2-(2-hexyloxyethoxy)ethanol

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug - Dec 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Materials and methods

Test guidelineopen allclose all

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALAC-approved laboratory in accordance with the German Anima! Weltare Act and the effective European Council Directive.
At least 5 male Wistar rats [Crl:Wl(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and ß-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm will be 12 hours: light from 6 am - 6 pm and darkness from 6 pm - 6 am.
Standardized pelleted feed and drinking water from bottles was available ad libitum.
24 hours after the last administration, the rats were sacrificed, and the induced livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were washed with 150 mM KCI solution. Afterwards, the livers were weighed and homogenized in three volumes of KCI solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, appropriate portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

S9 mix

The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawn at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors). The mixture of both components (S9 mix) was kept on ice until use. Following the concentrations of the cofactors:

MgCl2 8mM
KCI 33 mM
glucose-6-phosphate 5mM
NADP 4mM
phosphate buffer (pH 7.4) 15 mM

The phosphate buffer was prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.

Vehicle / solvent:

Due to the limited solubility of the test substance in medium, dimethyl sulfoxide (DMSO) was selected as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available.
The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Details on test system and experimental conditions:

Culture media
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (10000 IU / 10000 µg/ml)
- 1% (v/v) amphotericin B (250 µg/ml)

Culture medium
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).

Treatment medium (without S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) FCS.

Treatment medium (with S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine.

Pretreatment medium ("HAT" medium)
Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10-3 mg/ml)
- aminopterin (0.18 x 10-3 mg/ml)
- thymidine (3.88 x 10-3 mg/mL)
- 10% (v/v) FCS

Selection medium ("TG" medium)
Ham's F12 medium containing stable glutamine supplemented with:
- 6-thioguanine (10 µg/ml)
- 10% (v/v) fetal calf serum (FCS)

Cell culture
For cell cultivation, deep-frozen cell suspensions were thawed at 37°C in a water bath, and 0.5 ml were transferred into 25 cm2 plastic flasks containing 5.0 ml Ham's F12 medium supplemented with 10% (v/v) fetal calf serum (FCS). The cells were subcultured twice weekly (routine passage in 75 cm2 plastic flasks) and the cell cultures were incubated at 37°C with 5.0% (v/v) C02 and 90% relative humidity up to confluency.

TEST SUBSTANCE PREPARATION
The substance was dissolved in DMSO. The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
The further concentrations were diluted according to the planned doses.
All test substance solutions were prepared immediately before administration.

ANALYSIS OF TEST SUBSTANCE PREPARATION
The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was verified analytically.

Time schedule
Day 1: Seeding of the cells pretreated with "HAT" medium: in 300 cm² flasks (20x10^6 cells in 40 mL)
Day 2: Test substance incubation (approx. 20 – 24 hours after seeding); exposure period (4 hours); removal of test substance by intense washing; 1st passage of the treated cells in 175 cm2 flasks (2x10^6 cells in 20 mL medium) and seeding of the cloning efficiency 1 (survival) in 60 mm petri dishes (200 cells in 5 mL medium).
Day 5: 2nd passage of the treated cells (seeding of 2x10^6 cells in 20mL medium)
Day 7 - 9: Drying, fixation, staining and counting of the cloning efficiency 1
3rd passage of the treated cells; addition of selection medium ("TG" medium); and seeding of the cloning efficiency 2 (viability)
From day 16: Drying, fixation, staining and counting of the selected colonies and cloning efficiency 2

In this study, all incubations were performed at 37°C with a relative humidity of ≥ 90% in a 5% (v/v) CO2 atmosphere.

Preparation of test cultures
Cell stocks (1.0-mL portions) stored in the gas phase above the liquified nitrogen were thawed at 37°C in a water bath. 0.5 mL of stock cultures were pipetted into 25 cm2 plastic flasks containing 5 mL Ham's F12 medium (incl. 10% [v/v] FCS). After 24 hours, the medium was replaced to remove any dead cells. At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.

