2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxicity of dipentaerythritol was assessed in a weight of evidence approach.

 

Weight of evidence approach:

Dipentaerythritolwas tested negative in a bacterial reverse gene mutation test conducted in 1992 according to OECD 471, in a second bacterial reverse gene mutation test with an additional strain conducted in 2020 according to OECD 471, in an in vitro chromosome aberration assay conducted according to OECD 473 and in a mammalian cell gene mutation assay conducted according to OECD 476. Based on results, the test item was considered to not induce genetic toxicity .

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06.09.1991 to 04.02.1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): dipentaerythritol
- Substance type: white powder
- Physical state: solid

Target gene:

hisG46, hisC3076 and hisD3052.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent auxotrophic mutants

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: histidine dependent auxotrophic mutants

Metabolic activation:

with and without

Metabolic activation system:

-S9 mix obtained from the livers of Aroclor-1254 induced male Sprague-Dawley rats
- S9 mix contained: S-9 fraction (10% v/v), MgCl2 (8mgM), KCl (33mM). sodium phosphate buffer PH 7.4 (100mM), glucose-6-phosphate (5mM), NADP (4mM).
-the volume of S9 mix used in the culture is detailed in the "details on test system and experiment conditions" section below.

- quality controls of S9 : All the cofactors in S9 mix were filter sterilised before use. The S-9 fraction was tested with 7,12-dimethylbenxanthracene and 2-aminoanthracene before use.

Test concentrations with justification for top dose:

Range finding: solvent control, 5, 50, 500, 5000 µg/plate.
Mutation test: solvent control, 0, 50, 150, 500, 1500, 5000 µg/plate.

-Justification for the top dose: in the preliminary toxicity test, the test substance was not toxic towards the tester strains. therefore 5000 µg/plate was chose as the tope dose level in the mutation tests.

Vehicle / solvent:

dimethyl sulphoxide
- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test material in solvents compatible with the test system was assessed. The chosen solvent was dimethyl sulphoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9: 5 µg/plate for TA1535, and 3 µg/plate for TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9; 2 µg/plate for TA1538, 1 µg/plate for TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9: 2 µg/plate for TA1535 and TA1537, 0.5 µg/plate for TA1538 and TA98, 1 µg/plate for TA100

Details on test system and experimental conditions:

The plate incorporation method was used. The Salmonella strains used in the experiment were: TA1535 hisG46 rfa uvrB, TA1537 hisC3076 rfa uvrB, TA1538 hisD3052 rfa uvrB, TA98 hisD3052 rfa uvrB pKM101, and TA100 hisG46 rfa uvrB pKM101.
Batches of the strains were stored at -80°C. Each frozen batch was tested for cell membrane permeability and, where applicable, for ampicillin resistance. The response of the strain to a series of diagnostic mutagens was also assessed.
Fuse in tests, an aliquot of frozen culture was added to 25 ml of nutrient broth (DAB 7, Merck) and incubated, with shaking, at 37°C for 10 hours. This culture provided approximately 2x10exp9 cells per ml which was checked photometrically.

Preliminary toxicity test
Four concentrations of test substance were assessed for toxicity using the five tester strains. The highest concentration was 5000 µg/plate. The chosen solvent was used as the negative control. 0.1 ml bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1M phosphate buffer (pH7.4) were placed in glass bottles. 0.1 ml of the test substance solution was added, following immediately by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. A single pertri dish was used for each dose level. Plates were also prepared without the addition of bacteria in order to test the sterility of the test substance, S9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background lawn was examined. Revertant colonies were counted using a Biotran Automatic Colony Counter.

Concentrations for the main study were chosen based on toxic effects as assessed by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Mutation test
The test substance was added to cultures of the five tester strains at five concentrations separated by c. half-log10 intervals. The highest concentration used was 5000 µg/plate. The negative control was the chosen solvent. The plate incubation method was used as in the preliminary toxicity test. Three petri dishes were used per dose level. The mutation test was independently repeated using the same concentration of the test substance.

