance according to an internal protocol of the testing laboratory. Salmonella typhimurium strains TA98 and TA100 were treated with solutions of the test substance in a pre-incubation method at dose levels of 0, 20, 78, 313, 1250 and 5000 μg/plate with and without metabolic activation. Acetone was used as the negative control and furylfuramide and benzo-a-pyrene were used as the positive controls. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at any of the tested doses in the presence and absence of metabolic activation. The negative and positive controls produced expected results and were considered valid. Under the study conditions, the substance was considered to be non-mutagenic in bacterial reverse mutation assay (Oguma, 2004).

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

15-Jul-2010 to 24-Aug-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

RA study

Justification for type of information:

Refer to the section 13 of IUCLID dataset for details on the read across justification. The in vitro gene mutation study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: -RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: no- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test: Without and with S9-mix, 3 hours treatment: 1, 3, 10, 33 and 100 µg/mL Without S9-mix, 24 hours treatment: 1, 3, 10, 33 and 100 µg/mL
Experiment 1: Without and with S9-mix, 3 hours treatment: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL
Experiment 2: Without S9-mix, 24 hours treatment: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL
With S9-mix, 3 hours treatment: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Short-term treatment With and without S9-mix: 3 hours and Prolonged treatment period: Without S9-mix: 24 hours

- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY - Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.A test substance is considered negative (not mutagenic) in the mutation assay if:a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.b) The results are confirmed in an independently repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 33 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the precipitating dose of 100 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:In the absence and presence of S9-mix, no toxicity was observed up to and including the highest tested dose level in both experiments.

Conclusions:

Under the study conditions, the substance was considered to be non-mutagenic.

Executive summary:

A study was conducted to determine the mutagenic potential of the read-across substance CAS 7299-99-2 according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. The test substance was exposed to L5178Y mouse lymphoma cells in two repeated experiments for a duration of 3 and 24 hours in the presence and absence of metabolic activation at dose levels of 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL. Methyl methane sulfonate and cyclophosphamide were used as positive control substances. Both negative control and positive control substances induced appropriate responses. No treatment-related increases in the mutation frequencies were observed at either of the experiments, confirming the absence of mutagenic potential. Under the study conditions, the substance was considered to be non-mutagenic (Verbaan, 2010).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the mutagenic potential of the read-across substance CAS 7299 -99 -2 according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TAl00 and Escherichia coli strain WP2uvrA' were treated with solutions of the test substance using the Ames protocol at six dose levels with and without metabolic activation (10% liver S9 in standard co-factors). Acetone was used as the negative control and sodium azide, furylfuramide, 9 -aminoacridine and 2 -aminoanthracene were used as the positive controls for different test strains. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at any of the tested doses in the presence and absence of metabolic activation. The negative and positive controls produced expected results and were considered valid. Under the study conditions, the substance was considered to be non-mutagenic in bacterial reverse mutation assay (MHLW, 2005).

A study was conducted to determine the mutagenic potential of the substance according to an internal protocol of the testing laboratory. Only two strains of Salmonella typhimurium strains (TA98 and TA100) were treated with solutions of the test substance in a pre-incubation method at dose levels of 0, 20, 78, 313, 1250 and 5000μg/platewith and without metabolic activation. Acetone was used as the negative control and furylfuramide and benzo-a-pyrene were used as the positive controls. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at any of the tested doses in the presence and absence of metabolic activation. The negative and positive controls produced expected results and were considered valid. Under the study conditions, the substance was considered to be non-mutagenic in bacterial reverse mutation assay (Oguma, 2004).

A study was conducted to determine the clastogenic potential of the read-across substance CAS 7299 -99 -2 according to OECD Guideline 473, in compliance with GLP. A chromosome aberration assay using Chinese hamster lung cells (CHL/IU) was performed with the test substance in the presence and absence of metabolic activation at dose levels of 1250, 2500 and 5000 μg/mL. The cells were exposed to the test substance for a duration of 6 h in the presence and absence of metabolic activation and for a duration of 24 h in the absence of metabolic activation. No treatment-related increase in the frequency of chromosome aberrations were observed for both structural and numerical aberrations at any of the doses at either of the exposure durations. The negative and positive controls were considered valid in the assay. Under the study conditions, the substance was considered to be non-clastogenic (MHLW, 2005).

A study was conducted to determine the mutagenic potential of the read-across substance CAS 7299-99-2 according to OECD Guideline 476 and EU Method B.17, in compliance with GLP. The test substance was exposed to L5178Y mouse lymphoma cells in two repeated experiments for a duration of 3 and 24 hours in the presence and absence of metabolic activation at dose levels of 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 µg/mL. Methyl methane sulfonate and cyclophosphamide were used as positive control substances. Both negative control and positive control substances induced appropriate responses. No treatment-related increases in the mutation frequencies were observed at either of the experiments, confirming the absence of mutagenic potential. Under the study conditions, the substance was considered to be non-mutagenic (Verbaan, 2010).

Justification for classification or non-classification

Based on the in vitro genetic toxicity studies with the test substance and the read-across substance, no classification is warranted according to EU CLP (EC 1272/2008) criteria.