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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay / Ames test: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA: all negative with and without metabolic activation (OECD TG 471)

In vitro Mammalian Cell Gene Mutation Test (HGPRT): CHO cells: negative with and without metabolic activation (OECD TG 476)


In Vitro Sister Chromatid Exchange Assay in Mammalian Cells: CHO cells: negative with and without metabolic activation (analog OECD TG 479)

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
29 Jul 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO (Chinese hamster ovary) cell line is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes

Stocks of the CHO cell line (1ml portions) are maintained at -196°C in the gas phase above the liquified nitrogen using 7% (v/v) DMSO in culture as a cryoprotectant. Before being used, each batch was checked for mycoplasma contamination.
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).
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle control cultures with and without S9 mix contain the vehicle selected for the test substance at the same volume and concentration as used in the test cultures.
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
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.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
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).

Summary of results









































































































































































































































































Exp.Exposure Period (h)Test group (µg/ml)S9 mixPrec.*Genotoxicity** MFcorr. (per 10^6 cells)RS (%)CE2 (%)***
14Vehicle control1-n.d.2.48100.0100.0
  205.8--n.a.102.0n.a.
  370.4--4.42140.0101.0
  666.7--1.20205.8103.7
  1200.0--2.88113.577.4
  1600.0--0.97101.2102.7
  2400.0-+n.c.0.6n.c.
  Positive control2-n.d.144.85s80.074.7
        
24Vehicle control1+n.d.0.72100.0100.0
  205.8+-n.a.79.4n.a.
  370.4+-1.1882.680.9
  666.7+-0.7878.992.3
  1200.0+-0.5768.984.0
  1600.0+-3.46s48.983.0
  2400.0++n.c.0.3n.c.
  Positive control3+n.d.118.15s67.167.2
        
34Vehicle control1+n.d.1.33100.0100.0
  1000.0+-2.9172.791.4
  1200.0+-2.99103.089.0
  1600.0+-2.21103.490.4
  1800.0++0.4055.383.4
  2200.0++n.c.0.6n.c.
  Positive control3+n.d.23.68s99.675.7


* 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

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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2020
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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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 Animal Welfare Act and the effective European Council Directive. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-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 was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL 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 thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.
Test concentrations with justification for top dose:
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 6.0 mg/plate was used as top dose in all experiments.
Vehicle / solvent:
Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

TEST SUBSTANCE PREPARATIONS
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethyl sulfoxide (DMSO). To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before use.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene (2-AA); 4-nitro-o-phenylenediamine (NOPD)
Details on test system and experimental conditions:
TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.

Salmonella typhimurium
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined. The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Checking the tester strains
The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid).
E. coli WP2 uvrA was checked for UV sensitivity. Histidine and tryptophan auxotrophy were checked in each experiment via the spontaneous rate.


Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al. .
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al.. 0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Scope of tests and test conditions

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 3000 and 6000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 3000 and 6000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.
Evaluation criteria:
Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 6 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Toxicity
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 109 cells per mL were used.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: See "additional information on results"
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: See "additional information on results"
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: See "additional information on results"
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: See "additional information on results"
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: See "additional information on results"
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY
A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions at and above 1000 μg/plate.
In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions at and above 1000 μg/plate.

Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment S9 TA 1535 TA 100 TA 1537 TA 98 E.coli
1st-SPT Without - - 6000 1000, 6000 1000 – 6000
With - - 6000 6000 1000 – 6000
2nd-PIT Without - 6000 6000 3000 – 6000 1000 – 6000
With - 6000 3000 – 6000 - 6000

Reduced background growth was observed at following concentrations (μg/plate):

Experiment S9 TA 1535 TA 100 TA 1537 TA 98 E.coli
1st-SPT Without - - - - -
With - - - - -
2nd-PIT Without 6000 6000 6000 6000 6000
With 6000 6000 6000 6000 6000

SOLUBILITY
No test substance precipitation was observed with and without S9 mix.

For detailed results and historical control data in tabular form see the attached background material.

