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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro genotoxicity studies have been performed: C14 Alkyl ethoxylated glycidylether was shown to be non-mutagenic in the in vitro gene mutation study in bacteria (Ames). C12-14 Alkyl ethoxylated glycidylether is not clastogenic or aneugenic in human lymphocytes (OECD 478) but was concluded positive in the in vitro mutagenicity assay in L5178Y mouse lymphoma cells (MLA, OECD 490). The positive result was confined only to incubations without metabolic activation after prolonged treatment (24 hours) at the highest tested, toxic dose level of 50 µg/mL, demonstrated by a relative total growth of only 8-13%.

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Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 October 2018 - 24 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
No correction was made for the composition of the test item.
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
For lymphocytes:
- Sex, age and number of blood donors:
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2017) are presented below:

Dose-range finding study / First cytogenetic assay: age 32, AGT = 14.1 h
Dose-range finding study 2: age 26, AGT = 13.8 h
Cytogenetic assay 1A: age 26, AGT = 14.2 h
Cytogenetic assay 2: age 26, AGT = 15.8 h
Cytogenetic assay 2A: age 26, AGT = 14.4 h

Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum , L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.

Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.

Cytokinesis block (if used):
Cytochalasine B (5 μg/mL) for 24 hours (1.5 times normal cell cycle)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 :
Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix:
S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES.
The solution was filter (0.22 μm)-sterilized. S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration in exposure medium:
1.8% (v/v).
Test concentrations with justification for top dose:
Dose range finding test 1/Cytogenetic assay 1
Without S9-mix, 3 hr exposure; 27 hr harvest: 63, 125 and 250 μg/mL
Without S9-mix, 24 hr exposure, 24 hr harvest 15.6, 31.3, 63, 125 and 250 μg/mL
With S9-mix, 3 hr exposure; 27 hr harvest: 63, 125 and 250 μg/mL

Dose range finding test 2:
Without S9-mix, 3 hr exposure; 27 hr harvest: 3.1, 6.3, 12.5, 25, 50 and 100 μg/mL
Without S9-mix, 24 hr exposure, 24 hr harvest 0.4, 0.8, 1.6, 3.1, 6.3 and 12.5 μg/mL
With S9-mix, 3 hr exposure; 27 hr harvest: 3.1, 6.3, 12.5, 25, 50 and 100 μg/mL

Cytogenetic assay 1A:
Without S9-mix, 3 hr exposure; 27 hr harvest: 1, 20, 30, 40, 50 and 60 μg/mL
With S9-mix, 3 hr exposure; 27 hr harvest: 1, 20, 50, 60, 80 and 100 μg/mL

Cytogenetic assay 2:
Without S9-mix, 24 hr exposure, 24 hr harvest: 1, 10, 20, 40, 60, 80 and 100 μg/mL

Cytogenetic assay 2A:
Without S9-mix, 24 hr exposure, 24 hr harvest: 0.5, 2, 4, 6, 8, 10 and 12 μg/mL

Justification for top dose: based on the cytokinesis-block proliferation index of 55 ± 5%.
Vehicle / solvent:
Vehicle: DMSO
- Justification for choice of vehicle: The test item was soluble in dimethyl sulfoxide

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
solvent: Hanks’ Balanced Salt Solution without calcium and magnesium.
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Remarks:
Mitomycin C (without S9): final conc. 0.25 and 0.38 μg/mL for 3h and 0.15 and 0.23 μg/mL for 24 h. Colchicine (without S9) : final conc. of 0.1 μg/mL for 3h and 0.05 μg/mL for 24 h. Cyclophosphamide (with S9): final conc of 15 and 17.5 μg/mL for 3h.
Details on test system and experimental conditions:
Environmental conditions:
Humidity: 46 - 97%
CO2: 5.0 ± 0.5%
Temperature: 33.6 - 37.1°C

Test item preparation:
The test item was dissolved in DMSO and formed a clear colorless solution. Test item concentrations were used within 1.5 hours after preparation.The final concentration of DMSO in the culture medium was 1.0% (v/v).

