Pentaerythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pentaerythritol has been tested for in vitro genetic toxicity in a battery of tests. The bacterial mutation test, mammalian mutation test and chromosome aberration test were negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 September 1993 to 1 December 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Pentaerythritol (abbreviated to PE in the report), molecular weight 136.2, supplied as a white powder from Japan Chemical Industry Association, manufactured by Mitsubishi Gas Chemical Company. Purity is 92.7% (Lot number: 50825; Containing impurities such as Dipentaerythritol (2.2%), Bis-Pentaerythritol (4.9%), and unknown impurities of (0.2%).

Stability tests were carried out on the test solutions at the Hatano Research Institute. The lowest concentration (3.125 mg/ml) and the highest concentration (50 mg/ml) were sent for analysis. Three test solutions per concentration were stored at room temperature in the dark for 4 hours. The mean analytical concentrations were 103% (low concentration) and 101% (high concentration) of the starting value in the preliminary test, and were 97.1-99.8% (low concentration) and 95.2-98.7% (high concentration). All values were within the permissbale range stipulated by the laboratory, and thaht the test solution was stable for the duration of the test.

Target gene:

Histidine

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from the livers of male Sprague-Dawley rats induced with phenobarbitol and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary study: 50 to 5000 µg/plate, with a dose spacing factor of approximately 3.
Main study: 312.5 - 5000 µg/plate, with a dose spacing factor of approximately 2.

Vehicle / solvent:

'Pure water'. 50 mg PE was dissolved per ml of pure water, further dilutions were prepared from this stock solution for immediate use in the experiment.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Bacteria were frozen and stored at < -80°C. At the time of freezing, bacteria were examined for the absence or presence of amino acid requirement, UV radiation sensitivity, membrane mutation (rfa) and ampicillin resistance factor (pKM101). Bacterial cultures were prepared by inoculating the bacteria in an L-shaped test tube containing Nutrient Broth No. 2 (Oxoid), followed by approximately 10 hours incubation at 37°C.

The plate incorporation method was used. 2 ml top agar, 0.1 ml prepared test substance solution, 0.5 ml phosphate-buffered solution or 0.5 ml S9 mix, and 0.1 ml bacteria culture were mixed together in a small test tube and the synthetic culture medium was poured into a flat plate and solidified. In the control group, pure water or the positive control substance solution were used instead of the prepared test substance solution. The medium was cultured at 37°C for 48 hours and the number of mutant colonies counted. Bacterial toxicity was assessed bu examining the bacterial lawn with the naked eye or a microscope.
In the dose-finding study, 3 plates were employed for each of the negative and positive control groups and 1 plate was employed for each respective dose. In the main experiment, 3 plates were employed for each respective dose and for both control groups. The dose-finding study was conducted once and the main experiment was conducted twice.

Evaluation criteria:

The substance was deemed mutagenic if a reproducible doubling or dose-related increase in the number of revertant colonies was seen, compared to negative controls.

Statistics:

No information on statistics is provided in the report.

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:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

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:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no evidence of cytotoxicity in the preliminary dose-finding study, up to and including the highest concentration of 5000 µg/plate.
In the main study, there were no increases in the number of revertant colonies compared to solvent only controls and no dose-response relationship was evident in the number of revertants (tested up to 5000 µg/plate). This result was confirmed in the independent repeat assay.

All positive and solvent control results were reported to be within the range of historical control data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

There was no evidence of cytotoxicity in the preliminary dose-finding study, up to and including the highest concentration of 5000 µg/plate. In the main study, there were no increases in the number of revertant colonies compared to solvent only controls and no dose-respone relationship was evident in the number of revertants (tested up to 5000 µg/plate). This result was confirmed in the independent repeat assay.

All positive and solvent control results were reported to be within the range of historical control data.

Conclusions:

Pentaerythritol was found to be negative in the Ames test with and without metabolic activation, tested up to a concentration of 5000 µg/plate.