Conclusions:
Under the experimental conditions chosen here, it is concluded that 2-(2-Hexyloxyethoxy)ethanol is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. The number of revertant colonies in the negative controls, with and without S9 mix, were within the range of the historical negative control data for each tester strain. In addition, the positive control substances with and without S9 mix induced a significant increase in the number of revertant colonies compatible with the range of the historical positive control data.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Criteria for a positive response were not fully described.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells
Specific details on test material used for the study:
- Name of test material (as cited in study report):Diethylene glycol monohexyl ether (DEGHE)
- Analytical purity: 96.8%
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-lo). For chemical exposures of cells without metabolic activation, F12 medium with 5% (v/v) of dialyzed bovine serum (F12-D5) is used. For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, is employed.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 metabolic activation system
Test concentrations with justification for top dose:
0.75 to 2.0 mg/mL without S-9 and 1.0 to 2.5 mg/mL with S-9
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: For SCE assays, dimethylnitrosamine (+S9) (DMN)-CAS #62-75-9 and ethylmethanesulfonate (-S9) (EMS)-CAS #62-50-0 were used as positive control agents to assure the sensitivity and reliability of the test system.
Remarks:
none
Details on test system and experimental conditions:
METHOD OF APPLICATION: not applicable

DURATION
- Preincubation period: not applicable
- Exposure duration: For determination of direct genotoxic action, CHO cells were exposed to DEGHE and appropriate controls for 5 hours without S9 activation. Indirect activity, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period.

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 pg/ml) is added to culture flasks 1 to 2 hrs prior to harvesting to arrest cells in mitosis.
STAIN (for cytogenetic assays): Bromodeoxyuridine (BrdU), required to differentiate between the individual "sistern chromatids by SCE staining.
Chromosomes on the slides are stained for SCEs by treatment with 5.0 pglml of Hoechst 33258 dye for approximately 20 min, rinsed in distilled water, immersed in Sorenson's buffer and exposed to a high intensity sunlamp for 15 to 30 min., as required. Irradiated chromosomes are stained in Gurr's giemsa (diluted 1:25 with phosphate buffer), rinsed in water and dried before application of coverslips.

NUMBER OF REPLICATIONS: Duplicate cultures of CHO cells were exposed

NUMBER OF CELLS EVALUATED: A total of twenty-five cells/concentration was examined for SCE frequencies using duplicate cultures. At least 5 dose levels were tested both with and without metabolic activation.

DETERMINATION OF CYTOTOXICITY:The surviving fraction was determined at 18 to 24 hours after the removal of the test chemical using 4 plates/culture and 100 cell/plate.
-Cytotoxicity is determined by comparing the relative number of cells in control cultures (untreated cells) to the numbers of cells in cultures treated with various concentration8 of the test agent.
Evaluation criteria:
Interpretation of Data: The criteria for evaluation of a positive or negative response depend both on statistical analyses and scientific judgement. The key determinant is whether a dose-dependent increase in SCEs is induced by the test agent and if consistent responses are seen in duplicate cultures/dose. When no clear dose-response relationship is evident and when one or more responses of marginal indications of statistical differences are obtained, a careful examination of the data in comparison to the concurrent controls and the historical data base is necessary to
determine probable biological significance of the statistical indicators. Clearly positive responses will include any of the following:
(i) Doubling in the SCE frequency by one or more concentrations with both of the duplicate cultures/dose;
(ii) Statistically significant responses of p < 0.01 with one or more consecutive concentrations
(iii) Induction of a statistically significant, dose-related increase in the number of SCE.
Random statistical indications of positive increases which do not meet the criteria for a positive test result will be concluded to be negative indications of DNA damage potential.
Statistics:
Data were analyzed after Box–Cox transformation and those from the DEGHE and DMSO control groups were intercomposed for equality of variance by Levene’s test, analysis of variance (ANOVA) and t-tests. The t-tests were used when the F value from ANOVA was significant. When Levene’s test indicated similar variances and the ANOVA was significant, a pooled t-test was used for pairwise comparison. When Levene’s test indicated heterogeneous variances, all groups were intercomposed by an ANOVA for unequal variances, followed by a separate t-test for pairwise comparisons.
Species / strain:
Chinese hamster Ovary (CHO)
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:
RANGE-FINDING/SCREENING STUDIES: Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would kill no more than (approximately) 90% of the treated cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Test results with the test substance indicated that concentrations of 3 mg/mL or higher were lethal to CHO cells. The highest suitable concentrations tested were 2.0 mg/mL without S9 and 2.5 Mg/ml with S9 activation for the CHO test.

