Propanol, 1(or 2)-[methyl-2-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]ethoxy]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 17, 2010 to April 22, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline and EU Guideline No deviations GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996

Deviations:

not specified

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997

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:

-

EC Number:

470-680-5

EC Name:

-

Cas Number:

958872-63-4

Molecular formula:

C16H30O3

IUPAC Name:

1-[2-({1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}oxy)propoxy]propan-2-ol; 1-{[1-({1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}oxy)propan-2-yl]oxy}propan-2-ol; 2-[2-({1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}oxy)propoxy]propan-1-ol; 2-{[1-({1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}oxy)propan-2-yl]oxy}propan-1-ol

Details on test material:

- Name of test material (as cited in study report): Dipropylen Glycol Isobornyl Ether (mixture of isomers)
- Physical state: liquid
- Analytical purity: 98.8%
- Lot/batch No.: V07-017-9
- Expiration date of the lot/batch: 01 March 2013
- Storage condition of test material: Keep containers tightly closed in a dry, cool and well-ventilated place.

Method

Target gene:

tk locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Date of arrival: February 10, 2009.

Cells are stored as frozen stocks (Master Copies: MC) in liquid nitrogen. Each batch of frozen cells is purged of TK-/- mutants (producing Master Products) and checked for the absence of mycoplasma.

For the experiments, one vial was thawed rapidly, cells were diluted in RPMI 10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. Well growing cells, subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The following concentrations were investigated in the Assay 1:
3-hour treatment (-S9 Mix): 5; 10; 20; 40; 60 and 80 μg/mL.
3-hour treatment (+S9 Mix): 5; 10; 20; 40; 60 and 90 μg/mL.

The cytotoxicity results of the evaluated Assay 1 did not fulfill the assay acceptance criterion of the study regarding the number of evaluated test concentrations; therefore in the Completed Assay 1 the concentration range investigated in absence of metabolic activation was extended and additional concentration levels were examined with more narrow spacing above the concentration of 60 μg/mL.

The following concentrations were investigated in the Completed Assay 1:
3-hour treatment (-S9 Mix): 40, 60, 65 and 70 μg/mL.

The following concentrations were investigated in the Assay 2:
24-hour treatment (-S9 Mix): 1.25; 2.5; 5; 10; 20 and 25 μg/mL.
3-hour treatment (+S9 Mix): 10; 20; 40; 60; 75 and 85 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium: three types of RPMI 1640 medium (RPMI 5, RPMI 10 and RPMI 20)

DURATION
- Exposure duration:
Main Mutation Assay 1
A 3-hour treatment was performed in the presence and absence of S9 Mix. For the 3-hour treatment incubation, 1x10E07 cells were placed in each of a series of sterile flasks (culturing surface about 75 cm2). The treatment medium (RPMI 5) contained a reduced serum level of 5 % (v/v). After the 3-hour incubation at 37 °C ± 1 °C (approximately 5 % CO2 in air) with gentle shaking the cell cultures were centrifuged at 1200 rpm for 5 minutes, washed with 10 mL RPMI 10 Medium and suspended in 10 mL RPMI 10/tube. The cell densities were adjusted to a concentration of 2x10E05/mL. Cells were transferred to flasks for growth through the expression period1) and diluted to be plated for survival.

Completed Mutation Assay 1
A 3-hour treatment was performed in absence of S9 Mix. The concentration range investigated in absence of metabolic activation (-S9 Mix) had to extend and further concentration levels additionally examined, because of the toxicity properties of the test item observed in the Assay 1 after the treatment.

Main Mutation Assay 2
A 24-hour treatment was performed without metabolic activation, and a 3-hour treatment with addition of metabolic activation. The procedure for 3-hour treatment was the same as described above. For the 24-hour treatment 4x10E06 cells were placed in each of a series of sterile flasks (culturing surface 25 cm2). The treatment medium (RPMI 5) contained a reduced serum level of 5 % (v/v). After the 24-hour incubation at 37 °C ± 1 °C (approximately 5 % CO2 in air) the treated cultures were adjusted on the same way as it was performed at the 3-hour treatment.

- Expression time (cells in growth medium):
The cultures were maintained in flasks for 2 days, during this time the tk-/- mutation expressed. During the expression period, subculturing was performed daily with the aim of not exceeding 1x106 cells per mL and, where possible and retaining a total of at least 5x10E06 cells/flask. For this purpose cell densities were adjusted to a concentration of 2x10E05/mL and transferred to flasks for further growth.