Rationale for test conditions:

-Justification of the top does and vehicle choice were detailed in the relevant sections above.
-Justification of the number of plates per concentration: three petri dishes were used per does level which is aligned the OECD 471 suggestion.

Evaluation criteria:

The mean number of revertant colonies was compared between treated groups and the solvent control. The following criteria were used in assessing the mutagenic response:
- the substance was considered to be mutagenic if there was at least twice the number of revertant colonies compared to the control, with some evidence of a dose-response relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S 9 mix. No statistical analysis is performed.


- the substance was considered to be non-mutagenic if there was no reproducible increase of at least 1.5 times to control values at any dose with any bacterial strain. No statistical analysis is performed.

Statistics:

When the results obtained failed to satisfy the evaluation criteria above for a clear positive or negative response. the test data maybe subjected to statistical analysis according to the study description.

As in the study no substantial increase of revertant colony numbers of any strains were observed, statistical analysis were not performed in the study.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mean number of the revertant colonies obtained in the first mutation test are shown in Table 2 in attachment. Positive control mutability checks are shown in Table 3.

The mean number of the revertant colonies obtained in the second mutation test are shown in Table 4 in attachment. Positive control mutability checks are shown in Table 5.

There were no substantial increases in revertant colony numbers of any of the tester strains following treatment with dipentaerythritol at any concentration, in either the presence or absence of S9 mix.

Conclusions:

No evidence of mutagenic activity was seen at any dose level of dipentaerythritol both in the presence and absence of metabolic activation.

Executive summary:

In this in vitro assessment of the mutagenic potential of dipentaerythritol, histidine dependent auxotrophic mutants of Samonella typhimurium (strains TA1535, TA1537, TA1538, TA98 and TA100) were exposed to the test material, dissolved in dimethyl sulphoxide. In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254 -induced rats. No evidence of mutagenic activity was seen at any dose level of dipentaerythritol in either mutation test, when compared to the vehicle controls. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It was concluded that dipentaerythritol was not mutagenic in this bacterial test system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10.09.1991 to 23.03.1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): dipentaerythritol
- Substance type: white powder
- Physical state: solid
- Analytical purity: 96.2%
- Purity test date: Received 2 September 1991
- Lot/batch No.: 9404
- Expiration date of the lot/batch: 2 November 1992
- Storage condition of test material: room temperature in the dark

Target gene:

Not applicable - chromosome aberration study.

Species / strain / cell type:

lymphocytes: human primary culture) PBL

Details on mammalian cell type (if applicable):

Human blood was collected aseptically from healthy male donors, pooled and diluted with RPMI 1640 tissue culture medium. Lymphocytes were separated by centrifugation on a Histopaque 1077 gradient for 30 minutes at 400 g. After 3 ashed and sedimentation by centrifugation at 200 g, the cells were suspended at a concentration of 1 x 10exp6 cells/ml in RPMI 1640 + 20% foetal calf serum + phytohaemagglutin (PHA) at a final concentration of approximately 175 µg/ml. 5 ml aliquots of the cell suspension were placed in the 10 ml wells of Nunc multiwell tissue culture plates and incubated at 37°C in a humid atmosphere containing 5% CO2 for ca. 48 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix prepared from the livers of Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml. Concentrations chosen for metaphase analysis: 100, 400 and 800 µg/ml.

Vehicle / solvent:

Prior to commencing testing the solubility of the test substance in solvent compatible with the test system was assed. Due to its low solubility in all the solvents tested, dipentaerythritol was dissolved directly in RPMI 1640 + 20% foetal calf serum. It was found to be soluble at approximately 800 µg/ml, which was the highest concentration used in the test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

750 µg/ml, in the absence of S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

15 and 20 µg/ml, in the presence of S-9 mix

Details on test system and experimental conditions:

