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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial test and chromosomal aberration tests in vitro showed negative results with and without metabolic activation. A test to induce gene mutations in mouse lymphoma cells in both absence and presence of metabolic activation was negative. According to Column 2 in Annex VIII (Section 8.4), an in vivo test is not necessary given the consistently negative results in multiple in vitro studies.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate OECD test method and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 3-methyl-1,5-pentanediol (MPD)
- Molecular formula (if other than submission substance): C6H14O2
- Molecular weight (if other than submission substance): 118
- Physical state: transparent liquid
- Analytical purity: 99.3 %
- Purity test date: 2005-03-14
- Lot/batch No.: 62626 (supplier Kuraray Co. Ltd, Japan)
- Expiration date of the lot/batch: 2005-09-05
- Storage condition of test material: ambient temperature, dark
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
range finding test: 0.61, 1.2, 2.4, 4.9, 7.0, 10 (mmol/L) (with and without met. act.)
main test: 0, 1.2, 2.4, 4.9, 7.0, 10 (mmol/L) (with and without met. act.)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: the test substance was diluted in DMSO; the test was carried out in culture medium (RPMI 1640; with HEPES and Glutamax-I), supplemented with heat-inactivated horse serum (10 % v/v), sodium pyruvate and penicillin/streptomycin
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without activation: 100, 200 µmol/L
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with activation: 10 µl/L
Details on test system and experimental conditions:

DURATION
- Exposure duration: 24 hours at 37°C (without activation) and 4 hours at 37°C (with activation)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-14 days


SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: two


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 100 mutants per 1000000 clonable cells. A response was considered to be equivocal if the induced mutant frequency was more than 50 mutants per 1000000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90 % cytotoxicity was considered to be an artefact and not indicative of genotoxicity.
The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test points.
Statistics:
No statistical analysis was performed
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:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

In both the absence and presence of S9-mix no increases of the mean mutant frequency by more than 50 mutants per 1000000 clonable cells compared to negative control were observed. As exspected, the cultures treated with the positive controls exhibited significant increases in mutation frequency relative to their solvent control cultures.

Summary of the results:

 Dose (mmol/L)        absence of S9     presence of S9
 mean MF  Mean RTG  mean MF  RTG
 10  117  179  117  85
 7  154  135  83  80
 4.9  121  145  111  88
 2.4  103  139  145  90
 1.2  138  109  127  96
 0  171  100  118  100

Positive and negative controls

Methyl methanesulphonate (MMS) and 3 -methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of the S9 -mix, respectively. DMSO served as negative control. The negative controls were within acceptable ranges and treatment with the positive controls yielded the exspected significant increase in mutant frequency compared to the negative control.

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

In conclusion, 3-methyl-1,5-pentane-diol is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells under the conditions in this study.
Executive summary:

MPD was examined for its potential to induce gene mutataions at the TK-locus of cultured mouse lymphoma L5178Y cells in both absence and presence of metabolic activation system (S9 -mix). One assay was conducted; in the assay five concentrations were tested in duplicate in both absence and presence of S9 -mix. The test substance was diluted in DMSO prior to testing.

The highest concentration tested and evaluated for mutagenicity was 10 mmol/l in both the absence and presence of S9 -mix.

MPD was not cytotoxic to L5178Y. The relative total growth at the highest concentration was not decreased compared to the negative control. No increase of mutant frequency was observed at any dose level. Methyl methansulphonate MMS and 3 -methylcholanthrene were used as positive controls, respectively. Treatment with controls yielded the expected significant increase in mutant frequency.

Under the conditions of the study the test substance MPD is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

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

Additional information

Additional information from genetic toxicity in vitro:


Data are available from reliable studies for all the required in vitro endpoints. Where there was more than one reliable study for an endpoint the most recent study was selected as key study.


MPD has been tested in two reliable studies according to OECD 471 and 472 and under GLP in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA (Hiroshi Ono 1997) and S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli uvrA (Jonathan Kitching 1995) respectively. The test substance did not show any mutagenic activity in any of the tester strains with or without metabolic activation.


Two reliable studies are available for in vitro cytotoxicity in mammalian cells with and without metabolic activation, human lymphocytes and Chinese hamster CHL/IU cells respectively, according to OECD 473 and GLP (Huntingdon 2001 and Hiroshi Ono 1997). MPD did not show any evidence of clastogenic activity in this two in vitro cytogenetic test systems. Furthermore data published (Kusakabe, Hirokazu; Yamakage, Kohji; and other 2002) also shows that MPD did not induce structural chromosome aberration nor induce polyploidy.


Information on mutagenicity to mammalian cells is available for 3 -methylpentane-1,5 -diol from a reliable study conducted according to OECD 467 and under GLP, using mouse lymphoma L5178Y cells (M.J.S.T. Steenwinkel 2005). The highest concentration tested and evaluated for mutagenicity was 10 mmol/l in both the absence and presence of S9 -mix. The substance was not cytotoxic to L5178Y. The relative total growth at the highest concentration was not decreased compared to the negative control. No increase of mutant frequency was observed at any dose level. The test substance was observed as not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.



Justification for selection of genetic toxicity endpoint
Data are available from reliable studies for all the required in vitro endpoints. There are results of two bacterial reverse mutation assay, one experimental result and one published result of a in vitro mammalian chromosome aberration test and one result of a in vitro mammalian cell gene mutation test. No mutagenic effects were observed in all of the tests. The most recent study was selected.

Justification for classification or non-classification

The available information for the substance indicates that when testing in vitro, 3 -methylpentane-1,5 -diol does not induce mutations in bacterial or mammalian cells, nor chromosome aberrations in mammalian cells. Therefore it is considered that classification for mutagenicity is not required.