Executive summary:

The mutagenic potential of Pentaerythritol was determined in the bacterial reverse mutation assay (Ames test). Salmonella typhimurium strains TA100, TA1535, TA1537 and TA98 were tested along with Escherichia coli strain WP2 urvA in both the presence and absence of metabolic activation (S9 mix). A preliminary cytoxicity test was conducted, in which no evidence of bacterial toxicity was seen up to and including concentrations of 5000 µg/plate. In the main study, there were no increases in the number of revertant colonies compared to solvent only controls and no dose-respone relationship was evident in the number of revertants (tested up to 5000 µg/plate). This result was confirmed in the independent repeat assay.

No evidence of mutagenicity was seen under the conditions of this assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 September 1993 to 19 December 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Proprietary GLP guideline compliant study. A translation has been provided, however the raw data from the study are not included.

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 chromosome aberration test

Specific details on test material used for the study:

Pentaerythritol (abbreviated to PE in study report), manufactured by the Mitsubishi Gas Chemical Company Inc, supplied by the Japan Chemical Industry Association. PE has a molecular weight of 136.2, and was supplied as a white powder. The purity is 97.2%; containing the impurities bis-pentaerythritol (4.9 %) and dipentaerythritol (2.2 %). Storage was at room temperatire with an air-tight stopper, and protected from light.

Stability in solution (distilled water) was confirmed analytically for 4 hours. Analytical concentrations of the lowest and highest dose solutions were confirmed to be within the range permissable by the laboratory (90-110%).

Target gene:

Not applicable - chromosome aberration study

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Details on mammalian cell type (if applicable):

The cells were Chinese hamster lung cells (CHL/IU; abbreviated to CHL). The cells were obtained from the Research Resource Bank. Cells were used that were at passage 10 or less. The cells were stored frozen.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from the livers of Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary cell growth assay: 0.04 to 1.40 mg/ml.
Main assay: 0.4, 0.7 and 1.40 mg/ml.

Vehicle / solvent:

Water for injection. The test substance was dissolved in water for injection to give a stock solution of 14 mg/ml for further dilution. The test substance solution was adjusted to 10% v/v of the solvent in all tests. Solutions were prepared immediately prior to use.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Cells were cultured in Eagle MEM medium supplemented with 10% foetal calf serum. 2x10E4 cells were plated in a culture dish containing 5 ml culture medium and incubated at 37°C and 5% CO2 for 3 days.

Direct plating method: the medium was replaced with fresh culture medium (after the 3 day incubation) followed by addition of the test substance solution. Cells were incubated for 24 or 48 hours.

Metabolic activation: the medium was replaced with fresh culture medium (after the 3 day incubation), followed by addition of the test substance solution and S9 mix was added where appropriate. The cells were incubated for 6 hours with the test substance, then the culture medium was replaced with fresh medium and the cells were incubated for a further 18 hours.

A preliminary test was conducted to determine the effect of pentaerythritol on cell growth, according to the 48 hour direct plating method and the S9 presence/absence method. Concentrations of 0.04 to 1.40 mg/ml were tested, and plated in duplicate. After the 48 hour incubation period, the culture medium was discarded and the cells were fixed in position using 10% formalin. The cells were then stained with 0.1% crystal-violet.

In the main test, both the direct plating method and the metabolic activation method were used. In the case of direct plating, control included negative controls, solvent controls and the postive control mitomycin C, along with the 3 concentrations of test substance, all plated in duplicate. Treatment times were 24 or 48 hours (2 plates/incubation time/treatment). In the case of the metabolic activation method, negative controls, solvent controls and the positive controls (mitomycin C for cultures treated in the absence of S9, and cyclophosphamide in the presence of S9) along wiith 3 concentrations of the test substance were plated in duplicate, either in the presence of the absence of S9. Cultures were incubated for 6 hours, then transferred to fresh medium and incubated for a further 18 hours.