The cells were incubated in the presence of phytohaemagglutinin (see above) before being treated with the test substance. After 48 hours, cultures to be treated with the test substance and those to remain untreated were centrifuged at 200 g for 7 minutes. The supernatant was discarded and replaced with fresh culture medium which was either untreated or contained the various dilutions of test substance. Duplicate cultures were treated with the test susbtance, and four remained untreated. 1.25 ml S9 mix was added immediately to the relevant cultures. The positive controls were also cultured in duplicate. Three hours after dosing the cultures containing S9 mix were centrifuged and the cell pellets resuspended in fresh medium, and incubated for a further 21 hours.
22 hours after initiation of treatment with the test substance, mitotic activity was arrested by addition of colchicine to each culture at a final concentration of 0.25 µg/ml. After 2 hours incubation each cell suspension was transferred to a conical centrifuge tube and centrifuged at 200 g for 10 minutes. The cell pellets were then treated with 20% Hanks balanced salt solution for 10 minutes. The suspensions were then centrifuged at 110 g for 10 minutes and the cell pellets fixed by addition of freshly prepared fixative (3 parts methanol: 1 part glacial acetic acid). The pellets were allowed to fix for at least 2 hours.

The pellets were aspirated through a hypodermic needle, then centrifuged t 200 g for 10 minutes and finally resuspended in a small volume of fresh fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned microscope slides which were then allowed to air dry. The slides were then placed in buffered distilled water (pH 6.8) prior to staining in Giemsa. After rinsing in buffered distilled water, the slides were left to air-dry and then mounted in DPX.

The prepared slides were examined by light microscopy at a magnification of x160. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results, the dose level causing a decrease in mitotic index of 40-80% of the solvent control value, or if there was no decrease, the maximum achievable concentration was used as the highest dose level for the metaphase analysis. The intermediate and low doses were 50% and 12.5% of the highest concentration. The slides were coded. Metaphase figures were identified, approximately 100 metaphases were examined, where possible, from each culture, with normally a maxumum of 25 from each slide. Only cells with 44-46 chromosomes were analysed. The vernier reading of all aberrant metaphase figures were recorded.

Evaluation criteria:

The substance was considered to be mutagenic if it caused a statistically significant increase in the number of aberrant metaphases, in a dose-dependent manner, compared to the solvent control. Aberrations scored included: chromatid breaks (including single minutes and accentric fragments), exchange (including complex rearrangements, rings and dicentric chromosomes), chromatid gaps, chromosome gaps and cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes.

Statistics:

The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test.

Species / strain:

human lymphoblastoid cells (TK6)

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:

A precipitate was observed in cultures treated with 800 µg/ml on dosing, and in cultures treated with 200, 400 and 800 µg/ml at S9 change.

There was no substantial decrease in the mitotic index at any dose level when compared with the untreated negative control, both with and without S9. The dose levels selected for the metaphase analysis were 800, 400 and 100 µg/ml.

In the both the presence and asbsence of S9, the test substance caused no statistically significant increase in the proportion of aberrant metaphase figures at any dose level when compared with the untreated control. Both positive control compounds caused large statistically significant increases in the number of aberrant cells.

There was no increase in the proportion of aberrant metaphase figures at any dose level, when compared to the untreated control.

Conclusions:

Dipentaerythritol has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Executive summary:

A study conducted according to OECD Guideline 473 was performed to assess the ability of dipentaerythritol to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Untreated and positive control cultures were also prepared. After the appropriate treatment time cell division was arrested using colchicine, the cells harvested and slides prepared, so that metaphase figures could be examined.