Pre-treatment of cells with "HAT" medium
During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.
0.8 - 1x10^6 cells were seeded per flask (175 cm²) and incubated with "HAT" medium for 3 - 4 days. A subsequent passage in Ham's F12 medium incl. 10% (v/v) FCS was incubated for a further 3 - 4 days.

Attachment period
For each test group, about 20x10^6 logarithmically growing cells per flask (300 cm²) were seeded into about 40 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours.

Exposure period
After the attachment period, the medium was removed from the flasks and the treatment medium was added (see table below). The cultures were incubated for the respective exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity.

Treatment of the cultures
Test groups Ham's F12 medium Vehicle or test S9 mix
(with or without FCS)* substance preparation
[mL] in vehicle [mL] [mL]
Without S9 mix
Vehicle control 39.6 0.4 -
Test groups 39.6 0.4 -
Positive control
(EMS) 36.0 4.0 -
With S9 mix
Vehicle control 31.6 0.4 8.0
Test groups 31.6 0.4 8.0
Positive control
(DMBA) 31.6 0.4 8.0
* For exposure conditions without S9 mix, Ham's F12 medium with 10% (v/v) FCS was used.

Expression period
The exposure period was completed by rinsing several times with HBSS. This was directly followed by the 1st passage in which 2x10^6 cells were seeded in 20 mL medium (in 175 cm2 flasks). The flasks were left to stand in the incubator for about 3 days at 37°C, relative humidity of ≥ 90% and 5% (v/v) CO2 atmosphere. After about 3 days, the cells were passaged a 2nd time in 175 cm2 flasks with 2x10^6 cells. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (3rd passage).

Selection period
For selection of the mutants, two 175 cm2 flasks with 2x10^6 cells each from every treatment group, if possible, were seeded in 20 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 – 7 days. Only the cells resistant to 6-thioguanine that were assumed to be deficient of HPRT survived. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

Cytotoxicity determination
Cloning efficiency (CE) and Relative Survival (RS) (pre-experiment)
The determination of the cloning efficiency in the pre-experiment was similar to that described for the determination of the cloning efficiency 1 (CE1) in the main experiments, except that 1x10^6 cells were seeded in 25 cm2 flasks coated with 5 mL Ham´s F12 medium incl. 10% (v/v) FCS. After test substance incubation, 200 cells were transferred into petri dishes (60 mm diameter) with fresh Ham´s F12 medium incl. 10% (v/v) FCS. The RS was calculated based on CE values and cell numbers measured directly after treatment.

Cloning efficiency 1 (CE1) (main experiments)
For the determination of the influence of the test substance after the exposure period, 200 cells per concentration were reserved from the treated cells and were seeded in petri dishes (60 mm diameter) and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation. The RS was calculated based on CE values and cell numbers measured directly after treatment.

Cloning efficiency 2 (CE2; viability)
For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS. In all cases, after seeding the flasks or petri dishes were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group.

Rationale for test conditions:

Dose selection
Following the requirements of the current international guidelines and the ICPEMC Task Group a test substance should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10 - 20% relative survival (adjusted cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation
in culture medium at the end of the exposure period.
In the pre-test the pH value and the osmolality were not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
Precipitation of the test substance in the vehicle dimethyl sulfoxide (DMSO) was not observed in the stock solution (240.0 mg/mL). In culture medium, test substance precipitation occurred at the highest applied concentration (2400.0 μg/mL) by the end of treatment in the absence and presence of S9 mix.
After 4 hours treatment in the absence and presence of S9 mix, cytotoxicity was observed at 2400.0 μg/mL.

