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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genotoxicity assays according to OECD guidelines 471, 473, 476 and 482 are available for propylene glycol n-butyl ether. All studies have been conducted according to GLP.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06/1987-11/1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 471
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella/ mammalian-microsome bacterial mutagenicity assay. Strains TA98, TA100, TA1535, TA1537.
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate (aroclor 1254 induced)
Test concentrations with justification for top dose:
5.0, 15.8, 50, 158, 500, 1580, 5000 µg/plate
Vehicle / solvent:
none (test material solutions were prepared in distilled water)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (TA98, TA100, TA1535, TA1537/+S9); 2-nitrofluorene (TA98/-S9); sodium azide (TA100, TA1535/-S9); ICR-191 (TA1537/-S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min
- Incubation period: 48 hours

NUMBER OF REPLICATIONS:
3 replicates per dose / 2 independent repeats

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn evaluation
- no cytotoxcity observed
Evaluation criteria:
Concentrations of the test chemical which are overtly toxic are easily visualized as showing no bacterial growth on the plate (i.e. absence of background lawn). Lower levels of toxicity may be seen as a thin or sparse bacterial lawn, a reduction in the number of revertants, or the appearance of microcolonies (overgrown background lawn). Positive and negative controls were run concurrently with the test chemical, and appropriate responses for these controls are prerequesites for evaluating the response of the bacteria to the test chemical.

A test chemical is considered a bacterial mutagen if both the mean number of revertant colonies observed is at least three times higher than the mean of the negative (solvent) control and at the same time it produces a dose response relationship over several concentrations. If a chemical produces reproducible reversion rates in excess of 3x over background, but no definitive dose response relationship, it is considered to be a presumptive bacterial mutagen. If a chemical produces reproducible reversion rates greater than 2x but less than 3x over the negative controls, the results are considered to be equivocal or inconclusive. Test substances failing to meet the above criteria are considered non-mutagenic in this system.

Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

PnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation. Hence, the test material was classified as negative in the Ames test under the experimental conditions used.
Executive summary:

Propylene glycol n-butyl ether was evaluated in the Salmonella/mammalian-microsome bacterial mutagenicity assay (Ames test) using a pre-incubation modification of the standard assay. The test was conducted using Salmonella typhimurium bacetrial tester strains TA98, TA100, TA1535 and TA1537. The test agent was initially assayed in TA100 at concentrations of 0 (solvent control), 5.0, 15.8, 50.0, 158.0, 500.0, 1580.0 and 5000.0 micrograms/plate.

Based upon the results in TA100, the top five concentrations were repeated in TA98, TA1535 and TA1537. The assay was repeated independently at the same five concentrations using all four tester strains. The test material did not induce a mutagenic response in any of the tester strains in either of the assays as judged by the frequencies of histidine-independent (his+) revertants. Hence, the test material was classified as negative in the Ames under the experimental conditions used.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 473
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Induction of chromosomal damage (clastogenicity).
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 nutrient mix supplemented with 10% heat-inactivated fetal bovine serum 25 mM HEPES and antibiotic-antimycotics
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate prepared from AROCLOR 1254 treated Sprague-Dawley rats
Test concentrations with justification for top dose:
500, 1667, 5000 ug PnB/ml culture medium
Vehicle / solvent:
the test material was dissolved directly in the culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (-S9) & Cyclophosphamide (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): GIEMSA

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 (50/replicate)

DETERMINATION OF CYTOTOXICITY
- Method: relative total survival (colony formation)

Evaluation criteria:
Structural chromosomal abnormalities that were scored included chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, and chromosomal disintegration.  Chromatid and chromosome gaps were not included in the number of total aberrations.

The final interpretation of biological significance of the cytogenetic responses was based on both statistical outcome and sound scientific judgement.
Statistics:
The descriptive and comparative statistics on cytogenetoc aberrations considered each of the analyzed cells as an observational unit. Data from replicate cultures were pooled to calculated average scores. The frequencies of cytogenetic abnormalities, such as gaps/cell, total aberrations excluding gaps/cell, miscellaneous aberrations/cell, cells with gaps and cells with aberrations excluding gasp, and cells with miscellaneous aberrations, were analyzed by constructing two dimensional contingency tables. The total Chi-square was partitioned into components of interest.Specifically, statistics were generated to test the two global hypotheses of (1) no difference in average scores among the dose groups, and (2) no linear trend of increasing scores with increasing dose. If either statistic was found to be significant at alpha=0.01, pairwise tests (i.e., control vs. Treatment) were also performed at each dose level and evaluated at alpha=0.01.
Species / strain:
Chinese hamster Ovary (CHO)
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
Positive controls validity:
valid
Additional information on results:

Cultures treated with 1242 ug/ml ethylmethanesulfonate and 14 ug/ml cyclophosphamide served as positive controls for the non-activation and activation assays, respectively.  Negative control cultures were treated with culture medium (the solvent used to dissolve the test material).  No cytotoxicity was detected at the highest concentration tested (5000 ug).  The medium was tested for pH and osmolality and was found to be within normal limits at the highest concentration of test material.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results are shown in the table below: 