In order to assess the toxicity of dipentaerythritol to cultured human lymphocytes the mitotic index was calculated for all cultures treated with the test substance and the untreated control. On the basis of these data the dose levels selected for the metaphase analysis in both the presence and absence of S-9 mix were 800, 400 and 100 µg/ml. In both the presence and absence of S-9 mix dipentaerythritol caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the untreated control. Both positive control compounds, ethyl methanesulphonate and cyclophosphamide, caused large statistically significant increases in chromosomal damage. This demonstrates the sensitivity of the test system and the efficacy of the S-9 mix. It was concluded that dipentaerythritol showed no evidence of clastogenic activity in this in vitro cytogenetic test system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02.11.2009 to 17.02.2010

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:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Di-Penta 93 (Batch 3810066489), a white crystalline solid, was received from Perstorp Holding AB on 04 September 2009 and was stored, protected from light at ambient room temperature. The purity was quoted as 94.93% 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+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 tests: 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL.
Mutation assays: 125, 250, 500, 1000 and 2000 μg/mL.

Vehicle / solvent:

Di-Penta 93 was partially soluble in dimethylsulphoxide (DMSO), and less soluble in the basic tissue culture medium, R0P. For use as a vehicle, a maximum concentration of 1% DMSO (v/v) may be used in the test cultures, or 40% v/v R0P. In the initial toxicity test, DMSO alone was used, and a maximum concentration of 100 mg/mL was prepared as part solution/part suspension. When dosed, this gave 1000 μg/mL in the cell cultures. After 4 h exposure, all Di-Penta 93 was dissolved at the maximum concentration. When this concentration proved non-toxic, attempts were made to obtain a higher concentration. The following proved the most effective method and was used throughout the mutation tests:
DMSO was added to Di-Penta 93 to give a concentration of 200 mg/mL and the part solution/part suspension was vortex-mixed. After this, R0P was added to give a final concentration of 5 mg/mL. This preparation was vortex-mixed and diluted with a mixture of DMSO:R0P (1:39). When added to the cell cultures at 40% v/v, this gave a maximum concentration in the cultures of 2000 μg/mL. At the end of the exposure period (4 h and 24 h), a small amount of undissolved test item was present at the highest concentration and it was concluded that the highest concentration of 2000 μg/mL exceeded the limit of solubility of Di-Penta 93 in the test system.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

With S9

Details on test system and experimental conditions:

All toxicity and mutation assays included vehicle control cultures. In the initial toxicity test, the vehicle control cultures were treated with DMSO. In the mutation experiments, the vehicle control cultures were treated with a mixture of DMSO:R0P (1:39). Single cultures were treated in the toxicity tests. In the mutation assays, duplicate cultures were treated for all Di-Penta 93 concentrations. Four vehicle control cultures were treated in each mutation experiment. 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 one culture was prepared for each treatment. The selection in the mutation experiments of the treatments for final assessment is dependent on suspension growth following treatment. 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). 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 Di-Penta 93. Observations on the precipitation of Di-Penta 93 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 h 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 DMSO:R0P (1:39). 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 h exposure period: 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 DMSO:R0P (1:39). 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The results of the toxicity tests show that Di-Penta 93 did not cause an appreciable reduction in cell growth at the highest concentration of 1000 μg/mL in either the 4 h exposure system (absence and presence of S9 mix) or the 24 h exposure system. Following this result, the highest concentration was increased to 2000 μg/mL in the mutation tests.

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 and the test for linear trend was not significant (P = 0.48). In the presence of S9 mix, the overall survival (Hs) heterogeneity factor was statistically significantly higher than the historical control and therefore no results were reported for this assay. The mutant fractions obtained with the Di-Penta 93-treated cultures were all very close to the vehicle control value and it can be assumed that no mutagenic activity was present in these cultures. The second experiment in the presence of S9 mix was a straightforward repeat on which statistical analysis was performed, and is reported below. 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 was not reported as the slope was negative, while in the presence of S9 mix the test for linear trend was not significant (P = 0.94). There was no toxicity at any concentration in either experiment. Both experiments were classed 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.