Chromsome sample preparation:
Two hours before the end of the incubation period, Colcemid was added each culture. After completion of the incubation period, the cells with phosphoric acid buffer solution and adherent cells were detached by pipetting. The cells were gathered into a 10 ml centrifuge tube. The cells were centrifuged for 5 minutes, and the supernatant discarded. 3 ml of 0.075 M KCl aqueous solution was added to the cells and incubated for 30 minutes. 6 ml of fixative - glacial acetic acetic acid: methanol (1:3 v/v) was added, then the cells were centrifuged for a further 5 minutes. The upper layer of cells was discarded, and the cells were resuspended in fresh fixative. The fixative step was repeated several times. A small amount of cell suspension was dropped onto a previously-cleaned glass slide and allowed to air dry. Six slides were prepared from each culture. The dried slides were stained with Giemsa. The slides were coded prior to analysis by blinded observers. Chromosome abberations were viewed under a microscope and recorded for 200 cells per slide.

Evaluation criteria:

Growth inhibition was determined by measuring the optical density of the cells (Monocellater, Olympus). The growth index was calculated by calculating the ratio of the density of cells in treated groups to that of the solvent control.
Chromosome aberrations were recorded as follows: chromsome gaps, chromatid gaps, chromsome exchange, chromatid exchange, polyploidy. The evaluation criteria were that of Ishidate. A test substance was negative if less than 5% cells showed chromosome aberrations, between 5% and 10% was considered equivocal, and above 10% was considered positive.

Statistics:

Fisher's Exact Proability test

Species / strain:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no increases in the frequency or type of chromosomal aberrations in either treatment method, with or without metabolic activation, when compared to solvent controls.

There were no increases in the frequency or type of chromosomal aberrations in either treatment method, with or without metabolic activation, when compared to solvent controls.

Conclusions:

Pentaerythritol was negative in the chromosome aberration test with Chinese Hamster Lung cells cultured in vitro, both with and without metabolic activation.

Executive summary:

Pentaerythritol was tested for clastogenic properties in the chromosome aberration test, using cultured mammalian cells in vitro (Chinese Hamster Lung cells). The test substance was tested up to a concentration of 1.4 mg/ml, both in the presence and absence of metabolic activation. There were no increases in the number or type of chromosome aberrations in treated cultures, compared to solvent controls. No evidence of clastogenicity was seen under the conditions of this assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 October to 17 December 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Type of assay:

other: mammalian cell gene mutation assay

Specific details on test material used for the study:

Pentaerythritol Mono Grade (Batch 3910069389), a white crystalline solid, was received from Perstorp Holding AB on 04 September 2009 and was stored in the dark at ambient room temperature. The purity was quoted as 99% and the Sponsor supplied an expiry date of 03 September 2011. A Certificate of Analysis was provided.

Target gene:

tk+tk- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The cells used were from the tk+/- -3.7.2C mouse lymphoma L5178Y cell line obtained from Dr D Clive, Burroughs Wellcome & Company, Research Triangle Park, NC27709, USA, in December 1982. The cells were reported to grow in suspension culture, have a generation time of about 11 h, have a stable, near-diploid chromosome number and have a high cloning efficiency in serum-enriched cloning medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from the livers of Aroclor 1254-induced adult male Fischer rats

Test concentrations with justification for top dose:

Toxicity test: 0.14, 0.41, 1.36, 4.08, 13.61, 40.83, 136.1, 408.3 and 1361 μg/mL.
Mutation assays: 100, 500, 900 and 1361 μg/mL.

Vehicle / solvent:

Pentaerythritol Mono Grade was miscible with the basic tissue culture medium, R0P, which was therefore chosen as the vehicle to prepare the Pentaerythritol Mono Grade formulations throughout the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

Single cultures were treated for each concentration in the initial toxicity tests. In the mutation assays, duplicate cultures were treated for all Pentaerythritol Mono Grade concentrations. All experimental procedures were conducted using aseptic technique and under amber light. All water used in the preparation of reagents was produced in-house, by reverse osmosis followed by mixed-bed deionisation and sterilisation by autoclaving.