Dose Level   With/without   Cytotoxicity   Aberrations
(ug/ml)      S-9 
------------------------------------------------------
0 PnB            +/-        Negative       Negative
500 PnB          +/-        Negative       Negative
1667 PnB         +/-        Negative       Negative
5000 PnB         +/-        Negative       Negative
1242 EMS          -         N/A            Positive
14 CP             +         N/A            Positive

Conclusions:
Interpretation of results (migrated information):
negative

PnB was non-clastogenic to cultured CHO cells under the experimental conditions used.
Executive summary:

Propylene glycol n-butyl ether (PnB) was evaluated in an vitro chromosomal aberration assay utilizing Chinese hamster ovary (CHO) cells. The clastogenicity of the test material was assessed in the absence and presence of an externally supplied metabolic activation (S-9) system at dose levels of 500, 1667 and 5000 µg/ml of PnB/ml of culture medium. The dose levels of the test material were based upon the results of preliminary cytotoxicity assays. Cultures treated with 1242 µg/ml ethylmethane-sulfonate and 14 µg/ml cyclophosphamide served as positive controls for the non-activation and activation assays, respectively. Negative control cultures were treated with culture medium (the solvent used to dissolve the test material). There were no statistically significant increases in the frequencies of chromosomal aberrations in cultures treated with the test material either in the absence or presence of S-9 as compared to the negative control cultures. The positive control chemicals induced the epxected increases in aberration frequencies. Hence, under the experimental conditions used, PnB was considered to be non-clastogenic to CHO cells in culture.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 476
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
other: In vitro L5178Y TK+/- Mouse Lymphoma Cell Assay
Species / strain / cell type:
other: Mouse lymphoma cells deficient in thymidine kinase (-/-) may grow in the presence of the cell inhibitor, trifluorothymidine (TFT). A mutation from L5178Y TK+/- to -/- permits cell growth in the presence of TFT and detection of agents that c
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Up to 6000 microG/ml
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 5000 ug/ml and above.
Remarks on result:
other: other: Mouse lymphoma cells deficient in thymidine kinase (-/-) may grow in the presence of the cell inhibitor, trifluorothymidine (TFT). A mutation from L5178Y TK+/- to -/- permits cell growth in the presence of TFT and detection of agents that c
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

PnB is not mutagenic in the in vitro L5178Y +/- mouse lymphoma assay under the conditions of this test.
Executive summary:

T5178Y TK+/- lymphoma cells in logarithmic growth phase, grown to a density of 1 x 106 cells/ml, were used in the
assay.  To determine the doses to be used in the mutation assay, a range-finding cytotoxicity assay was first
performed with PnB concentrations of 100, 50, 10, 5.0, 1.0, 0.5, 0.1, 0.05, 0.01, or 0.005 µl PnB/ml.  From results of
the cytotoxicity assay, concentrations of 5.0, 4.0, 3.5, 2.5, 2.0, 1.5, 1.0, or 0.5 µl/ml were employed for both the
activation and non-activation phases of the main mutation assay. Appropriate solvent controls were used as were
positive control agents.  In the activation phase, dimethylbenzanthracene (DMBA), diluted in acetone, was the
positive control agent and ethylmethanesulfonate (EMS) in DMSO was the positive control in the non-activation phase.


In the mutation assay, cells were exposed in duplicate test tubes to PnB at the concentrations described above for 4
hours in a roller drum at 37°C in an atmosphere of 5% CO2 and 95% air.  A second identical set of test tubes was
prepared containing S-9 metabolic activation system (from Aroclor 1254-induced rat liver).  After incubation, cells
were pelleted by centrifugation, rinsed twice, resuspended in medium, and incubated for 20 and 44 hours to allow for
expression of potential mutations. After adjusting cell density, cells were incubated in cloning medium to select
for -/- revertants not inhibited by the presence of  the cell growth inhibitor, trifluorothymidine (TFT). Cells were
then plated on medium containing TFT and incubated at 37°C for 10 to 12 days to allow for expression of -/- colonies.  At the end of this period, colonies were enumerated for each
plate using an automatic colony counter.

In the range finding toxicity test, no growth occurred at PnB concentrations of 5 µl/ml and above, with or without S-9. 

In the first mutation assay, no increase in revertant frequencies were observed in cells treated with PnB, with or
without metabolic activation.  Cells at all PnB concentrations exhibited acceptable relative growth.  For
the EMS positive control (without activation), revertants were within historical ranges for the laboratory.  However,
for the DMBA control, revertants were below normal. Therefore, a repeat, second assay, only with S-9 activation,
was undertaken. Results from this second assay showed DMBA revertant rates that were ~8-fold higher than solvent
controls. Again, revertant rates for PnB samples were no higher than concurrent negative controls.

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

Additional information

Additional information from genetic toxicity in vitro:

All in vitro assays were conducted under GLP and according to OECD guidelines and are therefore valid without restrictions. Based on the consistent negative results from the in vitro assays no in vivo genotoxicity testing of propylene glycol n-butyl ether is required.


Justification for selection of genetic toxicity endpoint
GLP study according to OECD TG 473

Justification for classification or non-classification

All in vitro mutagenicity studies with the test substance were negative, therefore propylene glycol n-butyl ether is not classified for genotoxicity.