1,6-Octadien-3-ol, 3,7-dimethyl-, acid-isomerized

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro Ames test, in vitro HPRT test and the in vitro micronucleus test were all negative in the absence and present of S9-mix.

Therefore, no classification is warranted under the REACH guidance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch: 9000475443
Liquid

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitine and Beta Naphthoflavone

Test concentrations with justification for top dose:

Experiment 1: 33, 100, 333, 1000, 2500 and 5000 ug/plate.
Experiment 2: 10, 33, 100, 333, 1000 and 2500 ug/plate.
Base on bactericidal affects.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation); preincubation.

DURATION
- Exposure duration:3 days

SELECTION AGENT (mutation assays): Histidine and Biotin

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

Assess the potential of the test item to induce gene mutations.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshol of twice (TA98, TA100, TA102) of thrise (TA1535, TA1537) the colony count of the corresponding solvent control observed.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that during the mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshift in the genome of the strains used.

Executive summary:

Negative Ames test in the absence and presence of S9-mix in stains TA1535, TA1537, TA98, TA100 and TA102..

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammlian cell mutation

Specific details on test material used for the study:

The test article was a pale yellow liquid, batch number SC00011756.
Defined as a mixture.

Target gene:

Lime Oxide was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol.

Species / strain / cell type:

mammalian cell line, other: L5178Y

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37±1ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 50 to 1600 μg/mL (limited by solubility in culture medium).
Steep concentration-related toxicity was observed and the highest concentrations to give >10% relative survival (RS) were 50 μg/mL in the absence of S-9 and 100 μg/mL in the presence of S-9, which gave 89% and 86% RS, respectively.
In Experiment 1 twelve concentrations, ranging from 10 to 120 μg/mL in the absence of S-9 and from 25 to 250 μg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 60 μg/mL in the absence of S-9 and 120 μg/mL in the presence of S-9, which gave 3% and 16% RS, respectively. In the absence of S-9, no concentration gave 10-20% RS due to steep concentration-related toxicity.
In Experiment 2 eleven concentrations, ranging from 10 to 100 μg/mL in the absence of S-9, and from 20 to 200 μg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 55 μg/mL in the absence of S-9 and 120 μg/mL in the presence of S-9, which gave 8% and 18% RS, respectively.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

The test system was suitably labelled to clearly identify the study number, test article (if required), test article concentration (if applicable), positive and vehicle control, presence and absence of S-9.

Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiments. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium.
Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.

At least 107 cells in a volume of 18.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.
Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from these were diluted to 8 cells/mL
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance.

At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL.

Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (11 to 12 days). Wells containing viable clones were identified by eye using background illumination and counted.

At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 μg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (12 to 13 days) and wells containing clones were identified as above and counted.

Rationale for test conditions:

The assay was considered valid if both of the following criteria were met:
1. The MF in the vehicle control cultures fell within the normal range (not more than three times the historical mean value)
2. At least one concentration of each of the positive control chemicals induced a clear, unequivocal increase in mutant frequency.

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The MF at one or more concentrations was significantly greater than that of the vehicle control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity required careful interpretation when assessing their biological relevance.

Statistics:

None

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

It is concluded that Lime Oxide did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

Negative in the mammalian gene mutation hprt assay in the absence and presence of S9 -mix.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

CAS number 73018-51-6, batch number SC00011756, was a pale yellow liquid.

Target gene:

Clastogenic - Non-specific.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

DMSO was added to cultures designated as vehicle controls as described in the
methods section of this report. The positive control chemicals were supplied and used.

No donor was suspected of any virus infection or exposed to high levels of radiation
or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of
alcohol. Donors were not taking any form of medication. The measured cell cycle
time of the donors used at Covance, Harrogate falls within the range 13±2 hours. For
each experiment, an appropriate volume of whole blood was drawn from the
peripheral circulation into heparinised tubes within one day of culture initiation.
Blood was stored refrigerated and pooled using equal volumes from each donor prior
to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by
placing 0.4 mL of pooled heparinised blood into 9.0 mL pre-warmed (in an incubator
set to 371C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated
foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume
following addition of S-9 mix/KCl and the test article in its chosen vehicle was
10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the
culture medium at a concentration of approximately 2% of culture to stimulate the
lymphocytes to divide. Blood cultures were incubated at 371°C for approximately
48 hours and rocked continuously.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Sprague Dawley rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Based on toxicity.
3 hour -S9 = 1.814, 3.023, 5.039, 8.398, 14.0, 23.33, 38.88, 64.80, 108.0, 180.0, 300.0 and 500.0 ug/mL.
3 hour +S9 = 1.814, 3.023, 5.039, 8.398, 14.0, 23.33, 38.88, 64.80, 108.0, 180.0, 300.0 and 500.0 ug/mL.
24 hour -S9 = 1.814, 3.023, 5.039, 8.398, 14.0, 23.33, 38.88, 64.80, 108.0, 180.0, 300.0 and 500.0 ug/mL.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Vinblastine

Details on test system and experimental conditions:

The test system was suitably labelled (using a colour-coded procedure) to clearly identify the study number, assay type, experiment number, treatment time, donor sex, test article concentration (if applicable), positive and vehicle controls.
S-9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article or vehicle or positive controls (0.1 mL per culture).
The final culture volume was 10 mL. Cultures were incubated at 371°C for the designated exposure time.