Table 1: Mutation test in the absence of S9, 4 h exposure, summary of means

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
DMSO	(100 mL added)	100	62	N/A	0.90	N/A
EMS	250	50	735	673	0.57	*
MMS	10	35	594	532	2.69	*
Di-Penta 93	125	104	49	-	2.75	NS
	250	93	61	-	1.42	NS
	500	91	60	-	2.15	NS
	1000	101	66	4	2.30	NS
	2000	103	67	3	1.33	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 fracth concentration of Di-Penta 93 = not significant (P=0.48)

-  = IMF≤ 0

Table 2: Mutation test in the presence of S9, 4 h exposure, summary of means

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
DMSO	(100 mL added)	100	62	N/A	1.24	†
3-MC	2.5	87	466	404	2.26	†
	10	61	596	534	1.29	†
Di-Penta 93	125	111	63	1	0.85	†
	250	125	65	3	0.95	†
	500	105	60	-	1.32	†
	1000	131	54	-	2.47	†
	2000	105	56	-	2.31	†

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

N/A = Not Applicable

-  = IMF ≤ 0

† =Overall survival (Hs) heterogeneity factor statistically significantly higher than the historical control and

therefore no results were reported for this assay

Table 3: Mutation test in the absence of S9, 24 h exposure, summary of mean

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
DMSO	(100 mL added)	100	54	N/A	1.75	N/A
EMS	150	26	2278	2224	0.36	*
MMS	5	20	2038	1983	2.07	*
Di-Penta 93	125	95	62	8	1.54	NS
	250	80	63	9	0.68	NS
	500	83	47	-	1.26	NS
	1000	90	57	2	1.38	NS
	2000	93	39	-	5.50	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 Di-Penta 93 = not reported – slope negative

-  = IMF ≤ 0

Table 4: Mutation test in the presence of S9, 4 h exposure, summary of means

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
DMSO	(100 mL added)	100	59	N/A	1.44	N/A
3-MC	2.5	67	447	389	1.15	*
	10	61	703	644	1.57	*
Di-Penta 93	125	94	75	16	1.20	NS
	250	100	72	14	1.17	NS
	500	97	54	-	1.19	NS
	1000	109	66	7	0.92	NS
	2000	96	64	5	0.78	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 Di-Penta 93 = not significant (P=0.94)

-  = IMF ≤ 0

Conclusions:

Under the conditions of this study, Di-Penta 93 is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in dimethylsulphoxide up to and beyond its limit of solubility in the test system.

Executive summary:

Di-Penta 93 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+tk- to tk-tk-. Di-Penta 93 was dissolved and diluted in dimethylsulphoxide. 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, Di-Penta 93 was shown to be non-toxic at a concentration of 1000 μg/mL. 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 h exposure; Assay 4 - with S9, 4 h exposure.

The concentration of 2000 μg/mL exceeded the limit of solubility of Di-Penta 93 in the test system. 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. No evidence of mutagenic activity was obtained with Di-Penta 93 in any of the 4 assays. It was concluded that Di-Penta 93 is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in dimethylsulphoxide up to and beyond its limit of solubility in the test system.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 October 2020 - 15 Dec 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

updated 26 June 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: Di-Pentaerythritol; Di-Penta

Target gene:

Tryptophan locus

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

-10% liver microsomal preparation (S9-mix) prepared from Sprague-Dawley rats induced by phenobarbitone / β-Naphthoflavone.

- source of S9: purchased from Moltox.

- method of preparation of S9 mix: The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test. The S9 mix contains: S9 5.0 mL, 1.65 M KCl/0.4 M MgCl2 1.0 mL, 0.1 M Glucose-6-phosphate 2.5 mL, 0.1 M NADP 2.0 mL, 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL, Sterile distilled water 14.5 mL.

- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media.

- quality controls of S9:
S9 Quality Control and Production Certificate is presented in the report to demonstrate the S9 met the acceptance criteria in terms of microorganisms contamination, the ability to activate promutagens and alkoxyresorufin-0-dealkylase activities.

During the study, a 0.5 mL aliquot of S9-mix and 2 mL of molten, trace tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Test concentration in Exp. 1 (Plate Incorporation Method), with and without metabolic activation: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Test concentration in Exp. 2 (Pre-Incubation Method), with and without metabolic activation:1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was employed in the second mutation test (pre-incubation method).