There was no effect of treatment on the incidence of sister chromatid exchanges (SCEs) in the absence of metabolic activation.  

In the presence of metabolic activation, a significant  increase in SCEs was seen in one culture at 1.5 mg/ml (0.77 +/- 0.24 vs. 0.49 +/- 0.14 in DMSO control). 
This effect was not dose-related. There was no mitotic inhibition in the absence of S-9, and only the high dose (2 mg/ml) caused mitotic inhibition in the presence of S-9. The test was valid, as the positive controls induced significant increases in the frequency of SCEs.

Conclusions:
The SCE test results showed no significant increases of DNA damage in the test without S9 activation but one random statistically significant increase was observed in the test with S9. No pattern of a dose-related trend was evident and the single positive increase was not seen at higher doses or with the duplicate culture/dose. Because of the absence of consistent and dose related effects, the test substance was judged to possess at most, only a very weak genotoxic activity under the conditions of the present tests. However, the marginal increases seen in the CHO mutation test and SCE test, both with S9 activation, suggest that metabolism may play a critical role in biological effectiveness of the test substance. The present study suggests that the test substance lacks significant genotoxic potential but it must be considered a limited evaluation of possible metabolic effects upon biological activity. However, the presence of an active mutagenic contaminant responsible for the low level increases cannot be eliminated with current data.
Executive summary:

The test substace was evaluated for potential genotoxic activity using the Sister Chromatid Exchange (SCE) test.

Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in

which the highest concentration would kill no more than (approximately) 90% of the treated cells. Test concentrations for the SCE test were chosen in a range of 40 to 60% growth inhibition so that sufficient numbers of cells in the second division (M2) would be available for determination of SCEs. Test results with DEGHE indicated that concentrations of 3 mg/ml or higher were lethal to CHO cells. The highest suitable concentrations tested were 2.0 mg/ml without S9 and 2.5 rng/ml with S9 activation for the CHO test.

For the SCE test maximum concentrations tested were 2.0 mg/ml both with S9 and without S9 activation.

For determination of direct genotoxic action, CHO cells were exposed to DEGHE and appropriate controls for 5 hours without S9 activation. Indirect activity, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period. Bromodeoxyuridine (BrdU), required to differentiate between the individual "sistern chromatids by SCE staining, was present at a concentration of 3 pg/ml in the growth medium during treatment and during the culture period following exposure period. A total of twenty-five cells/concentration was examined for SCE frequencies using duplicate cultures. At least 5 dose levels were tested both with and without metabolic activation. SCE production was determined for the highest 3 doses which did not produce excessive cytotoxic inhibition of cell division. The number of SCEs/cell, mean number of SCEs/chromosome and the level of statistical significance of the increases above the concurrent solvent control values were scored.

The SCE test results showed no significant increases of DNA damage in the test without S9 activation but one random statistically significant increase was observed in the test with S9. No pattern of a dose-related trend was evident and the single positive increase was not seen at higher doses or with the duplicate culture/dose. Because of the absence of consistent and dose related effects, DEGHE was judged to possess at most, only a very weak genotoxic activity under the conditions of the present tests. However, the marginal increases seen in the CHO mutation test and SCE test, both with S9 activation, suggest that metabolism may play a critical role in biological effectiveness of DEGHE.

The present study suggests that the test substance lacks significant genotoxic potential but it must be considered a limited evaluation of possible metabolic effects upon biological activity. However, the presence of an active mutagenic contaminant responsible for the low level increases cannot be eliminated with current data.