In the Assay 1 following the 3-hour treatment (+S9 Mix) at the concentration of 90 μg/mL and in the Completed Assay 1, following the 3-hour treatment (-S9 Mix) at the concentration of 70 μg/mL high toxicity was observed during the expression period. These concentration levels were plated for survival, and the cell suspensions were maintained for the expression period, however the cell density at the first day of the expression period did not allow the further culturing and the viability, mutagenicity experiments.

SELECTION AGENT (mutation assays):
5-trifluorothymidine (TFT) Resistance

NUMBER OF REPLICATIONS: Duplicates

DETERMINATION OF CYTOTOXICITY
- Method: At the end of the expression period, the cell density in the cultures was determined and adjusted to nominally 1x10E04/mL with RPMI 20 for plating for a viability test. Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microtiter plates (192 wells) resulting in an average of 1.6 cells per well. The microtiter plates were incubated at 37 ºC ± 1 °C containing approximately 5 % (v/v) CO2 in air for 11-12 days. Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:

The test item is considered to be mutagenic in this assay if all the following criteria are met (based on Moore et al.):
1. The assay is valid;
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding vehicle control values at one or more concentrations;
3. The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
4. There is a significant dose-relationship as indicated by the adequate trend analysis;
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 10E06 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.
[e.g.: If the vehicle control MF is 50 x 10-6, then one of the test cultures must have an MF of at least (50+126) x 10 -6 = 176 x 10 -6 to meet the GEF criterion for a positive call.]

Results, which only partially satisfied the criteria, should be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity should require careful interpretation when assessing their biological significance. Indeed, extreme caution should be exercised with positive results obtained at levels of survival lower than 10 %.

There is no requirement for verification of a clear positive response. Equivocal or negative results need to be verified in a follow-up experiment. Modification of study parameters in the follow-up experiment should be considered.

Statistics:

The heterogeneity of the obtained data was tested. The statistical significance of mutant frequencies (total wells with clones) was carried out using Dunnett’s Test, using TOXSTAT statistical software. The positive control data were compared with the respective vehicle control or untreated control data with 2 Sample t-Test, using TOXSTAT statistical software. The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Additional information on results:

Results of the Assay 1
3h treatment in absence of S9 Mix:
Because of the high toxicity of the test item, the highest concentration level of 80 μg/mL was not plated for survival after the treatment and the cultures of this concentration level were not further carried on to the expression period. Based on the harmonised relative survival (harmonised RS) data 74 % toxicity was observed at the concentration of 60 μg/mL and 24 % at 40 μg/mL. No significant cytotoxicity was observed in the concentration range of 20-5 μg/mL.

The cytotoxic effect of the test item was evaluated with the calculation of relative total growth (RTG) data as well. The daily cell growth (during the expression period) and the viability of the cultures have influence on the RTG data. 77 % toxicity was obtained at the concentration level of 60 μg/mL based on the RTG data, and no cytotoxic effect of the test item was observed at the lower concentrations (40-5 μg/mL).

No statistically significant increase of the mutation frequencies and no dose-response relationship were observed at the examined concentrations (Dunnett’s Test, α=0.05).

3h treatment in presence of S9 Mix:
The highest examined concentration level of 90 μg/mL was plated for survival and carried on the expression period, however because of its highly toxicity, (~100 % toxicity was observed based on the harmonised RS data) the subculturing of this treatment was terminated during the expression period. The 52 % and 21 % toxicity (based on the harmonised RS and RTG values), at the concentration level of 60 μg/mL was not high enough to fulfil the validity criterion in connection with the cytotoxicity level; therefore in the Assay 2 the concentration range was modified. The most important modification was the choosing of more narrow spacing at the higher concentration levels.

Similarly to the non-activated part of the assay (-S9 Mix) the mutation frequencies did not show dose-response relationship and statistically significant differences from that of the control in the whole analysed concentration range (Dunnett’s Test, α=0.05).

Results of the Completed Assay 1
The harmonised relative survival (harmonised RS) and relative total growth data showed 97-98 % toxicity (above the acceptable 80-90 % range) at 65 μg/mL, and 82 % toxicity (18 % relative survival) based on the harmonised RS and RTG at the concentration level of 60 μg/mL. No cytotoxicity was obtained at the concentration level of 40 μg/mL.

The observed difference between the results obtained in the Assay 1 and Completed Assay 1 at the concentration level of 60 μg/mL (5 % difference, based on the RTG and 8 %, based on the harmonised RS) was considered as acceptable and in the biological variability range of the applied test system.