For removal of the test article, cells were pelleted by centrifugation (approximately 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 371C), and resuspended in fresh pre-warmed medium containing foetal calf
serum and penicillin / streptomycin. Cytochalasin B (Cyto-B, formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 g/mL per culture.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in chromosome aberrations (Scott et al.,1991; Brusick, 1986). Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment.

At the defined sampling time, cultures were centrifuged at approximately 300 g for 10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL (hypotonic) 0.075 M KCl at 371C for 4 minutes to allow cell swelling to occur.
Cells were then fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as
necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean.

Rationale for test conditions:

Scoring was carried out using fluorescence microscopy.
Binucleate cells were only included in the analysis if all of the following criteria were
met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology
to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a
main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the
diameter of the main nuclei.

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if: 1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed 3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Statistics:

The proportion of MNBN cells for each treatment condition were compared with the
proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989).
Probability values of p0.05 were accepted as significant. Additionally, the number of
micronuclei per binucleate cell were obtained and recorded.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Lime Oxide was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single
experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic
activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO) and the highest
concentrations tested in the Micronucleus Experiment (limited by toxicity), were determined following a preliminary cytotoxicity Range-Finder Experiment.
Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article
concentrations for micronucleus analysis were selected by evaluating the effect of Lime Oxide on the replication index (RI). Micronuclei were analysed at four
concentrations.
Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells
in the vehicle cultures fell within or were very close to the current 95th percentile of the observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and
Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was
employed as a clastogenic positive control chemical in the presence of rat liver S-9.
Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control
compounds induced statistically significant increases in the proportion of cells with micronuclei.
All acceptance criteria were considered met and the study was therefore accepted as valid.
Pulse (3+21 hour) treatment of cells with Lime Oxide in the absence and presence of S-9 resulted in frequencies of MNBN cells which were similar to and not significantly
(p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all treated cultures fell within
the normal ranges. Following 24 hour treatment (with 24 hour recovery) in the absence of S-9, frequencies of MNBN cells were similar to and not significantly (p≤0.05) higher than
those observed in concurrent vehicle controls for the majority of concentrations
analysed. The exception to this was observed at a single high concentration
(50 μg/mL, inducing 51% cytotoxicity) where a small increase (p≤0.01) was apparent
with both replicate cultures exhibiting MNBN cell frequencies that exceeded the normal range. However, no such statistically significant increase was observed at a
slightly lower concentration of 45 μg/mL inducing similar cytotoxicity (48%), or for lower concentrations analysed of 40 and 35 μg/mL (inducing 16% and 3%
cytotoxicity, respectively). Marginal increases in MNBN cell frequency above the calculated normal range were observed in single cultures at 35 μg/mL and 45 μg/mL
and in both cultures at 40 μg/mL, but these were small, did not exceed the observed historical vehicle control range or the maximum frequency observed in the concurrent
vehicle control. As such, these marginal increases (above the normal range) were not considered of biological importance.
Overall, and in line with published guidance on interpretation of in vitro genotoxicity data, the small and isolated statistically significant increase in micronucleus frequency
observed at 50 μg/mL (with no evidence of any concentration-related effect) was considered to be of highly questionable biological importance.
It is concluded that Lime Oxide did not induce micronuclei in cultured human peripheral blood lymphocytes following pulse (3+21 hour) treatments in the absence
and presence of an Aroclor-induced rat liver metabolic activation system (S-9).
Maximum concentrations analysed induced 54% and 60% cytotoxicity, respectively.
Following extended (24 hour) treatment with 24 hour recovery (24+24 hour) in the absence of S-9, an isolated statistically significant increase was observed at a single
high concentration of 50 μg/mL inducing 51% cytotoxicity. However, as this increase was small and similar findings were not observed at slightly lower concentrations
including one inducing a similar level of cytotoxicity (45 μg/mL inducing 48% cytotoxicity), the observed increase was considered of highly questionable biological importance.

Executive summary:

Negative in the MNvit in the absence and presence of S9-mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The in vitro Ames test, in vitro HPRT test and the in vitro micronucleus test were all negative in the absence and present of S9-mix.

Therefore, no classification is warranted under the REACH guidance.