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

Genetic toxicity in vivo

Description of key information

Mammalian Erythrocyte Micronucleus Test: mouse: negative (analog OECD TG 474)
Mammalian Bone Marrow Chromosome Aberration Test: rat: negative (analog OECD TG 475)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: Chromosome aberration assay
Specific details on test material used for the study:
- Name of test material (as cited in study report):Diethylene glycol monohexyl ether (DEGHE)
- Analytical purity: 96.8%
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s): corn oil
- Justification for choice of solvent/vehicle: not specified in the report
- Concentration of test material in vehicle: not specified in the report
- Amount of vehicle (if gavage or dermal): not specified in the report
- Type and concentration of dispersant aid (if powder): not specified in the report
- Lot/batch no. (if required): not specified in the report
- Purity: not specified in the report
Duration of treatment / exposure:
up to 48 hours
Frequency of treatment:
once
Post exposure period:
Animals treated with test material were terminated at preassigned time intervals of 12 hr, 24 hr or 48 hours for the first study, and 24 or 48 hours for the second study. Vehicle controls were terminated at 24 hours in the first study and at 24 and 48 hours in the second study. Positive control animals were killed at 24 hours in both studies.
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
first study / females
Dose / conc.:
900 mg/kg bw/day (actual dose received)
Remarks:
first study / females
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
first study / females
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
first study / males
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
first study / males
Dose / conc.:
2 400 mg/kg bw/day (actual dose received)
Remarks:
first study / males
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Remarks:
second study / males
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
second study / males
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
second study / males
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
second study / males
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Remarks:
second study / males
No. of animals per sex per dose:
In the first test, groups of 5 animals/sex/test interval were dosed with test material by gavage at 25% (750 mg/kg for males, 450 mg/kg for females), 50% (1500 mg/kg for males and 900 mg/kg for females), and 80% (2400 mg/kg for males and 1500 mg/kg for females) of the LD50 values.
An additional dose of 375 mg/kg (12.5% of LD50 value) was given to 5 males/ test interval (two of these rats were not within 2 standard deviations of the mean) due to high mortality at the high dose. Groups of 5/sex/interval were administered corn oil vehicle by gavage.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): historical control
- Route of administration: i. p.
- Doses / concentrations:30 mg/kg
Tissues and cell types examined:
When possible, 50 metaphase cells per animal were evaluated blindly for chromosome number, specific type of chromosome or chromatid-type aberrations, deletions and exchanges, gaps, endoreplicated chromosomes and polyploid cells were noted but were not included as statistically evaluated aberrations. Severely damaged cells (> = 10 breakage events) and pulverized cells were recorded as severely damaged, but no attempt was made to classify the types of damage in these cells.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary test acute peroral test was conducted as follows: males, 3016 (2496–3616) mg kg−1; females, 1823 (1927–2295) mg kg−1. Dose levels, selected on the basis of 25%, 50% and 80% of the LD50, were 750, 1500 and 2400 mg kg−1 for males and 450, 900 and 1500 mg kg−1 for females. Because of mortality at the high dose with males, an additional dose group of 375 mg kg−1 was added to the male groups.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals treated with test material were terminated at preassigned time intervals of 12 hr, 24 hr or 48 hours for the first study, and 24 or 48 hours for the second study. Vehicle controls were terminated at 24 hours in the first study and at 24 and 48 hours in the second study. Positive control animals were killed at 24 hours in both studies. Colchicine (4 mg/kg) was injected i.p. 2-3 hours prior to euthanization.

DETAILS OF SLIDE PREPARATION: A femur was removed from each animal. Bone marrow cells were collected and fixed. Slides were prepared and chromosomes were stained with Giemsa.