Vehicle / solvent:

Vehicle: dimethyl sulphoxide (with and without metabolic activation)

-Justification for choice of vehicle/solvent: The Sponsor stated that test item is fully soluble in dimethyl sulphoxide and was confirmed up to 50 mg/ml in solubility checks performed in-house of the laboratory

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

controls used in the presence of S9-mix.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Controls used in the absence of S9-mix.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2
- Negative (untreated) controls were performed on the same day as the experiment 1 and 2. All testing were performed in triplicate.

METHOD OF TREATMENT/ EXPOSURE:
- First experiment: in agar (plate incorporation)
- Second experiment: pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period (Exp. 2): incubated at 37 ± 3°C for 20 minutes, with shaking
- Exposure duration (Exp 1 and 2): Plates were incubated at 37 ± 3°C for between 48 and 72 hours

OTHER EXAMINATIONS:
- Other: Observation of the number of revertant colonies and bacterial lawns

Rationale for test conditions:

-Rationale for selection of test concentrations were detailed above in section "Test concentrations with justification for top dose ".
-Justification of number of plates per concentration: triplicate plating as suggested in the OECD TG 471.

Evaluation criteria:

The test item is considered non-mutagenic (negative) in the test system if the below criteria are not met.

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).

2. A reproducible increase at one or more concentrations.

3. Biological relevance against in-house historical control ranges.

4. A fold increase greater than two times the concurrent solvent control (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Statistics:

Dunnett’s Regression Analysis (* = p < 0.05) to confirm statistical significance in increases in frequency of revertant colonies compared to the concurrent solvent control

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Solubility in vehicle: Sponsor stated that substance was completely soluble in dimethyl sulphoxide. This was confirmed up to 50 mg/ml in solubility checks performed in-house of the laboratory
- Precipitation and time of the determination: no precipitation occurred
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES:
No evidence of toxicity was obtained following exposure to Di-Penta. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in both experiments.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle controls were within range of the current historical control range of the laboratory (see historical control data in the attachment)

Results for the negative controls (spontaneous mutation rates) and viability are presented in Table 1 and were considered to be acceptable.

Experiment 1 (plate incorporation):
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). (see table 2).

Experiment 2 (pre-incubation):
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

Small, but statistically significant increases in WP2uvrA pKM101 revertant colony frequency were observed in the absence of S9-mix at 5000 µg/plate and in the presence of S9-mix at 15, 1500 and 5000 µg/plate. These increases were considered to be of no biological relevance because there was no evidence of a dose response relationship nor reproducibility. Additionally, the maximum fold increase was only 1.3 times the concurrent vehicle controls. (See table 3).

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls)

	Mean number of colonies per plate	Viability – Bacterial cells 10^9 per mL
Experiment 1	123	2,9
Experiment 2	119	2,2

 

Table 2 Test Results: Experiment 1 – Without and With Metabolic Activation (Plate Incorporation)

	Number of revertants (mean) +/- SD
Dose level per plate	Without S9-mix		With S9-mix
	Mean	SD	Mean	SD
Solvent Control(DMSO)	152	1,5	160	16,1
Positive control (ENNG)_0.5 μg	457	147,3	-	-
Positive control (2AA)_10 μg	-	-	1278	165,0
Test Item_1.5μg	142	8,1	162	10,8
Test Item_5μg	133	4,0	174	9,5
Test Item_15μg	141	12,0	160	4,7
Test Item_50μg	148	3,5	161	19,1
Test Item_150μg	123	13,5	172	7,5
Test Item_500μg	149	5,1	166	23,2
Test Item_1500μg	133	13,7	158	11,4
Test Item_5000μg	150	22,1	154	10,0

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
2AA: 2-Aminoanthracene

 

Table 3 Test Results: Experiment 2 – With and Without Metabolic Activation (Pre-Incubation)