Toxicity test: Only 1 culture was prepared for each treatment. The toxicity test was performed using the standard 4 h exposure period in the absence and presence of S9 mix. An additional toxicity test was performed in the absence of S9 mix with 24 h exposure to Pentaerythritol Mono Grade, as a contingency against the later requirement for a full experiment using this extended exposure period. Observations on the precipitation of Pentaerythritol Mono Grade were made after dosing and at the end of the exposure period. Observations of pH change (colour change in indicator in RPMI medium) were made and if any change was noted, pH measurements were made.

Mutation tests.
4 hour exposure period: On the day of the test (Day 0), samples of cell culture (in 5 mL R10P) were dispensed to sterile tubes. Freshly prepared S9 mix or R0P (1 mL) was added to each tube followed by 4 mL of test solution. Vehicle control cultures received 4 mL R0P. Positive control cultures received 0.1 mL of the appropriate solution plus 3.9 mL R0P. The final reaction mixture in all cultures contained 10 mL of cells, at a population density of ca 6.0 x 105 cells/mL, in R5P medium. All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 4 h. After this, the cells were gently sedimented by centrifugation at ca 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated to give a cell density of ca 3 x 105/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3 x 105 cells/mL on Day 1.
24 hour exposure: On the day of the test (Day 0), samples of cell culture (in 10 mL R10P) were dispensed to sterile tubes. R50P (R0P:serum, 50:50) (2 mL) was added to each tube followed by 8 mL of the test solution. Vehicle control cultures received 8 mL R0P. Positive control cultures received 0.2 mL of the appropriate solution plus 7.8 mL R0P. The final reaction mixture in all cultures contained 20 mL of cells, at a population density of ca 3 x 105 cells/mL, in R10P medium. (The larger volumes allow the same numbers of cells to be treated as in the experiments conducted at 4 h exposure, but at half the density. The lower density is required to allow cell growth during the exposure period. The serum concentration is not lowered, as some essential nutrients can become exhausted during the exposure period.) All tubes were incubated on a rotating drum at ca 37°C, 10 r.p.m. for 24 h. After this (on Day 1), the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in R10P medium (20 mL). This step was repeated. Cell counts were made and the densities adjusted (where higher) to give ca 3 x 105 cells/mL. The cells were returned to the rotating drum and allowed to express their genetic lesions at ca 37°C for 2 days. Cell numbers were adjusted, after counting, to ca 3 x 105 cells/mL on Day 2.

On Day 2 (4 h exposure) or Day 3 (24 h exposure), cell counts were determined. The cell counts over the 2 or 3 days of the experiments provided a measure of suspension growth. This in turn provided a measure of RSG. The cultures were then assessed for expression of genetic damage. This was determined by performing two parallel cloning assays: the cloning efficiency assay and the mutant selection assay. For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well. For the mutant selection assay, TFT stock solution was added to cloning medium to give a final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an estimated 1 x 104 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 2000 cells per well. All dishes were incubated at ca 37°C in an atmosphere of 5% CO2:95% air (v/v) until the colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay).
The plates were scored using a dissecting microscope fitted to a light box with dark field illumination. The number of empty wells in each plate in the cloning efficiency assay was counted. When scoring the mutant selection assay, separate counts were made of the numbers of wells containing large type and small type colonies. Large colonies are defined as covering greater than ¼ of the floor of the well, while small colonies cover less than ¼ of the well. Any wells containing both colony types were scored as a large type.

Evaluation criteria:

The measure used to assess toxicity in the Toxicity Test was therefore relative suspension growth. The cell population densities were recorded over 2 days (following treatment) using a haemocytometer, then the total suspension growths were expressed as percentages of the vehicle control group (= relative suspension growth, or RSG).
Mutagenic activity: assessment of log mutant fractions, relative total growth and colony size fractions. Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value

Statistics:

Survival and mutant data were available for 4 experiments, two without S9 mix (Assays 1 and 3) and two with S9 (Assays 2 and 4). Assays were measured over an exposure period of 4 hours or 24 hours. Data were analysed using methods outlined in Robinson et al (1989).