Number of replicates:
2

Duration:
Without and with S9-mix: 3 hour treatment, 27 hour harvest time
The exposure period was followed by centrifugation, rinsing, a second centrifugation and incubation for another 24 hours with cytokinase block.

Without S9-mix: 24 hour treatment, 24 hour harvest time.
The exposure period was followed by immediate fixation without rinsing.

Cytokinase block:
5 µg/mL Cytochalasine B for 24 hours

Preparation of slides:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol: acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated.

Cytotoxicity:
Cytotoxicity was determined using the cytokinesis-block proliferation index (CBPI index).

NUMBER OF CELLS EVALUATED
At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei.
In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately
Evaluation criteria:
ACCEPTABILITY CRITERIA
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

The following criteria for scoring of binucleated cells were used (1 - 2, 6):
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Key result
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose-range Finding Test and CYTOGENETIC ASSAY 1
The test item precipitated at a concentration of 250 μg/mL.
All concentrations were cytotoxic thus the dose-range finding test was repeated.

Dose-range Finding Test 2
Based on the results of dose range finding 2 dose levels were selected for performing cytogenetic assay 1A

CYTOGENETIC ASSAY 1A
Doses selected for scoring (without S9): 1, 30 and 50 μg/mL culture medium
Doses selected for scoring (with S9): 1, 50 and 80 μg/mL culture medium
Both in the absence and presence of S9-mix, C12-14 Alkyl ethoxylated glycidylether did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

CYTOGENETIC ASSAY 2
No appropriate dose levels could be selected for scoring of micronuclei since the concentration of 10 μg/mL was already too toxic for scoring (69%).
Therefore, this part of the experiment was repeated in cytogenetic assay 2A

CYTOGENETIC ASSAY 2A
Doses selected for scoring (without S9): 0.5, 6 and 10 μg/mL culture medium
C12-14 Alkyl ethoxylated glycidylether induced a statistically significant increase in the number of mononucleated cells with micronuclei at the highest concentration tested. three mononucleated cells with micronuclei were counted compared to the vehicle control (0 cells). However, the number of mononucleated cells with micronuclei was within the 95% control limits of the distribution of the historical negative control database. Furthermore, since the Cochran Armitage trend test was negative (p = 0.115), the increase was considered not biologically relevant. No statistically significant increases were observed in the number of binucleated cells with micronuclei.

VALIDITIY
The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei.
The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated an binucleated cells with micronuclei.
In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Conclusions:
An in vitro micronucleus assay performed according to OECD 487 and GLP principles showed that C12-14 Alkyl ethoxylated glycidylether is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described.
Executive summary:

The objective of this study was to evaluate C12-14 Alkyl ethoxylated glycidylether for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of C12-14 Alkyl ethoxylated glycidylether was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

Batch 1658866 of C12-14 Alkyl ethoxylated glycidylether was a clear colourless liquid. The vehicle of the test item was dimethyl sulfoxide.

In the first cytogenetic assay, C12-14 Alkyl ethoxylated glycidylether was tested up to
50 µg/mL and 80 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at these dose levels.

In the second cytogenetic assay, C12-14 Alkyl ethoxylated glycidylether was tested up to
100 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of
S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system
(S9-mix) functioned properly.

C12-14 Alkyl ethoxylated glycidylether did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, this test is valid and C12-14 Alkyl ethoxylated glycidylether is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Horse serum: inactivated by incubation at 56°C for at least 30 minutes.
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth media:
Basic medium: supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: For 3 hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium); for 24 hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT).
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-150°C).
- Periodically checked for Mycoplasma contamination: yes
- Cleansed against high spontaneous background: yes
Environmental conditions: All incubations were carried out in a humid atmosphere (31 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 35.4 - 37.5°C.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
Dose-range finding test (with and without S9-mix, 3 hour treatment; without S9-mix, 24 hour treatment): 15.6, 31.3, 63, 125 and 250 µg/mL.
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 250 μg/mL.

