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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical appearance: clear colourless liquid
- Storage conditions: at room temperature
- Test item name as cited in the report: C12-14 Alcohol ethoxylated (EO<2.5), reaction products with epichlorohydrin
- Purity: UVCB
- Purity test date: 21 Sept 2018
- Batch: 1658866
- Expiry date of batch: 15 August 2019
Specific details on test material used for the study:
No correction was made for the composition of the test item.

Method

Species / strain
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

Controls
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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.