Mutation frequencies were calculated for the 60 μg/mL and 40 μg/mL treatments. The mutation frequencies did not differed statistically significantly from the control and remained far below the GEF criterion for positive call (Dunnett’s Test, α = 0.05).

Results of the Assay 2
24h treatment in absence of S9 Mix:
The toxicity caused by the test item was 86 % based on the harmonised RS data and 84 % based on the RTG (both were in the acceptable 80-90 % toxicity range) at the highest examined concentration level, at 25 μg/mL.

The toxicity was 52 and 56 % (based on the harmonised RS and the RTG) at the concentration of 20 μg/mL. The harmonised RS and RTG data did not show any toxic effect of the test item in the concentration range of 1.25-10 μg/mL [or the results showed a slight inhibition or even stimulation (the harmonised RS was 122 % at 10 μg/mL) in the range of the biological variability of the applied test system].

The calculated mutation frequencies did not differ statistically significantly from the mutation frequency of the vehicle control (Dunnett’s Test, α = 0.05) in the whole concentration range. The mutation frequencies of all test item treatments were below the GEF criterion for positive call.

3h treatment in presence of S9 Mix:
The obtained cytotoxicity showed a dose-related tendency, and the highest values: 86 % and 84 % (based on the harmonised RS and RTG data, in the acceptable 80-90 % range) were observed at the highest examined concentration level of 85 μg/mL.

The outcomes of the Assay 2 were similar (in the range of the biological variability of the applied test system) to the results of the Assay 1.

The mutation frequencies were slightly higher at the treatments of 85, 75, 60, 20 and 10 μg/mL than the mutation frequency of the vehicle control, but these mutation frequencies were not statistically significantly different from the control value (Dunnett’s Test, α = 0.05), and the remained far below the GEF threshold for a positive call.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test item Dipropylen Glycol Isobornyl Ether (mixture of isomers) did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of Dipropylen Glycol Isobornyl Ether (mixture of isomers) to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix). The appropriate vehicle, Dimethyl sulfoxide (DMSO) was chosen based on the results of the preliminary Solubility Test. The test item solutions were prepared in DMSO and diluted prior to treatment. This vehicle was compatible with the survival of the cells and the S9 activity. The concentrations applied in the Assay 1 were chosen according to the solubility and cytotoxicity results of the pre-experiments. The following concentrations were investigated in the Assay 1: 3-hour treatment (-S9 Mix): 5; 10; 20; 40; 60 and 80 μg/mL. 3-hour treatment (+S9 Mix): 5; 10; 20; 40; 60 and 90 μg/mL. The investigation of the cytotoxic effect of the test item, consequently the concentration selection was difficult because of the narrow effect range of the test item that was supported in the Completed Assay 1 as well as in Assay 2. The cytotoxicity results of the evaluated Assay 1 did not fulfill the assay acceptance criterion of the study regarding the number of evaluated test concentrations; therefore in the Completed Assay 1 the concentration range investigated in absence of metabolic activation was extended and additional concentration levels were examined with more narrow spacing above the concentration of 60 μg/mL. The following concentrations were investigated in the Completed Assay 1: 3-hour treatment (-S9 Mix): 40, 60, 65 and 70 μg/mL. The following concentrations were investigated in the Assay 2: 24-hour treatment (-S9 Mix): 1.25; 2.5; 5; 10; 20 and 25 μg/mL. 3-hour treatment (+S9 Mix): 10; 20; 40; 60; 75 and 85 μg/mL.

Due to the narrow cytotoxic effect range of the test item the concentration levels were slightly modified in the Assay 2 at the 3-hour treatment in presence of metabolic activation (+S9 Mix). In the performed mutation assays the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x10E05/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) or diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability. The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments. The Assay 1 with the Completed Assay 1 and the phases of the Assay 2 accomplished the necessary concentration ranges for a valid assay. There were at least four analysable concentration levels, where the highest concentration level was based on the required level of the caused cytotoxicity (80-90 %), based on the harmonised relative survival and relative total growth. In the performed assays the obtained mutation frequencies (neither in presence nor in absence of exogeneous metabolic activation) were not statistically significantly different from that of the corresponding vehicle controls (Dunnett’s Test, α = 0.05), did not show dose-related tendencies, furthermore remained far below the relevant GEF thresholds for positive call. Under the conditions of this study, the test item Dipropylen Glycol Isobornyl Ether (mixture of isomers) did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.