METHOD OF ANALYSIS: When possible, 500 cells per animal were scored for the proportion of mitotic cells. When possible, 50 metaphase cells per animal were evaluated blindly for chromosome number, specific type of chromosome or chromatid-type aberrations, deletions and exchanges, gaps, endoreplicated chromosomes and polyploid cells were noted but were not included as statistically evaluated aberrations. Severely damaged cells (> = 10 breakage events) and pulverized cells were recorded as severely damaged, but no attempt was made to classify the types of damage in these cells.
Evaluation criteria:
A positive effect will be detected by production of a statistically significant, dose-related increase in the frequency of structural chromosome aberrations. Alternatively, production of statistically significant increase for at least one dose level will be considered an equivocal effect if there is no evidence of a dose-related response.
For test with such random statistical indication, effects will not be considered to be biologically significant if increases are within the range of variation of the historical control data. Additional testing may be required to clarify suspect findings. A test substances which does not produce positive increases as described in the preceding paragraph will be considered inactive as a clastogenic agent in vivo.
Statistics:
Data were analyzed by Fisher’s Exact Test to determine any statistical increase of aberrant cells between the DEGHE and CP groups compared with the corn oil controls.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None of the the test substance dose levels tested produced statistically significant or dose-related increases in relative numbers of chromosomal aberrations compared to control values among female Sprague-Dawley rats.
Similarly, male Sprague-Dawley rats in the 12 hr and 24 hr post-treatment sacrifice groups had no statistically significant or dose related increases in the incidence of chromosomal aberrations. However, male Sprague-Dawley rats in the 48 hr post-treatment sacrifice group treated at 375 mg/kg or at 750 mg/kg test substance did have statistically significant increases in the incidence of chromosomal aberrations. The incidence of chromosomal aberrations increased with increasing concentrations over these two dose levels. Due to the magnitude of the increases, their biological significance could not be ruled out.
Therefore, the study was repeated in part. Male Sprague-Dawley rats (5/dose/sacrifice) were dosed at 375, 600, 750, 1000 or 1500 mg/kg test substance and sacrificed at either 24 hr or 48 hr post-treatment. There were no statistically significant or dose-related increases in the incidence of chromosomal aberrations among male Sprague-Dawley rats in the repeat test. Consequently, the test substance was not  considered clastogenic to Sprague-Dawley rats under the conditions of this in vivo test.

Table 1: Summary of chromosomal aberrations in femoral bone marrow of Sprague-Dawley rats at various times after dosing perorally with DEGHE and corn oil and following an intraperitoneal injection of cyclophosphamide

Sample time (h)

Materiala

Dose (mg/kg)

Mitotic index (%)b

Total aberrant cells for whole groupc

Aberrant cells as mean± SD

 

First study - males

12

DEGHE

375

1.7 ± 0.4

8

3.2 ± 2.7

 

~

750

1.7 ± 0.9

4

1.6 ± 1.7

 

~

1500

3.1 ± 0.8

9

3.6 ± 3.0

24

DEGHE

375

4.1 ± 1.5

4

1.6 ± 1.70

 

~

750

5.8 ± 1.4

3

1.2 ± 1.8

 

~

1500

6.7 ± 2.8

3

1.2 ± 1.1

 

Corn oil

 

4.8 ± 1.7

3

1.2 ± 1.1

 

CP

30

2.3 ± 0.4

63

25.2 ± 3.6d

48

DEGHE

375

4.6 ± 2.2

17

6.8 ± 4.6e

 

~

750

6.3 ± 1.4

58

34.3 ± 30.7f

 

~

1500

4.8 ± 1.3

5

2.0 ± 2.0

First study - females

12

DEGHE

450

5.8 ± 1.2

4

1.6 ± 2.2

 

~

900

6.0 ± 1.0

0

0

 

~

1500

5.5 ± 0.7

9

3.6 ± 2.6

24

DEGHE

450

5.4 ± 1.6

13

5.2 ± 2.3

 

~

900

5.2 ± 1.7

13

5.2 ± 4.8

 

~

1500

6.4 ± 2.1

10

4.0 ± 2.8

 

Corn oil

 

3.7 ± 1.0

8

3.2 ± 1.8

 

CP

30

2.5 ± 1.4

49

24.2 ± 11.0

48

DEGHE

450

4.1 ± 1.9

6

2.4 ± 0.9

 

~

900

3.7 ± 1.4

6

2.4 ± 0.9

 

