Sulfuric acid, C16-C18 (even numbered) alkyl esters, sodium salts and C16-18 (even numbered) alcohols

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted July 29, 2016.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Sulfuric acid, C16-C18 (even numbered) alkyl esters, sodium salts and C16-18 (even numbered) alcohols

EC Number:

944-399-8

Cas Number:

not yet assigned

Molecular formula:

see information in structural formula

IUPAC Name:

Sulfuric acid, C16-C18 (even numbered) alkyl esters, sodium salts and C16-18 (even numbered) alcohols

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: OE70403001
- Expiration date of the lot/batch: April 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At +10°C to +25°C

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Remarks:

cultured human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human peripheral blood was obtained by venipuncture and collected in heparinised vessels.
- Sex, age and number of blood donors if applicable: young, healthy, non-smoking individuals with
no known recent exposures to genotoxic chemicals or radiation.
- Methods for maintenance in cell culture if applicable: Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin
* Ham’s F10 supplemented with 10% fetal calf serum (FCS)
* Chromosome complete medium with 5 μg/mL Cytochalasin B.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

CytoB used in a concentration 5 μg/mL

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver, Aroclor 1254 induced)

Test concentrations with justification for top dose:

-Preliminary experiment (without and with metabolic activation): 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/mL medium.
-Main Test:
4 h exposure with and without S9: 18.75, 37.5, 75, 150 and 300 μg/mL medium.
24h exposure without S9: 6.25, 12.5, 25, 50 and 100 µg/mL medium.
In the preliminary test cytotoxicity was noted starting at a concentration of 100 µg/mL medium in the experiment without metabolic activation (24-hour exposure). Test item precipitation and cytotoxicity were noted at concentrations of 316 and 1000 µg/mL medium in both experiments (24-hour or 4-hour exposure).
Hence, 300 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour exposure and 100 µg/mL medium for the 24-hour exposure experiment.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 was completely dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg/mL

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

mitomycin C

Remarks:

0.1 µg/mL and 0.2 µg/mL without S9

Controls 2

Positive controls:

yes

Remarks:

clastogen

Positive control substance:

cyclophosphamide

Remarks:

10 µg/mL and 20 µg/mL with S9

Controls 3

Positive controls:

yes

Remarks:

aneugen

Positive control substance:

other: colchicine

Remarks:

0.01 µg/mL and 0.02 µg/mL without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
Without S9: 4 hours and 24 hours.
With S9: 4 hours
- Exposure duration: 20 h incubation with spindle inhibitor Cytochalasin B
- After exposure: 20 h incubation with spindle inhibitor Cytochalasin B

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: duplicate (main study), one culture per concentration in the preliminary test

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Each culture was harvested and processed separately. After the test item incubation, mitotic activity was arrested by the addition of CytoB to each culture at a final concentration of 5 μg/mL. Afteran additional incubation of 20 hours the cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%). After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide. The slides were then stained using 10% Giemsa and left to air-dry at room temperature.

NUMBER OF CELLS EVALUATED:
-At least 500 cells per replicate cell culture were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity.
-The micronucleus frequencies were analysed in at least 2000 binucleate cells per concentration (at least 1000 binucleate cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

DETERMINATION OF CYTOTOXICITY
- Method: other:
The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI). The CBPI indicates the average number of nuclei per cell during the period of exposure to CytoB, and is used to calculate cell proliferation. The RI indicates the relative number of cell cycles in treated cultures compared to control cultures and can be used to calculate the % cytostasis:
CBPI =((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells)) / (Total num ber of cells).
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
Cytostasis = 100 - RI
RI = [((No. binucleate cells)+(2×No. multinucleate cells))÷(Total number of cells)T / ((No. binucleate cells)+(2×No. multinucleate cells))÷(Total number of cells)C ] × 100
T= treated cultures
C= control cultures
Thus, an RI of 53% means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53% of this number divided in the treated culture, i.e. 47% cytostasis.
- Any supplementary information relevant to cytotoxicity: Treatment of cultures with CytoB, and measurement of the relative frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture, provides an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and ensures that only cells that divided during or after treatment are scored.

Evaluation criteria:

Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction.Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined.Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if: at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control; the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test; any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits).Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if: none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control; there is no concentration-related increase when evaluated with an appropriate trend test; all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).Equivocal results may be clarified by analysis of another 1000 cells from all the cultures to avoid loss of blinding.If this approach does not resolve the result, further testing would be necessary.Modification of study parameters over an extended or narrowed range of conditions, as appropriate, would be considered in follow-up experiments.Study parameters that might be modified include the test concentration spacing, the timing of treatment and cell harvest, and/or the metabolic activation conditions.Although most experiments give clearly positive or negative results, in some cases the data set would preclude making a definite judgement about the activity of the test item.These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Complete cytotoxicity and test item precipitation (macroscopically) were noted in the 4-hour exposure experiments with metabolic activation at concentrations of 300 µg/mL medium.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

vehicle control = negative control

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted starting at concentrations of 75 µg/mL medium. Complete cytotoxicity and test item precipitation (macroscopically) were noted at concentrations of 150 µg/mL medium (4 h-exposure without S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

vehicle control = negative control

Positive controls validity:

valid

Test results 3

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was noted starting at concentrations of 25 µg/mL medium in the experiments without metabolic activation (24-hour exposure).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

vehicle control = negative control

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant changes in pH of the test item formulations compared to the negative control were noted up to the top concentration of 1000 μg/mL medium. Digital pH meter type SevenCompact S210 Mettler-Toledo GmbH, 35396 Gießen, Germany was used.
- Effects of osmolality: No relevant changes in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 1000 μg/mL medium. Semi-micro osmometer Typ ML A0299 Knauer GmbH, 14163 Berlin, Germany was used.
- Precipitation of the test item was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.
- Definition of acceptable cells for analysis: Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. S
coring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
For the last 27 studies (most recent background data of the years 2015 to 2017, not audited by the QAU-department):
WITHOUT METABOLIC ACTIVATION
*Vehicle controls 4 hour exposure
Mean: 5.4
SD: 2.6
Range:1.0 - 16
95% Confidence interval: 4.8 - 6.1
*Vehicle controls 24 hour exposure
Mean: 5.2
SD: 2.4
Range: 1.0 - 13
95% Confidence interval: 4.6 – 5.8
WITH METABOLIC ACTIVATION
*Vehicle control
Mean: 4.7
SD: 2.1
Range: 1 - 9
95% Confidence interval: 4.2 – 5.4
- Positive historical control data: For the last 27 studies (most recent background data of the years 2015 to 2017, not audited by the QAU-department):
*Mitomycin C
Mean: 26.0
SD: 10.3
Range: 12 - 61
*Colchicine
Mean : 31.2
SD: 20.6
Range:12 - 125
*Cyclophosphamide
Mean : 22.5
SD: 9.1
Range: 12 - 55
- Negative (solvent/vehicle) historical control data: The results for the vehicle controls were within the historical control range.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: . The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

Remarks on result:

other: 4-hour exposure

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.
The results for the vehicle controls were within the historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Executive summary:

Test samples of Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 was completely dissolved in dimethyl sulfoxide (DMSO) to a concentration of 100 mg/mL. The test item was soluble only by warming up to 37°C for 30 minutes and by carefully stirring after addition to the treatment medium to a final concentration of 1000 µg/mL medium. 1000 µg/mL medium was the maximum feasible concentration.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium were employed. Cytotoxicity was noted starting at a concentration of 100 µg/mL medium in the experiment without metabolic activation (24-hour exposure). Test item precipitation and cytotoxicity were noted at concentrations of 316 and 1000 µg/mL medium in both experiments (24-hour or 4-hour exposure). No changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 1000 µg/mL medium.

Hence, 300 µg/mL medium were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour exposure and 100 µg/mL medium for the 24-hour exposure experiment.

In the main study cytotoxicity was noted starting at concentrations of 75 or 25 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium in the experiments without metabolic activation (4-hour or 24-hour exposure, respectively). Complete cytotoxicity and test item precipitation (macroscopically) were noted in the 4-hour exposure experiments without and with metabolic activation at concentrations of 150 and 300 µg/mL medium.

Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 18.75, 37.5, 75, 150 and 300 or 6.25, 12.5, 25, 50 and 100 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 3.5 to 6.5 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 75 or 25 µg/mL medium (4- and 24-hour exposure, respectively). No binucleated cells could be evaluated at the higher concentrations due to cytotoxicity and/or test item precipitation. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test mean frequencies of 5.5 or 4.0 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively, were observed. The vehicle result was within the historical control ranges.

In the positive control cultures the mean micronucleus frequencies were increased to 17.5 or 18.5 micronucleate cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively.This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The mean micronucleus frequencies of cultures treated with the concentrations of 18.75, 37.5, 75, 150 and 300 µg Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18/mL medium (4-h exposure) in the presence of metabolic activation ranged from 2.0 to 3.5 micronucleate cells per 1000 binucleate cells. There was no dose-related increase in micronuclei up to the top concentration of 75 µg/mL medium. No binucleate cells could be evaluated at the higher concentrations due to cytotoxicity and/or test item precipitation. The frequency of micronucleate cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 4.5 micronucleate cells per 1000 binucleate cells was observed. The vehicle result was within the historical control ranges.

In the positive control culture the mean micronucleus frequency was increased to 23.5 micronucleate cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.

Conclusion

Under the present test conditions, Reaction mass of Sulfuric acid, C16-18-alkylesters, neutralized and Alcohols, C16-18 tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

The results for the vehicle controls were within the historical control range.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.