Main experiment 1:
3 hours treatment (without S9-mix): 0.6, 1.3, 2.5, 5, 10, 20, 40, 45, 50, 55 and 60 μg/mL exposure medium.
3 hours treatment (with S9-mix): 0.6, 1.3, 2.5, 10, 20, 50, 70, 80, 90, 100, 110 and 120 μg/mL exposure medium.
Additional first experiment (experiment 1A): 1.3, 2.5, 5, 10, 20, 50, 55, 60, 65, 70 and 80 μg/mL exposure medium.

Main experiment 2:
24 hours treatment (without S9-mix): 0.63, 1.3, 2.5, 5, 10, 20, 30, 35, 40, 45, 50 and 60 μg/mL exposure medium.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: DMSO was selected as vehicle based on a solubility test.
- The final concentration of DMSO in exposure medium was 1% (v/v)



Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; in DMSO: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9: 7.5 µg/mL, in Hanks’ balanced salt solution (HBSS) without calcium and magnesium.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cell density: 10^6 cells/mL for 3 hour treatment or 1.25 x 10^5 cells/mL for 24 hour treatment.

CLEANSING:
Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

EXPOSURE:
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix)
- Expression time (cells in growth medium): 2 days; at least 4 x 10^6 cells were subcultured every day.
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

CELL STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for 1.5-2 hours.

NUMBER OF REPLICATIONS:
- Solvent controls: 2
- Test concentrations and positive control: 1

DEERMINATION OF THE MUTATION FREQUENCY:
For determination of the Cloning Efficiency (CEday2) the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).
The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. The plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF THE MUTANT COLONIES
The colonies were divided into small and large colonies. The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

ACCEPTABILITY CRITERIA
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6)



Evaluation criteria:
- Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
mammalian cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
3 hours treatment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Relative total growth at the highest test item concentration was 8% and 14%, respectively without and with metabolic activation compared to the total growth of the solvent control
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
mammalian cell line
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
24 hours treatment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Relative total growth of the highest test item was 13% compared to the total growth of the solvent controls
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE-RANGE FINDING TEST:
Precipitation observed:
125 μg/mL and above directly after addition to the exposure media.
250 μg/mL and above after 3 and 24 hours treatment.
Cells were treated with a concentration range of 15.6 to 250 μg/mL during 3 hours (with and without S9-mix) and during 24 hours (without S9-mix).

Relative suspension growth:
- 3 hours of treatment, with S9-mix: the relative suspension growth was 49% at the test item concentration of 63 μg/mL compared to the relative suspension growth of the solvent control.No cell survival was observed at test item concentrations of 125 μg/mL and above.
- 3 hours of treatment, without of S9-mix: the relative suspension growth was 60% at the test item concentration of 31.3 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test item concentrations of 63 μg/mL and above.
- 24 hours of treatment, without S9-mix: the relative suspension growth was 3 % at the test item concentration of 63 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 125 μg/mL and above.

MUTATION EXPERIMENT 1
In the 3 hours treatment without S9-mix, the dose levels of 0.6 to 10 μg/mL showed no cytotoxicity. Therefore, the dose level of 10 μg/mL was not regarded relevant for mutation frequency measurement.
The dose levels of 55 and 60 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
In the 3 hours treatment with S9-mix, no appropriate levels of toxicity were observed and this part of the experiment was repeated (experiment 1A). The following dose-range was selected for the additional first experiment: 1.3, 2.5, 5, 10, 20, 50, 55, 60, 65, 70 and 80 μg/mL exposure medium. The dose levels of 1.3 to 50 μg/mL showed no cytotoxicity. Therefore, the dose levels of 1.3 and 2.5 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 80 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 0.6, 1.3, 2.5, 5, 20, 40, 45 and 50 μg/mL exposure medium.
With S9-mix: 5, 10, 20, 50, 55, 60, 65 and 70 μg/mL exposure medium.
The relative total growth of the highest test item concentration compared to the total growth of the solvent controls were 8% (without S9-mix) and 14% (without S9-mix).

Evaluation of mutagenicity: No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

MUTATION EXPERIMENT 2
In the 24 hours treatment without S9-mix, the dose levels of 30 to 40 μg/mL and 45 and 50 μg/mL showed similar cell growth delay. Therefore, the dose levels of 30, 40 and 45 μg/mL were not regarded relevant for mutation frequency measurement.
The dose level of 60 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were: 0.63, 1.3, 2.5, 5, 10, 20, 35, and 50 μg/mL exposure medium.
The relative total growth of the highest test item was 13% compared to the total growth of the solvent controls.