Test groups and doses
Based on the data and the observations from the pre-test and taking into account the current guidelines, the following doses were selected in this study:
Dose selection of the 1st Experiment
Without S9 mix With S9 mix
205.8 μg/mL 205.8 μg/mL
370.4 μg/mL 370.4 μg/mL
666.7 μg/mL 666.7 μg/mL
1200.0 μg/mL 1200.0 μg/mL
1600.0 μg/mL 1600.0 μg/mL
2400.0 μg/mL 2400.0 μg/mL

For clarification of the results from the first experiment in the presence of S9 mix, a second experiment was performed in the presence of S9 mix using the following concentrations.

Dose selection of the 2nd Experiment
With S9 mix
1000.0 μg/mL
1200.0 μg/mL
1600.0 μg/mL
1800.0 μg/mL
2200.0 μg/mL

At least four concentrations were evaluated to describe a possible dose-response relationship.

Evaluation criteria:

Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• Concurrent positive controls both with and without S9 mix should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase in mutant frequencies compared with the concurrent vehicle control.

Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose.
The calculation was performed using EXCEL function RGP.
The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using EXCEL function HYPGEOM.VERT.
Both, biological and statistical significance are considered together.

Results and discussion

Additional information on results:

TREATMENT CONDITIONS
Osmolality and pH values were not influenced by test substance treatment.
In the 1st Experiment in the absence of S9 mix, test substance precipitation was observed macroscopically in culture medium at the end of treatment at 2400 μg/mL.
In the presence of S9 mix precipitation was observed at 2400.0 μg/mL in the 1st Experiment and at 1800.0 μg/mL and above in the 2nd Experiment.

CELL MORPHOLOGY
In the 1st Experiment in the absence of S9 mix the morphology and attachment of the cells was adversely influenced (grade > 2) at 2400.0 μg/mL.
In the presence of S9 mix the morphology and attachment of the cells was adversely influenced (grade > 2) at 2400.0 μg/mL in the 1st Experiment and at 2200.0 μg/mL in the 2nd Experiment.

CYTOTOXICITY
In the absence of S9 mix in the 1st Experiment there was a no decrease in the relative survival (RS) up to 1600.0 μg/mL (RS: 101.2 – 205.8%). At 2400.0 a strong decrease of RS was observed (0.6%).
In the presence of S9 mix in the 1st Experiment a dose dependent decrease in the relative survival was observed up to 1600.0 μg/mL. The values ranged between 48.9 - 82.6%. At 2400.0 mg/mL the RS was strongly decreased (0.3%). In the 2nd Experiment the relative survival values were not relevantly decreased up to 1800.0 μg/mL. The RS at 1800 μg/mL was 55.3%; at the next higher concentration (2200.0 μg/mL) the RS dropped to 0.6%.

MUTANT FREQUENCY
In the 1st Experiment in the absence of metabolic activation, the values for the corrected mutation frequencies (MFcorr.) ranged between 0.97 – 4.42 per 10^6 cells, the respective vehicle control value had 2.48 per 10^6 cells. All values were neither statistically significant nor dose dependently increased and within the within the range of the 95% vehicle control limit (without S9 mix: MFcorr.: 0.00 – 6.08 per 10^6 cells).
In the 1st Experiment in the presence of metabolic activation, the values for the corrected mutation frequencies (MFcorr.) ranged between 0.57 – 3.46 per 10^6 cells, the respective vehicle control value had 0.72 per 10^6 cells. The value in the test group 1600.0 μg/mL (MFcorr.: 3.46 per 10^6 cells) was statistically significantly increased. The value was, however, within the range of the 95% vehicle control limit (with S9 mix: MFcorr.: 0.00 – 6.96 per 10^6 cells; and a concentration related increase in the mutant frequencies was also not observed. Nevertheless, the relevance of this observation was assessed in a 2nd Experiment.
In the 2nd Experiment in the presence of metabolic activation, the values for the corrected mutation frequencies ranged between MFcorr.: 0.40 – 2.99 per 10^6 cells; the respective vehicle control value had 1.33 mutants per 10^6 cells. All values were within the range of the 95% vehicle control limit (with S9 mix: MFcorr.: 0.00 – 6.96 per 10^6 cells). A statistically significant increase or dose dependency was not observed.
The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 144.85 mutants per 10^6 cells; with S9 mix: MFcorr.: 23.68 – 118.5 mutants per 106 cells) were compatible with the historical positive control data range (without S9 mix: MFcorr.: 42.12 – 438.29 mutants per 10^6 cells; with
S9 mix: MFcorr.: 21.52 – 296.74 mutants per 10^6 cells).

