Pentaerythritol tetrakis(3-mercaptopropionate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2008 - 04 January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pentaerythritol tetrakis(3-mercaptopropionate)

EC Number:

231-472-8

EC Name:

Pentaerythritol tetrakis(3-mercaptopropionate)

Cas Number:

7575-23-7

Molecular formula:

C17H28O8S4

IUPAC Name:

3-[(3-sulfanylpropanoyl)oxy]-2,2-bis({[(3-sulfanylpropanoyl)oxy]methyl})propyl 3-sulfanylpropanoate

Details on test material:

- Name of test material (as cited in study report): Pentaerythritol tetra(3-mercaptoproprionate)
- Physical state: liquid
- Analytical purity: 96.6% (w/w)
- Purity test date: 2008-08-26
- Lot/batch No.: 24318
- Expiration date of the lot/batch: 2009-08-14
- Stability under test conditions: sensitive to oxidation
- Storage condition of test material: room temperature

Method

Target gene:

Thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from male Wistar rats induced with phenobarbital (80 mg/kg bw) and ß-naphtoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

Experiment I
with metabolic activation: 40, 200, 300, 400, 450, 500, 550, 600 µg/mL
without metabolic activation: 10, 20, 30, 40, 50, 55, 60, 65 µg/mL

Experiment II
with metabolic activation: 130, 180, 230, 280, 480, 530, 630, 675 µg/mL
without metabolic activation: 20, 40, 50, 60, 80, 100, 110, 120 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with low water solubility

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethanesulphonate 200 and 500 µg/mL, methylmethanesulfonate 10 µg/mL (without metabolic activation)

Controls 2

Positive control substance:

other: benzo(a)pyrene 3.5 µg/mL (with metabolic activation)

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4, 24 h
- Expression time (cells in growth medium): 72 or 48 h (5 h or 24 h exposure time)
- Selection time (if incubation with a selection agent): 6 days

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

NUMBER OF REPLICATIONS: 4

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

OTHER EXAMINATIONS:
- Other: colony sizing

OTHER:

Evaluation criteria:

There are several criteria for determining a positive result:
- clear and dose-reIated increase in the mutant frequency,
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher
than the historical range of negative controls) for at least one of the dose groups.
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low
Large/small colonies ratio (smaller than or equal to 1.5 times the ratio of clastogenic controls MMS and/or B[a]P) is an indication for potential
clastogenic effects rtnd/or chromosomal aberrations.

Statistics:

not necessary

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >= 40 µg/mL (-S9); >=313 µg/mL (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [µg/ mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	Mutation factor	Quotient large / small colonies
0 (DMSO)	100	100	60.71	1	1.25
40	99.45	98.54	44.95	0.74	–
200	99.45	71.69	43.25	0.71	–
300	96.70	62.66	68.92	1.14	–
400	91.76	54.59	107.48	1.77	–
450	98.90	48.87	64.57	1.06	–
500	89.01	32.29	112.17	1.85	0.87
550	95.60	27.59	85.69	1.41	1.26
600	90.11	13.21	108.15	1.78	1.10
B[a]P, 3.5	98.90	68.85	243.73	4.01	0.80

B[a]P  Benzo[a]pyrene

 

 

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration [µg/ mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	Mutation factor	Quotient large / small colonies
0 (DMSO)	100	100	46.33	1	2.23
10	81.87	75.76	89.19	1.93	–
20	97.25	82.59	54.28	1.17	–
30	95.05	69.19	43.11	0.93	–
40	65.60	57.56	68.01	1.47	–
50	86.81	38.86	78.51	1.69	–
55	100.00	34.93	57.96	1.25	1.31
60	95.60	25.04	48.16	1.04	2.19
65	96.70	16.34	79.48	1.72	1.47
EMS, 500	83.52	61.99	769.08	16.60	–
MMS, 10	93.41	68.16	332.63	7.18	0.84

EMS   Ethyl methane sulphonate

MMS  Methyl methane sulphonate

 

 

Table 3: Experiment II - 24 h Exposure - With Metabolic Activation

Concentration [µg/ mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	Mutation factor	Quotient large / small colonies
0 (DMSO)	100	100	95.25	1	5.45
130	111.60	93.47	65.40	0.69	–
180	111.60	86.53	46.30	0.49	–
230	107.84	77.71	71.18	0.75	–
280	96.55	66.27	93.14	0.98	–
480	114.11	40.44	49.36	0.52	–
530	110.97	30.48	67.17	0.71	1.39
630	105.96	18.22	79.49	0.83	2.04
675	89.03	13.89	164.89	1.73	2.58
B[a]P, 3.5	96.55	67.52	562.23	5.90	1.06

B[a]P  Benzo[a]pyrene

 

 

Table 4: Experiment II - 24 h exposure - Without Metabolic Activation

Concentration [µg/ mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	Mutation factor	Quotient large / small colonies
0 (DMSO)	100	100	58.93	1	3.13
20	92.39	95.48	109.56	1.86	–
40	92.39	89.64	102.58	1.74	–
50	99.15	77.81	72.05	1.22	–
60	95.77	57.17	79.05	1.34	–
80	93.52	34.35	108.22	1.84	–
100	98.03	29.14	72.35	1.23	3.93
110	103.10	22.42	82.57	1.40	3.00
120	96.90	10.59	63.38	1.08	2.15
EMS, 200	72.68	24.95	1539.16	26.12	–
MMS, 10	73.80	31.79	1207.82	20.50	0.90

EMS   Ethyl methane sulphonate

MMS  Methyl methane sulphonate

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without activation

PETMP is negative in the mouse lymphoma assay, with and without metabolic activation

Executive summary:

The test item PETMP was assessed for its potential to induce mutations at the thymidine kinase locus using the mouse lymphoma cellline L5178Y in accordance with OECD Guideline 476. The se1ection of the concentrations was based on data from the pre-experiment. In experiment I, 600 µg/mL (with metabolic activation) and 65 µg/mL (without metabolic activation) were selected as the highest

concentrations. In experiment II, 675 µg/ml. (with metabolic activation) and 120 µg/mL (without metabolic activation) were selected as the highest concentrations. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I

with metabolic activation:

40, 200,300,400, 450, 500, 550, 600 µg/mL

and without metabolic activation:

10, 20, 30, 40, 50, 55, 60, 65 µg/mL

Experiment II

with metabolic activation:

130,180,230,280,480,530,630,675 µg/mL

and without metabolic activation:

20,40,50,60,80,100,110, 120 µg/mL

Growth inhibition was observed in experiment land II with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 13.21% for the highest concentration (600 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 65 µg/mL with a RTG of 16.34%. In experiment II with metabolic activation the relative total growth (RTG) was 13.89% for the highest concentration (675 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 120 µg/mL with a RTG of 10.59%.

In experiment land II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

The positive controls and showed distinct and biologically relevant effects in mutation frequency.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item PETMP is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cellline L5178Y.