Evaluation of mutagenicity: An increase above the positive threshold of MF(controls) + 126 (GEF= 227 x 10^-6) was observed at the top dose level of 50 μg/ml.

CONTROLS
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. The mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:
An in vitro mammalian cell gene mutation test was performed according to the OECD guideline 490 and following GLP principles with C12-14 Alkyl ethoxylated glycidylether. Based on the results, the test item is mutagenic in the mouse lymphoma L5178Y test system. The mutagenicity was confined only to incubations without metabolic activation after prolonged treatment (24 hours) at a single toxic dose level of 50 µg/mL, showing a relative total growth of only 8-13%.
Executive summary:

The mutagenic potential of C12-14 Alkyl ethoxylated glycidylether (GE) was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence (3 and 24 hour treatment) or presence (3 hour treatment) of a metabolic system (S9-mix).

In the dose-range finding test, in the absence of S9-mix, the relative suspension growth was 60% at the test item concentration of 31.3 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at the next test item concentrations of 63 μg/mL and above. In the presence of S9-mix, the relative suspension growth was 49% at the test item concentration of 63 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 125 μg/mL and above. 

In the first experiment GE was tested for 3 hours exposure up to concentrations of 50 (-S9) and 70 µg/mL (+S9) in DMSO. Relative total growth (RTG) was reduced to 8 and 14%, respectively. The test item did not precipitated in the culture medium up to the dose level of 70 µg/mL.

In the second experiment was tested for 24 hours up to concentrations of 50 µg/mL (-S9). The RTG was reduced to 13%. The test item did not precipitated in the culture medium up to the dose level of 50 µg/mL.

Results from positive controls and solvent control were within the acceptability criteria of the assay.

 

In the first mutation experiment in the absence and presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency (MF).

In the second mutation experiment in the absence of S9-mix, the test item induced increases in the MF, with a MF just above the positive threshold at the top dose level of 50 µg/ml only.

Although the increase in the mutation frequency at the TK locus was only observed at a toxic dose level (RTG of only 13% at 50 µg/mL, with no growth from 55 µg/mL, whereas in experiment 1 the same concentration lead to a RTG of 8%, which is below the lowest recommended level for toxicity stated in the guideline), the mutation frequency at this concentration was above the GEF and the result is therefore considered to be biological relevance and the test item mutagenic at toxic dose levels after prolonged treatment.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
20-22 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
HYPOTHESIS FOR THE ANALOGUE APPROACH
Read across is done from C14-Alkyl polyethoxy glycidylether (C14-GEE) to C12-14 Alkyl polyethoxy glycidylether (C12-14-GEE)

The production of both substances is identical: Alkyl alcohol ethoxylate (2EO) oligomers are reacted with epichlorohydrin (in slight molar excess) in the presence of a catalyst to form the corresponding epoxides. The substances are in principle UVCB because of variation in alkyl chain, number of EO (range 0 to 4, average 2) and possibility of 0 to two glycidyl (most 1) and all possible combinations.

However, the only difference between the two substances is the alkyl chain lengths distribution of the Alkyl alcohol (2EO) starting material, being either a (predominantly) 14-carbon chain length or a mixture of 12 and 14-carbon chain length in the ratio of 3:1 (C12:C14).
As consequence, these products are for 25% completely identical, whereas for the other 75% the C12-14-GEE has a slightly shorter alkyl chain compared to the C14-GEE.
For all other aspects, the substances are identical. As both have the same chemical structure they both have identical functional groups, show the same chemical reactivity and have very comparable physicochemical properties as can be seen in the comparative profiling included to the endpoint summary of chapter 7.1 on toxicokinetics. Consequently, they share the same mechanism of action. This is similarly applicable to all constituents of these UVCBs.