Any other information on results incl. tables

Summary of results

Exp.	Exposure Period (h)	Test group (µg/ml)	S9 mix	Prec.*	Genotoxicity** MFcorr. (per 10^6 cells)	RS (%)	CE2 (%)***
1	4	Vehicle control1	-	n.d.	2.48	100.0	100.0
		205.8	-	-	n.a.	102.0	n.a.
		370.4	-	-	4.42	140.0	101.0
		666.7	-	-	1.20	205.8	103.7
		1200.0	-	-	2.88	113.5	77.4
		1600.0	-	-	0.97	101.2	102.7
		2400.0	-	+	n.c.	0.6	n.c.
		Positive control2	-	n.d.	144.85s	80.0	74.7
2	4	Vehicle control1	+	n.d.	0.72	100.0	100.0
		205.8	+	-	n.a.	79.4	n.a.
		370.4	+	-	1.18	82.6	80.9
		666.7	+	-	0.78	78.9	92.3
		1200.0	+	-	0.57	68.9	84.0
		1600.0	+	-	3.46s	48.9	83.0
		2400.0	+	+	n.c.	0.3	n.c.
		Positive control3	+	n.d.	118.15s	67.1	67.2
3	4	Vehicle control1	+	n.d.	1.33	100.0	100.0
		1000.0	+	-	2.91	72.7	91.4
		1200.0	+	-	2.99	103.0	89.0
		1600.0	+	-	2.21	103.4	90.4
		1800.0	+	+	0.40	55.3	83.4
		2200.0	+	+	n.c.	0.6	n.c.
		Positive control3	+	n.d.	23.68s	99.6	75.7

* macroscopically visible precipitation in culture medium at the end of exposure period
** Mutant frequency MFcorr.: mutant colonies per 10^6 cells corrected with the CE2 value
*** Cloning efficiency related to the respective vehicle control
s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)
n.a. Culture was not continued since a minimum of only four analysable concentrations is required
n.c. Culture was not continued due to strong cytotoxicity
n.d. not examined

Controls:
1 DMSO 1% (v/v) 2 EMS 400 μg/mL 3 DMBA 1.25 μg/mL

Applicant's summary and conclusion

Conclusions:

In the absence and the presence of metabolic activation, the test substance was not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The dose selection of this study was based on the solubility properties of the test substance in an appropriate vehicle and in culture medium in accordance to the recommendations of the current guidelines.
According to the results of the present in vitro study, in two experiments performed independently of each other the test substance 2-(2-Hexyloxyethoxy)ethanol did not lead to a biologically relevant increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the 95% control limit of the
historical negative control data.
In the 1st Experiment in the presence of metabolic activation, one value at 1600.0 μg/mL (MFcorr.: 3.46 mutants per 10^6 cells) was statistically increased compared to the concurrent vehicle control value. The value was, however, within the 95% control limit of the historical data base. The statistical significance was most probably due to the low rate of mutant colonies in the concurrent vehicle control group. Nevertheless, for clarification of the observation a 2nd Experiment was performed with a narrower dose range (1800.0 μg/mL). In this experiment the increase observed in the first experiment at (1600.0 μg/mL) could not be  reproduced.
Furthermore, in both experiments a dose related increase in the mutation frequencies could not be observed. Since the finding was not reproducible, it is regarded as biologically irrelevant.
The mutation frequencies of the vehicle control groups were within the historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study.
The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data.

The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.