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:

not applicable

Positive controls validity:

valid

Additional information on results:

The results of the toxicity tests show that Pentaerythritol Mono Grade did not cause an appreciable reduction in cell growth at the predetermined maximum concentration of 1361 μg/mL (= 10 mM) in either the 4 h exposure system (absence and presence of S9 mix) or the 24 h exposure system.

In mutation assays 1 (without S9) and 2 (with S9), 4 hour exposure (Tables 1 and 2 respectively): None of the assessed concentrations tested significant for increase in log mutant fraction in either experiment. In both experiments the test for linear trend was not reported as the slope was negative. There was no toxicity at any concentration in either experiment. Both experiments were classed negative, and therefore the second experiment in the absence of S9 mix was conducted with the extended, 24 h exposure period.

In mutation assays 3 (without S9, 24 hour exposure) and 4 (with S9, 4 hour exposure) (Tables 3 and 4, respectively): None of the assessed concentrations tested significant for increase in log mutant fraction in either experiment. In the absence of S9 mix, the test for linear trend showed a low level of significance (P = 0.040). This was dismissed as coincidence as the largest increase in mutant fraction at any concentration was only 30 mutants per million above the vehicle control group
and the mutant fraction at that concentration (84 mutants per million) was well within the normal vehicle control range of 51-129 and was also within the mean +1 standard deviation of the historical vehicle control range. In the presence of S9 mix, the test for linear trend was not reported as the slope was negative. There was no toxicity at any concentration in either experiment. Both experiments were classed negative.

A combined statistical analysis of Assays 2 and 4 (where all treatment conditions and concentrations of test item were identical) was made. There were no statistically significant differences between the Pentaerythritol Mono Grade-treated groups and the vehicle controls. The test for linear trend was not reported as the slope was negative.

The solvent control mean mutant fractions were within the normal ranges experienced in the test laboratory. All positive control log mutant fractions were significantly higher than the vehicle controls at P<0.05.

Mutation test in the absence of S9, 4 h exposure, summary of means (Assay 1)

Chemical	Concentration (µg/mL)	Relative Total Growth (%)	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies	Statistical Comparison
Vehicle (R0P)	(4 mL added)	100	68	N/A	1.11	N/A
EMS	250	52	460	392	0.57	*
MMS	10	27	707	640	3.41	*
Pentaerythritol	100	88	86	19	0.90	NS
	500	86	41	-	1.11	NS
	900	95	55	-	1.73	NS
	1361	82	65	-	1.05	NS

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

∗= Significant difference in log mutant fraction compared with vehicle control (P<0.05)

NS = Not Significant

Test for linear trend of mutant fraction with concentration of Pentaerythritol Mono Grade = not reported – slope

negative

-  = IMF≤ 0

Mutation test in the presence of S9, 4 h exposure, summary of means (Assay 2)

Chemical	Concentration (µg/mL)	Relative Total Growth (%)	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies	Statistical Comparison
Vehicle (R0P)	(4 mL added)	100	84	N/A	1.36	N/A
3-MC	2.5	79	540	457	1.37	*
	10	60	685	601	1.61	*
Pentaerythritol	100	97	93	9	1.14	NS
	500	126	78	-	2.05	NS
	900	97	106	22	1.07	NS
	1361	113	73	-	0.64	NS

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

∗= Significant difference in log mutant fraction compared with vehicle control (P<0.05)

NS = Not Significant

Test for linear trend of mutant fraction with concentration of Pentaerythritol Mono Grade = not reported – slope

negative

-  = IMF≤ 0

Mutation test in the absence of S9, 24 h exposure, summary of means (Assay 3)