Related to the (small) difference in average alkyl chain-lengths between the two substances, the C12-14-GEE with on average a slightly shorter chain-length than the C14-GEE, is expected to be slightly better bioavailable (slightly smaller molecule also leading to more moles per unit of weight, slightly higher solubility, slightly lower Kow, and slightly higher vp) and is expected to have (a slightly) higher toxicity profile compared to C14-GEE.

Specifically for mutagenicity, the molecular structure with functional groups are essential with respect to reactivity to proteins and DNA. In that respect, C12-14-GEE and C14-GEE are identical. As dosing of the bacteria was limited by level of toxicity, it means that the exposure of the bacteria to C14-GEE was adequately, and therefore its results are also fully applicable to C12-14-GEE.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100
experiment 1 and 2: All tester strains in triplicate:
Dose range ranged between 1.5 and 5000 µg/plate depending on bacterial tester strain type and presence or absence of S9-mix.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene and 1,8-dihydroxyanthraquinone (DANTHRON)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett’s method of linear regression
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the bacterial tester strains both with and without metabolic activation. The first indication of toxicity was observed at 150 ug/plate.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Conclusions:
Not mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

Introduction.

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 “Reverse Mutation Study”, Method B 14 of Commission Directive 92/69/EEC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

 

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 were treated with the test material using the Ames plate incorporation method up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 1.5 to 5000 µg/plate depending on tester strain type. The experiment was repeated on a separate day using a similar dose range to Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

 

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the bacterial tester strains both with and without metabolic activation. The first indication of toxicity was observed at 150 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose of 5000 µg/plate or its toxic limit, depending on bacterial tester strain type and the presence or absence of S9-mix.

An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Alkyl ethoxylated glycidylether is an epoxide. The epoxide ring of glycidyl derivatives contains electrophilic carbon atoms which can react with nucleophiles at rates dependent on the substitution pattern on the ring and the nature of the nucleophile. The epoxide ring opening can occur either via an SN2 nucleophilic reaction or an SN1 reaction which proceeds via a reactive carbenium ion. Such reactions can have the potential to alkylate nucleophilic centres in DNA.

A large number of glycidyl derivatives have been found to give positive responses in the Ames test, usually with the strains TA1535 and TA100 of Salmonella typhimurium. Also, positive results in the in vitro chromosome aberration test have been reported. In in vivo assays, however, responses are usually negative, with a few weakly positive or equivocal results.

The lack of positive responses in vivo appears to be largely the result of detoxification, mainly by epoxide hydrolase, but also by conjugation with glutathione

 

Considering that Alkyl ethoxylated glycidylether was clearly negative in the Ames and in vitro micronucleus assay, indicates that, although being a glycidylether, its genotoxic potency is compared to other glycidylethers low. Alkyl ethoxylated glycidyle resulted to a positive results in an in vitro Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells (MLA). However, the increase in the mutation frequency at the TK locus was a little above the threshold and was only observed at the highest, toxic dose level (RTG of 13%), and only in the experiment involving 24-hr exposure without S9. The same concentration without S9 resulted to a RTG of 8% in the short-time exposure, which is below the lowest recommended level for toxicity stated in the guideline. This single, marginal positive result at toxic level therefore also indicates a low potency.

Considering that most positive genotoxic acting glycidyl ethers are negative in vivo, a negative in vivo result can also be expected for Alkyl ethoxylated glycidylether.

Additional information

In vitro gene mutation study in bacteria (Ames, OECD 471):

C14 Alkyl ethoxylated glycidylether was tested in the bacterial Reverse Mutation Study “Ames test” using Salmonella typhimurium. Strains TA1535, TA1537, TA98, TA100 and TA102 were treated with the test material using the plate incorporation method up to seven dose levels, in triplicate, both with and without S9-mix. The dose range for the first experiment was determined in a preliminary toxicity assay and ranged between 1.5 to 5000 µg/plate depending on tester strain type. An independent repeated of the assay was performed on a separate day.

Results: Results from positive controls and solvent control were within the acceptability criteria of the assay.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies in all of the bacterial tester strains both with and without metabolic activation. The first indication of toxicity was observed at 150 µg/plate. The test material was, therefore, tested up to either the maximum recommended dose of 5000 µg/plate or its toxic limit, depending on bacterial tester strain type and the presence or absence of S9-mix.

