Reaction mass of Pentaerythritol bis (2-ethylhexanoate) bis (3,5,5-trimethylhexanoate) and Pentaerythritol tris (2-ethylhexanoate) 3,5,5-trimethylhexanoate and Pentaerythritol 2-ethylhexanoate tris (3,5,5-trimethylhexanoate) and Pentaeryhthritol tetrakis(2-ethylhexanoate) and Pentaerythritol tetrakis (3,5,5-trimethylhexanoate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 September 2019 - 26 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

415-650-4

EC Name:

-

Cas Number:

153965-54-9

Molecular formula:

not applicable (multi-constituent)

IUPAC Name:

3-[(2-ethylhexanoyl)oxy]-2,2-bis({[(2-ethylhexanoyl)oxy]methyl})propyl 2-ethylhexanoate; 3-[(2-ethylhexanoyl)oxy]-2,2-bis({[(2-ethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-ethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-ethylhexanoyl)oxy]-2-{[(2-ethylhexanoyl)oxy]methyl}-2-{[(3,5,5-trimethylhexanoyl)oxy]methyl}propyl 3,5,5-trimethylhexanoate; 3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate

Test material form:

liquid

Details on test material:

- Appearance: Light yellow liquid
- Storage condition: At room temperature

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

Direct plate assay:
Experiment 1
Preliminary test (without and with S9-mix) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Main study:
TA1535, TA1537 and TA98 (without and with S9-mix): 52, 164, 512, 1600 and 5000 μg/plate
Pre-incubation assay:
Main study:
TA1535, TA1537, TA98, TA100 and WP2uvrA (without and with S9-mix): 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of vehicle: The test item formed a clear (colourless) solution in ethanol. Test item concentrations were used within 3.5 hours after preparation.

Controls

Controls 1

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:

Remarks:

Without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA) in DMSO, 1 μg for TA98 and TA100 (Direct plate assay), 2.5 μg for TA1535 and TA1537, 5 μg for TA100 (Pre-incubation assay), 15 μg for WP2uvrA

Remarks:

With S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

METHOD OF TREATMENT/ EXPOSURE:
in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Amount of revertant colonies.

OTHER:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 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 TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not performed.

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of RB68 on the plates was observed at the concentration of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES (if applicable): RB68 was initially tested in the tester strains TA100 and WP2uvrA 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 relevant decrease in the number of revertants were observed.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The negative control values were within the laboratory historical control data ranges, except the response for TA1537 in the absence of S9-mix in the first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (2 revertant colonies) when compared against relevant historical control data (3 revertant colonies), the validity of the test was considered to be not affected.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Ames test:
- Signs of toxicity : There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
- Genotoxicity results : All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 128 – 1530 73 – 1481 58 – 1422 54 – 1239 365 – 1978 250 – 2018
Mean 919 256 802 328 1305 910
SD 172 122 362 154 236 355
n 3215 3122 2777 3187 3202 3216

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1993 408 - 2379 93 – 1999 109 - 1968
Mean 907 1308 1073 437
SD 167 386 537 158
n 3231 3179 2923 2987
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Apr 2016 and Apr 2019.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 3 – 27 3 – 20 3 – 23 8 - 61 8 – 60 61 – 176 60 - 176 10 – 61 9 - 68
Mean 10 11 6 6 16 22 112 108 27 33
SD 3 3 2 3 5 7 18 21 8 9
n 3303 3265 3232 3212 3251 3326 3336 3246 3021 2993
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Apr 2016 and Apr 2019.

Any other information on results incl. tables

 Formulation Analysis

Accuracy

In the vehicle, no test item was detected.

The concentrations analyzed in the dose formulation samples at low concentration level were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

For the dose formulation samples at intermediate and high concentration level the mean accuracy was slightly below the target concentration (i.e. 87% and 88% of target, respectively). This is just below the criterion range of 90-110% but comparable to the recovery of the QC samples at comparable levels. An out of specification investigation was performed. No explanation for the low accuracies could be found. The results were accepted.

Homogeneity

The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).

Applicant's summary and conclusion

Conclusions:

In an Ames test, performed according to OECD 471 guideline and GLP principles, RB68 was found not to be mutagenic with or without metabolic activation.

Executive summary:

An Ames test was performed according to OECD 471 guideline and GLP principles.

RB68 was initially tested in the tester strains TA100 and WP2uvrA 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. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

RB68 precipitated on the plates at the highest dose level only. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, RB68 was tested up to the dose level of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

All other bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

In conclusion, based on the results of this study it is concluded that RB68 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.