~

1500

4.5 ± 2.0

10

4.0 ± 3.2

Second study - males

24

DEGHE

375

5.9 ± 0.7

3

1.2 ± 1.8

 

 

600

6.8 ± 1.6

4

1.6 ± 1.7

 

 

750

6.2 ± 1.2

4

1.6 ± 2.6

 

 

1000

5.9 ± 2.9

4

1.6 ± 1.7

 

 

1500

6.4 ± 2.3

2

0.8 ± 1.1

 

Corn oil

 

6.8 ± 2.3

3

1.2 ± 1.8

 

CP

30

2.0 ± 0.5

41

16.4 ± 7.3d

48

DEGHE

375

7.2 ± 1.3

0

0

 

 

600

6.7 ± 1.7

2

0.8 ± 1.1

 

 

750

8.4 ± 1.2

0

0

 

 

1000

7.2 ± 1.5

3

1.2 ± 1.8

 

 

1500

8.0 ± 1.6

0

0

 

Corn oil

 

6.7 ± 1.9

2

0.8 ± 1.1

aDEGHE = diethylene glycol monohexyl ether; CP = cyclophosphamide.

bMean ± SD.

c50 cells scored for each animal; n = five of each gender per group.

dP < 0.001 compared with corn oil controls.

eP < 0.01 compared with corn oil controls.

Conclusions:
The test substance was not considered clastogenic to Sprague-Dawley rats under the conditions of this in vivo test.
Executive summary:

The test substance was evaluated for the chromosomal abberation test in rats. The test substance was given to both male and female Sprague-Dawley rats as a single oral dose by gavage. Bone marrow cells were harvested and evaluated for potential chromosomal damage. Preliminary testing (LD50) indicated that the substance produced mortality in all of the female rats at dose levels between 3200 and 5000 mg/kg, in 4 out of 5 male rats at 4000 mg/kg and in all of the male rats dosed at 5000 mg/kg. The LD50 was 3016 mg/kg (2496 to 3616, 95% confidence interval) for the male rats and 1823 mg/kg (1297 to 2295, 95% confidence interval) for the female rats. Dose levels for the definitive bone marrow cytogenetics assay were approximately 80% (2400 mg/kg for males, 1500 mg/kg for females), 50% (1500 mg/kg for males, 900 mg/kg for females) and 25% (750 mg/kg for males, 450 mg/kg for females) of the LD50. An additional dose level of 12.5% of the LD50 (male rats only) was added to the study on the day of dosing since 9 out of 15 male rats dosed at 80% of the LD50 (2400 mg/kg) died within 4.5 hours of dosing.
None of the test substance dose levels tested produced statistically significant or dose-related increases in relative numbers of chromosomal aberrations compared to control values among female Sprague-Dawley rats.
Similarly, male Sprague-Dawley rats in the 12 hr and 24 hr post-treatment sacrifice groups had no statistically significant or dose related increases in the incidence of chromosomal aberrations. However, male Sprague-Dawley rats in the 48 hr post-treatment sacrifice group treated at 375 mg/kg or at 750 mg/kg the test substance did have statistically significant increases in the incidence of chromosomal aberrations. The incidence of chromosomal aberrations increased with increasing the test substance concentrations over these two dose levels. Due to the magnitude of the increases, their biological significance could not be ruled out.
Therefore, the study was repeated in part. Male Sprague-Dawley rats (5/dose/sacrifice) were dosed at 375, 600, 750, 1000 or 1500 mg/kg the test substance and sacrificed at either 24 hr or 48 hr post-treatment. There were no statistically significant or dose-related increases in the incidence of chromosomal aberrations among male Sprague-Dawley rats in the repeat test. Consequently, the test substance was not considered clastogenic to Sprague-Dawley rats under the conditions of this in vivo test.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Remarks:
Likely to have been conducted under GLP
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test
Specific details on test material used for the study:
- Analytical purity: 96.8% (by capillary gas chromatography)
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Weight at study initiation: 23.8 - 26.9 g for males and 20.6 - 22.0 g for females
- Housing: five of each gender per cage in shoe-box-type plastic cages
- Assigned to test groups randomly: yes
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period:5 - 6 days
Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: corn oil
Details on exposure:
intraperitoneal
Duration of treatment / exposure:
single i.p. injection
Frequency of treatment:
once
Post exposure period:
30, 48 and 72 hours
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
640 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 male and 5 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg
Tissues and cell types examined:
erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
used doses represented 25%, 50% and 80% of LD50 determined in preliminary test (LD50 in males: 894 mg/kg; in females: 715 mg/kg)