An oily precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

In vitro mammalian cytogenicity - Micronucleus Assay (OECD 487):

C12-14 Alkyl ethoxylated glycidylether (GE) was evaluated for its ability to induce micronuclei in cultured human lymphocytes, either in the absence (3 and 24-hour treatment) or presence (3-hour treatment) of a metabolic system (S9-mix).

In the first cytogenetic assay, GE was tested for 3 hours exposure up to 50 (-S9) and 80 µg/mL (+S9) in DMSO. In the second cytogenetic assay, GE was tested for 24 hours up to 100 µg/mL. Appropriate toxicity was reached in both assays.

Results from positive controls and solvent control were within the acceptability criteria of the assay.

GE did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments

 

In vitro mammalian mutagenicity - MLA (OECD 490, GLP):

The mutagenic potential of C12-14 Alkyl ethoxylated glycidylether (GE) was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence (3 and 24 hour treatment) or presence (3 hour treatment) of a metabolic system (S9-mix).

In the first experiment GE was tested for 3 hours exposure up to concentrations of 50 (-S9) and 70 µg/mL (+S9) in DMSO. Relative total growth (RTG) was reduced to 8 and 14%, respectively. The test item did not precipitated in the culture medium up to the dose level of 70 µg/mL.

In the second experiment was tested for 24 hours up to concentrations of 50 µg/mL (-S9). The RTG was reduced to 13%. The test item did not precipitate in the culture medium up to the dose level of 50 µg/mL.

Results from positive controls and solvent control were within the acceptability criteria of the assay.

 

In the first mutation experiment in the absence and presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency (MF).

In the second mutation experiment in the absence of S9-mix, the test item induced increases in the MF, with a MF just above the positive threshold at the top dose level of 50 µg/ml only.

 

Although the increase in the mutation frequency at the TK locus was only observed at a toxic dose level (RTG of only 13% at 50 µg/mL, with no growth from 55 µg/mL, whereas in experiment 1 the 50 µg/mL concentration lead to a RTG of 8%, below the lowest recommended level for toxicity stated in the guideline), the mutation frequency at this concentration was above the GEF and the result is therefore considered to be biological relevance and the test item mutagenic at toxic dose levels after prolonged treatment.

 

 

Overall evaluation:

Considering that Alkyl ethoxylated glycidylether was clearly negative in the Ames and in vitro micronucleus assay, indicates that, for a glycidylether, its genotoxic potency is compared to other glycidylethers low. Alkyl ethoxylated glycidyle resulted to a positive results in an in vitro Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells (MLA). However, the increase in the mutation frequency at the TK locus was a little above the threshold and was only observed at the highest, toxic dose level (RTG of 13%), and only in the experiment involving 24-hr exposure without S9. The same concentration without S9 resulted to a RTG of 8% in the short-time exposure, which is belowthe lowest recommended level for toxicity stated in the guideline.

This single, marginal positive result at toxic level also indicates a general weak potency.

Considering further that even most positive genotoxic acting glycidyl ethers are negative in vivo, a negative in vivo result can thus be expected for Alkyl ethoxylated glycidylether.

 

There is a low likelihood of exposures following the industrial use only as intermediate in the production of chemicals involving well controlled conditions, involving closed automated processes, in which the glycidyl ether iscompletely consumed. The low vp (≤ 0.070 Pa. at 25°C) and the use of PPE, also related to its skin sensitizing properties, contribute further to its low potential of exposure. Considering the low likelihood of exposures, the low concerns and predicted negative outcome from in vivo testing, a subsequent in vivo assay is not proposed.

Justification for classification or non-classification

Alkyl ethoxylated glycidylether was clearly negative in the Ames andin vitromicronucleus assay, and only positive in an MLA, where the positive result was confined only to incubations without metabolic activation after prolonged treatment (24 hours) at the highest tested, severely toxic dose level of 50 µg/mL, giving a relative total growth of only 8-13%.

Considering lack of exposures, and that furtherin vivostudies can be expected to result to negative results, there is not insufficient basis for classification.