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Genetic toxicity in vitro

Description of key information

The endpoint conclusion is based on three relevant in vitro studies: an AMES test, a chromosome aberration study and a mouse lymphoma assay. All tests were performed with PEMP product and according to OECD/EC guidelines and GLP principles (Klimisch 1 studies). All three studies resulted in a negative outcome, independent of metabolic activation.

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01MAY2015 - 16JUL2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted September 26, 2014
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3hr exposure; 24 hr fixation: 17, 52 and 164 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1.7, 5.4, 17, 52 and 164 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 17, 52 and 164 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 17, 52 and 164 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 17, 52 and 164 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 17, 52, 89 and 164 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 17, 52, 89 and 164 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: Choice of solvent was based on a solubility test.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Mitomycin C: at 0.5 and 0.75 μg/ml (3 h exposure period), 0.2 and 0.3 μg/ml (24 h exposure period) and 0.1 and 0.15 μg/ml (48 h exposure period).
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Cyclophosphamide: 10 μg/ml (3 h exposure period; 24 h fixation time).
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitating concentration = 164 μg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 164 µg/ml and above
- No toxicity was observed up to and including the highest precipitating tested dose

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.
Conclusions:
A chromosome aberration study with PEMP product was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the test substance is not clastogenic in human lymphocytes.
Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of PEMP product (dissolved in DMSO), in the presence and absence of S9-mix according to OECD/ EC guidelines and GLP principles. In the first cytogenetic assay, PEMP product was tested up to precipitating concentration for a 3 h exposure time with a 24 h fixation time (≥ 164 μg/ml). In the second cytogenetic assay, PEMP product was tested up to precipitating concentrations for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. PEMP product did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of PEMP product on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that PEMP product does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
25AUG2015 - 29SEP2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(adopted July 21, 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(31 May 2008)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24 hours treatment: 5.4, 17, 52, 164 and 512 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 1, 5, 10, 25, 50, 65, 80 and 100 µg/mL
With S9-mix, 3 hours treatment: 0.5, 1, 5, 10, 50, 100, 250 and 500 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 1.7, 5.4, 20, 25, 50, 70, 90 and 110 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Based on solubility test performed in WIL Research project 508563.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
at 15 and 5 μg/mL for the 3 and 24 hours treatment periods, respectively.
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
at 7.5 μg/mL..
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

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

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

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

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

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

Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 90 µg/mL and above.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 250 µg/mL and above.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 164 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at the precipitating dose levels of 164 µg/mL in the absence of S9-mix. No toxicity was observed in presence of metabolic activation (up to and above precipitating concentrations).

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest tested dose level in the main experiments in the absence and presence of S9-mix. In absence of metabolic activation, test item precipitated in the exposure medium at 90 µg/mL and above. With metabolic activation, precipitation was observed at 250 µg/mL and above.

In the first experiment, values of the absolute cloning efficiency of one of the solvent controls in the absence and presence of S9-mix was not within the range mentioned in the study plan. Values of the absolute cloning efficiency (50 and 51) were below the limit of the range (65). Although the values were outside the limit of the ranges, the deviation was minor and clear negative results were obtained and therefore this deviation in the absolute cloning efficiency was considered to have no effect on the results of the study.

Conclusions:
Based on the results of a mouse lymphoma assay conducted according to OECD 476 guideline and GLP principles, it is concluded that PEMP product is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

A mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. The test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and the metabolic activation system (S9-mix) functioned properly. In the absence and presence of S9-mix, PEMP product did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment. In these experiments, no cytotoxicity was seen, but cells were exposed up to and including precipitating concentrations.

Based on these results, it is concluded that PEMP product is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01MAR1990 - 03APR1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1991)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test was performed with PEMP product, the registered substance.
Target gene:
Histidine (Salmonella strains);
Tryptophan (E.coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction from male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
Dose finding test: 50, 100, 500, 1000 and 5000 µg/ plate;
Main study: 313, 625, 1250, 2500 and 5000 µg/ plate.
Vehicle / solvent:
- Solvent used: Dimethylsulfoxide;
- Justification for choice of solvent: Based on the results of a solubility test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide for TA100 and WP2uvrA (0.01 µg/ plate); Sodium azide for TA1535 (0.5 µg/ plate); 2-Nitrofluorene for TA98 (1 µg/ plate) and TA1538 (2 µg/ plate); 2-Aminoanthracene for TA1537 (80 µg/ plate).
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Benzo[a]pyrene for TA100, TA98, TA1537 and TA1538 (5 µg/ plate); 2-Aminoanthracene for TA1535 (2 µg/ plate) and WP2uvrA (20 µg/ plate).
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) following preincubation of 20 minutes

DETERMINATION OF CYTOTOXICITY
- Method: toxic activation was observed with a stereoscopic microscope

REPLICATES
- Duplicate (dose finding test) and triplicate (main test and independent repeat test)
Statistics:
The number of revertant colonies of all test substance treated groups were compared with those obtained from negative control groups. Non parametric multiple range tets (Ryan) was employed for analyzing the data. A reproducible and statistical significant dose related increase in revertant colonies for at least one strain is considered positive.
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538) and E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
tested up to and including 5000µg/ plate.
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The number of revertant colonies in positive controls increased markedly compared to the negative control indicating that the assay was carried out properly.
Conclusions:
In an AMES test, performed according to OECD guideline and GLP principles, pentaerythritol tetrakis (3-mercaptopropionate) was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed according to OECD guideline and GLP principles. All bacterial strains showed negative responses up to and including 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. The number of revertant colonies in positive controls increased markedly compared to the negative control indicating that the assay was carried out properly. An independent repeat experiment resulted in the same results as the first test. Based on the results of this study it is concluded that pentaerythritol tetrakis (3-mercaptopropionate) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

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

Additional information

In an AMES test, all bacterial strains showed negative responses up to and including 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. The number of revertant colonies in positive controls increased markedly compared to the negative control indicating that the assay was carried out properly. An independent repeat experiment resulted in the same results as the first test.

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of PEMP product (dissolved in DMSO), in the presence and absence of S9-mix. In the first cytogenetic assay, PEMP product was tested up to precipitating concentration for a 3 h exposure time with a 24 h fixation time (≥ 164 μg/ml). In the second cytogenetic assay, PEMP product was tested up to precipitating concentrations for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. PEMP product did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of PEMP product on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that PEMP product does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

Furthermore, in a mouse lymphoma assay in the absence and presence of S9-mix, PEMP product did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment. In these experiments, no cytotoxicity was seen, but cells were exposed up to and including precipitating concentrations. Based on the results of all three in vitro tests it is concluded that there are no indications of genotoxic properties of PEMP.

Justification for classification or non-classification

Based on the available data, PEMP is not classified for genotoxic properties according to CLP Regulation (EC) No. 1272/2008.