DETAILS OF SLIDE PREPARATION:
at each time time period on or two drops of blood were collected from each animal by tail nicking and blood smears were prepared. Slides were stained with Gurr's R-66 Giemsa diluted with phosphate buffer and coded by animal number.

METHOD OF ANALYSIS:
blind reading, PCE/NCE ratio for 1000 total cells and the number of micronucleated polychromatic erythrocytes (MN-PCE) for 1000 PCE were determined.
Evaluation criteria:
statistically significant and dose-related increase in MN-PCE
Statistics:
Fisher's exact test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 410-1000 mg/kg
- Harvest times: 48 and 72 h post-injection
- Other: intraperitoneal LD50 in males: 894 (664-1203) mg/kg; in females: 715 (623-821) mg/kg)

RESULTS OF DEFINITIVE STUDY
No positive or dose-related increases in the frequency of micronucleated PCE of the bone marrow were observed after intraperitoneal injection of the test substance to Swiss-Webster mice.
The test substance was considered to be inactive as a clastogenic agent.

Dosages, mortality and PCE/NCE ratios for mice given an acute i.p. injection of DEGHE or corn oil :

Materiala Dosage Group size Number
dyingb
Mortality (%) PCE/1000 NCEc      
48 h    72 h   
          Mean ± SD %Control Mean ± SD %Control
Males
DEGHE 410 mg/kg 5 0 0
DEGHE 512 mg/kg 5 0 0
DEGHE 640 mg/kg 5 1 20 33 ± 5.3 124 26.0 ± 3.7 88.4
DEGHE 800 mg/kg 5 4 80
DEGHE 1000 mg/kg 5 5 100
Corn oil 10 ml/kg 5 0 0 26.6 ± 4.0  - 29.4 ± 3.4
Females
DEGHE 410 mg/kg 5 0 0
DEGHE 512 mg/kg 5 0 0
DEGHE 640 mg/kg 5 0 0
DEGHE 800 mg/kg 5 2 40 24.3 ± 2.5d 79.9 24.0 ± 1.0 68.2
DEGHE 1000 mg/kg 5 3 60
Corn oil 10 ml/kg 5 0 0 30.4 ± 2.1  - 35.2 ± 8.1  -

aDEGHE = diethylene glycol monohexyl ether; corn oil is solvent control

bThree-day mortality value

cPCE = polychromatic erythrocyte; NCE = normochromatic erythrocyte

dP<0.05 compared with solvent control

Polychromatic erythrocyte to normochromatic erythrocyte ratios in peripheral blood samples from Swiss Webster mice at various times after an intraperitoneal injection of DEGHE or control substances:

Materiala Dosage Sex 30 hb  48 hb  70 hb 
PCE/1000 NCE % of controls PCE/1000 NCE % of controls PCE/1000 NCE % of controls
Corn oil 10 ml/kg M 37.8 ± 4.82 44.6 ± 6.02 39.4 ± 2.70
F 36.0 ± 3.39 41.4 ± 4.51 40.6 ± 6.80
TEM 0.3 mg/kg M 33.0 ± 7.07 87.3 NE NE
F 35.4 ± 3.05 98.3 NE NE
DEGHE 200 mg/kg M 37.0 ± 7.28 97.7 41.8 ± 4.32 93.7 38.4 ± 3.21 97.5
F 38.4 ± 3.85 106.7 42.4 ± 8.44 102.4 41.2 ± 6.02 101.5
DEGHE 400 mg/kg M 39.0 ± 8.51 103.2 41.0 ± 2.12 91.9 35.6 ± 3.21 90.4
F 42.6 ± 7.02 118.3 45.4 ± 4.98 109.7 41.5 ± 6.35 102.2
DEGHE 640 mg/kg M 35.6 ± 9.42 94.2 39.8 ± 6.53 89.2 33.0 ± 1.58 83.8
F 40.2 ± 6.34 111.7 40.2 ± 3.83 97.1 35.6 ± 2.97 87.7