	Mean number of revertants ( SD)
Dose level per plate	Without S9-mix		With S9-mix
	Mean	SD	Mean	SD
Solvent Control(DMSO)	106	14,4	130	4,5
Positive control (ENNG)_0.5 μg	638	25,5	-	-
Positive control (2AA)_10 μg	-	-	1177	180,6
Test Item_1.5μg	108	8,1	139	10,6
Test Item_5μg	89	22,9	140	14,7
Test Item_15μg	118	15,9	153*	11,5
Test Item_50μg	118	11,2	142	2,9
Test Item_150μg	116	8,9	143	8,2
Test Item_500μg	126	4,4	144	7,5
Test Item_1500μg	131	13,6	158**	9,7
Test Item_5000μg	137*	7,2	159**	5,0

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
2AA: 2-Aminoanthracene
* p≤0.05
** p≤0.01

Conclusions:

Negative: under the conditions of this test Di-Pentaerythritol was considered to be non-mutagenic.

Executive summary:

A bacterial reverse mutation test was performed to determine the potential of the test substance Di-Pentaerythritol (Di-Penta) to induce gene mutation. The test was performed in accordance with OECD, EC, US EPA, Japanese, and other international test guidelines, and the test was carried out to GLP. Only one strain of Escherichia coli (WP2uvrA pKM101) was tested in this study. No evidence of cytotoxicity or mutagenic activity of biological relevance was observed during two tests, either in the presence or absence of S9 metabolic activation at concentrations up to and including the limit concentration of 5000 ug/plate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

 

The mutagenic potential of di-pentaerythritol was evaluated in a GLP study according to OECD method 471 (Jones & Gant, 1992). Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were exposed to the test material, dissolved in DMSO. No toxicity was observed in a preliminary study conducted at dose levels up to 5000 µg/plate therefore this dose was chosen as the top dose for the main study. No evidence of mutagenic activity was seen at any dose level of di-pentaerythritol in either of two independent mutation tests (in the presence and absence of metabolic activation), when compared to the vehicle controls. It was concluded that di-pentaerythritol was not mutagenic in the in vitro bacterial test system.

 

An additional bacterial reverse mutation test was performed (Wisher, 2020) to determine the potential of the test substance Di-Pentaerythritol (Di-Penta) to induce gene mutation in Escherichia coli (WP2uvrA pKM101). The test was performed in accordance with OECD, EC, US EPA, Japanese, and other international test guidelines, and the test was carried out according to GLP. The test material was dissolved in Dimethyl sulphoxide. No evidence of cytotoxicity or mutagenic activity of biological relevance was observed during two independent mutation tests, either in the presence or absence of S9 metabolic activation at concentrations up to and including the limit concentration of 5000 µg/plate compared to vehicle control. It was concluded that di-pentaerythritol was not mutagenic in the in vitro bacterial test system.

 

Clastogenicity assay

 

A GLP study was performed to assess the ability of di-pentaerythritol to induce chromosomal aberrations in human lymphocytes cultured in vitro, according to OECD method 473 (Jones et al, 1992). The mitotic index was calculated for all cultures treated with the test substance and the untreated control, on the basis of these data the dose levels selected for the metaphase analysis in both the presence and absence of metabolic activation were 100, 400 and 800 µg/ml. The test material caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the untreated control. It was concluded that di-pentaerythritol showed no evidence of clastogenic activity in this in vitro cytogenetic test system.

 

Mammalian cell mutation assay

 

Di-penta 93 was assayed for mutagenic potential in the mouse lymphoma L5178Y cell line, according to GLP and OECD method 476 (Riach, 2010). The concentration of 2000 μg/mL exceeded the limit of solubility of Di-Penta 93 in the test system. No evidence of mutagenic activity was obtained with Di-Penta 93 in any of the 4 assays. It was concluded that Di-Penta 93 is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in dimethylsulphoxide up to and beyond its limit of solubility in the test system.

 

Di-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 classification or non-classification

The results of the available genetic toxicity studies are negative; no classification is therefore required according to the CLP Regulation 1272/2008.