Chemical	Concentration (µg/mL)	Relative Total Growth (%)	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies	Statistical Comparison
Vehicle (R0P)	(8 mL added)	100	54	N/A	1.24	N/A
EMS	150	30	1881	1827	0.31	*
MMS	5	26	1604	1550	1.81	*
Pentaerythritol	100	112	60	6	1.00	NS
	500	97	50	-	1.08	NS
	900	91	76	22	0.94	NS
	1361	89	84	30	0.89	NS

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

∗= Significant difference in log mutant fraction compared with vehicle control (P<0.05)

NS = Not Significant

Test for linear trend of mutant fraction with concentration of Pentaerythritol Mono Grade = significant (P=0.040)

-  = IMF≤ 0

Mutation test in the presence of S9, 4 h exposure, summary of means (Assay 4)

Chemical	Concentration (µg/mL)	Relative Total Growth (%)	Mutant Fraction (x 10^-6)	IMF (Induced Mutant Fraction x 10^-6)	Ratio of Small to Large Colonies	Statistical Comparison
Vehicle (R0P)	(4 mL added)	100	125	N/A	1.46	N/A
3-MC	2.5	76	565	439	1.13	*
	10	70	764	638	0.93	*
Pentaerythritol	100	107	117	-	0.95	NS
	500	108	107	-	1.56	NS
	900	114	118	-	1.30	NS
	1361	107	111	-	0.99	NS

IMF = Mutant fraction of treatment minus mutant fraction of vehicle control group

N/A = Not Applicable

∗= Significant difference in log mutant fraction compared with vehicle control (P<0.05)

NS = Not Significant

Test for linear trend of mutant fraction with concentration of Pentaerythritol Mono Grade = not reported – slope

negative

-  = IMF≤ 0

Conclusions:

No evidence of mutagenicity was seen under the conditions of this assay.

Executive summary:

Pentaerythritol Mono Grade was assayed for mutagenic potential in the mouse lymphoma L5178Y cell line, clone -3.7.2C, scoring for forward mutations at the thymidine kinase locus: tk+/- to tk-/-.

Pentaerythritol Mono Grade was dissolved and diluted in tissue culture medium. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix). In preliminary cytotoxicity tests, Pentaerythritol Mono Grade was shown was shown to be non-toxic at the predetermined maximum concentration of 1361 μg/mL (10 mM). Positive control cultures were included, and the resultant mutant fractions from these provided the expected increase and proof of adequate recovery of ‘small’ type colonies. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate.

Four independent mutation assays were conducted: Assay 1 - without S9 4 h exposure; Assay 2 - with S9, 4 h exposure; Assay 3 - without S9, 24 hour exposure; Assay 4 - with S9 4 hour exposure. The only significant finding was a low level linear trend of mutant fraction with concentration of Pentaerythritol Mono Grade in the experiment conducted in the absence of S9 mix with a 24 h exposure. This result was dismissed as coincidence, as the increases recorded throughout the concentrations were very small and the mutant fractions were all comparable to normal vehicle control values. No other evidence of mutagenic activity was obtained with Pentaerythritol Mono Grade in any of the 4 assays. It was concluded that Pentaerythritol Mono Grade is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested to the predetermined maximum concentration of 1361 μg/mL (10 mM)

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study, conduted according to scientifically acceptable methods.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The study followed an adapted Ames method, however it does not appear that concentrations were plated in duplicate.

GLP compliance:

no

Remarks:

published Japanese study

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Pentaerythritol, purity 90%, chemical structure C(CH2OH)4, obtained from Wako Pure Chemical Industries, Ltd., Japan.

Target gene:

Not specified

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:

S9 fraction obtained from the livers of male Sprague-Dawley rat pretreated with polychrlorinated biphenyl (KC 500).

Test concentrations with justification for top dose:

0, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate.