aCorn oil was solvent control; TEM = triethylenemelamine; DEGHE = diethyl glycol monohexyl ether

bPCE = polychromatic erythrocyte; NCE = normochromatic erythrocyte; % change in ratio calculated against corn oil controls

cP<0.05 compared with corn oil control

Frequency of micronucleated polychromatophils in peripheral blood samples from Swiss Webster mice at various times after the intraperitoneal injection of DEGHE and control substances

Sample
time (h)
Materiala Dose Sex MN-PCE/1000 PCEb  Mean % MN-PCEc
Per animal Mean ± SD
30 Corn oil 10 ml/kg M 4,3,4,1,1 2.6 ± 1.52 0.26
F 1,2,2,1,0 1.2 ± 0.84 0.12
TEM 0.3 mg/kg M 27,28,31,13,39 27.6 ± 9.42 2.76c
F 44,35,30,30,15 30.8 ± 10.52 3.08c
DEGHE 200 mg/kg M 3,3,2,3,1 2.4 ± 0.89 0.24
F 2,4,1,1,1 1.8 ± 1.30 0.18
DEGHE 400 mg/kg M 5,1,1,3,1 2.2 ± 1.79 0.22
F 0,2,2,0,0 0.8 ± 1.10 0.08
DEGHE 640 mg/kg M 0,1,2,0,3 1.2 ± 1.30 0.12
F 2,1,1,2,0 1.2 ± 0.84 0.12
48 Corn oil 10 ml/kg M 3,5,3,4,2 3.4 ± 1.14 0.34
F 1,1,2,1,2 1.4 ± 0.55 0.14
DEGHE 200 mg/kg M 2,1,0,3,3 1.8 ± 1.30 0.18
F 2,1,1,1,3 1.6 ± 0.89 0.16
DEGHE 400 mg/kg M 5,2,4,1,2 2.8 ± 1.64 0.28
F 1,2,3,0,0 1.2 ± 1.30 0.12
DEGHE 640 mg/kg M 0,2,4,4,2 2.4 ± 1.67 0.24
F 0,0,1,0,0 0.2 ± 0.45 0.20
72 Corn oil 10 ml/kg M 6,0,2,2,1 2.2 ± 2.28 0.22
F 2,2,1,2,0 1.4 ± 0.89 0.14
DEGHE 200 mg/kg M 4,2,1,2,1 2.0 ± 1.22 0.20
F 3,3,2,2,8 3.6 ± 2.51 0.36
DEGHE 400 mg/kg M 2,0,1,4,3 2.0 ± 1.58 0.20
F 3,1,2,1,0 1.8 ± 0.96 0.18
DEGHE 640 mg/kg M 3,2,0,4,0 1.8 ± 1.79 0.18
F 1,1,1,1,0 0.8 ± 0.45 0.08

aCorn oil = solvent control; TEM = triethylenemelamine; DEGHE = diethylene glycol monohexy ether; MN-PCE = micronucleated polychromatophilic erythrocyte

b1000 PCE counted per animal (n = five of each sex per group)

cP<0.001 compared with corn oil control

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

Additional information

The test substance was not mutagenic in the Bacterial reverse mutation assay (S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvrA) or mammalian cells (HPGRT assay), and did not produce an increase in SCEs in CHO cells (both tests with and without metablic activation). In vivo, the mouse peripheral blood micronucleus test and the rat femoral bone marrow chromosomal aberration test showed no evidence for a clastogenic potential.





Justification for classification or non-classification

Due to the fact that the test substance was not mutagenic in all in vitro and in vivo assays, classification is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.