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl_-3-(5-nitro-2-furyl)acrylamide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: 2AA

Details on test system and experimental conditions:

The preincubation method was used. Histidine and biotin were used in the top agar for Salmonella strains, whilst tryptophan was used in the top agar for E. coli. The test substance was dissolved in water to achieve the required test concentrations. 0.1 ml of bacterial strain and 0.5 ml of S9 mix or sodium phosphate buffer (pH 7.4) were added to a sterile test tube containing 0.1 ml of the test substance dilution. The mixture was preincubated in a shaker water bath at 37°C for 20 minutes, and then added to 2 ml of molten top agar (45°C). The contents of each tube were mixed and poured onto a minimal glucose agar plate immediately. The plates were incubated at 37°C for 24 hours, and then the number of revertant colonies on each plate was scored with an automated colony counter. All positive control tests were performed in duplicate. A contamination test was carried out in each experiment; the background bacterial lawn was checked routinely by microsope.

Evaluation criteria:

The number of revertant colonies was compared between treated plates and controls.

Statistics:

None reported.

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:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

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:

not applicable

Positive controls validity:

valid

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 valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no evidence of mutagenicity at any concentration or strain tested, and there was no evidence of cytotoxicity at the limit concentration. The authors report the results for a numbre of chemicals, indicating the sensitivity of the assay in detecting negative and positve results.

Number of revertant colonies per plate

Dose µg/plate	Revertant colonies/plate
	TA100		TA1535		WP2uvrA		TA98		TA1537		TA1538
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0 (H2O control)	149±17.1	161±16.2	28±6.9	15±3.6	32±7.3	33±10.3	29±6.2	39±8.6	16±6.4	21±8.1	21±5.5	28±7.0
5	147	130	30	17	27	30	29	35	19	26	13	19
10	132	131	33	20	28	31	28	39	15	34	24	29
50	134	137	31	13	31	28	15	37	15	24	26	19
100	131	132	27	14	30	34	30	28	17	21	20	27
500	132	131	31	18	30	30	27	35	19	21	21	26
1000	159	130	28	13	31	28	21	33	15	26	18	34
5000	145	133	33	14	34	30	23	33	16	24	25	27

Control data presented as mean±SD

Conclusions:

Pentaerythritol was not mutagenic in the Ames test with and without metabolic activation up to concentrations of 5000 µg/plate.

Executive summary:

The mutagenic potential of pentaerythritol was investigated in the bacterial reverse mutation assay. Salmonella typhimurium strains TA100, TA98, TA1535, TA1537 and TA1538, and Escherichia coli strain WP2uvrA were tested in the preincubation method, both with and without metabolic activation (S9 mix obtained from the livers of male Sprague-Dawley rats pretreated with polychlorinated biphenyl). The substance was diluted in water and tested at concentrations of 0, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate (concentrations were not plated in duplicate). After incubation the number of revertant colonies were scored per plate. Pentaerythritol did not cause an increase in the number of colonies in any strain, at any dose tested both with or without S9 activation, and there were no signs of cytotoxicity at the limit concentration.

No evidence of mutagenicity was seen under the condtions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Pentaerythritol was found to be negative in a good quality Ames test performed in S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 (Shimizu, 1994) and in a further good quality performed in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in E. coli WP2 (Shibuya, 1994). No evidence of mutagencitiy was seen in cultured CHL cells (Tanaka, 1994). A modern, proprietary GLP and guideline-compliant mouse lymphoma assay also reports a negative result (Riach, 2010).

Pentaerythritol has therefore been adequately tested for genetic toxicity according to REACH requirements. The results of the available in vitro studies are all clearly negative, therefore additional testing in vivo is not required.

Justification for selection of genetic toxicity endpoint
A weight of evidence approach is taken to this endpoint. No single study is identified as key.

Short description of key information:
No evidence of mutagenicity was seen in two Ames tests, a study of clastogenicity in cultured CHL cells and in a mouse lymphoma assay performed with pentaerythritol.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The results of the available genetic toxicity studies are negative. No classification is therefore required according to Regulation (EC) number 1272/2008.