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Toxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Reference substance name:
Reaction product aqueous Phosphoric Acid, 2-Butoxyethanol and 4,4'-Isopropylidenediphenol-Epichlorohydrin
EC Number:
944-181-2
Molecular formula:
Not possible to assign for a complex UVCB substance
IUPAC Name:
Reaction product aqueous Phosphoric Acid, 2-Butoxyethanol and 4,4'-Isopropylidenediphenol-Epichlorohydrin
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human blood donations
- Suitability of cells: Considered suitable
- Sex, age and number of blood donors if applicable: 4 donors male and female at 31 to 32 years old. Donors were healthy and non-smokers without any recent exposure to drugs or radiation.

MEDIA USED
The culture medium for the lymphocytes had the following composition:
RPMI 1640 1x (Dutch modification): 500mL
Foetal Calf Serum: 100mL
L-Glutamine (200mM): 6.25mL
Antibiotic solution: 1.25mL
The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the
initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used
in the following proportion: 10mL of PHA was added to 500mL of medium.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of Sprague Dawley rats treated with phenobarbital and benzoflavone
Test concentrations with justification for top dose:
Main phase 1:
3 hour treatment in the absence and presence of S9 mix: 0, 58.5, 87.8, 132, 198, 296, 444, 667, 1000 and 1500 µg/mL.
Continuous treatment in the absence of S9 mix: 0, 39, 58.5, 87.8, 132, 198, 296, 444, 667, 1000 and 1500 µg/mL.

Main phase 2:
3 hour treatment presence of S9 mix: 0, 199, 229, 263, 303, 348, 400, 460, 529, 609 and 700 µg/mL.
Continuous treatment in the absence of S9 mix: 0, 0.206, 0.308, 0.462, 0.694, 1.04, 1.56, 2.34, 3.51, 5.27, 7.90, 11.9 17.8, 26.7 and 40 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Selected during a preliminary solubility test. This solvent was selected since it is compatible with the survival of the cells and the S9 metabolic activity.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Colchicine in the absence of S9 Mix
Details on test system and experimental conditions:
Formulation procedure:
Solutions of the test item, as received, were prepared immediately before use in DMSO on a weight/volume basis without correction for the displacement due to the volume of the test item. For each Main Assay, a clear solution without any visible precipitation was prepared at 300mg/mL, following vortexing for approximately 5 minutes, and used to prepare serial dilutions. All test item solutions were used within 25 minutes from the initial formulation.

Test culture preparation:
One Main Experiment was performed including solvent and positive controls. Two cultures were prepared at each test point. Lymphocyte cultures were treated fourty-eight hours after they were initiated. Before treatment, cultures were centrifuged for 10 minutes and the culture medium was decanted and replaced with treatment medium. For the short term treatment the composition of the media included the test item or control solution, S9 mix (with and without) and culture medium without PHA. For the continuous treatment this was similar although no S9 mix was included.

For the short term exposure, the treatment media were added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with PBS Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 µg/mL. For the continuous treatment, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.

Experimental design:
For the first Main Assay, dose levels were selected on the basis of the solubility of the test item in the culture medium. Since no adequate cytotoxicity levels were obtained in the treatment series in the presence of S9 metabolic activation and using the continuous treatment in its absence, an additional Main Assay was performed. The dose range used was modified to take into account the toxicity observed in the previous experiment. Appropriate negative and positive control cultures were included in both Main Assays. Using the short treatment time, since tests with and without metabolic activation were done concurrently, positive control cultures were treated only with Cyclophosphamide at the dose levels of 20.0 and 15.0 µg/mL. Using the long treatment time, in the absence of S9 metabolism, the positive control cultures were treated with Colchicine at the dose levels of 80 and 40 ng/mL.

Cell harvesting and slide preparation:
The lymphocyte cultures were centrifuged for and the supernatant was removed. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.
A few drops of the cell suspension obtained in this way were dropped onto clean,wet, grease free glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

Slide evaluation:
The cytokinesis-block proliferation index (CBPI) and % cytotoxicity was calculated. Five hundred cells per cell culture were analysed. At least five dose levels were analysed and when negligible cytotoxicity was observed, scoring was interrupted. The highest dose level for genotoxicity assessment should be selected as a dose which produces a substantial cytotoxicity (approximately 55±5%) compared with the negative control. If the test item does not induce relevant toxicity at any concentration, then the highest treatment level is selected as the highest dose level for scoring. Two lower dose levels are also selected for the scoring of micronuclei. For the three selected doses, for the solvent and positive control Cyclophosphamide, at least 1000 binucleated cells per cell culturewere scored to assess the frequency of micronucleated cells. Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.
Rationale for test conditions:
Standard as per OECD guideline
Evaluation criteria:
Positive:
- Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
- The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
- There is a significant dose effect relationship.

Negative:
- None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
- There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
- All the results are inside the distribution of the historical control data.
Statistics:
For the statistical analysis, a modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures. Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Results and discussion

Test results
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
Osmolality and pH effects:
Following treatment with the test item, no relevant variations of pH or osmolality values over the concurrent controls were observed.

Selection of doses for scoring:
Based on the cytotoxicity observed the following doses were selected for micro-nuclei scoring:
First assay: 3 hour treatment (without S9 mix): 132, 198 and 296 µg/mL and with S9 mix: 296, 444 and 667 µg/mL.
Second assay: 3 hour treatment (with S9 mix): 303, 348 and 400 µg/mL.
Second assay: continuous treatment (without S9 mix): 7.9, 11.9 and 17.8 µg/mL.

Result analysis:
Results show that the incidence of micronucleated cells of the negative controls in Main Assay I was within the distribution range of our historical control values. In Main Assay II, the observed values for both treatment series slightly exceeded the 95% control limits of the historical control distribution. However, the incidences were consistent with those reported in literature. In addition, the effect of gender on micronucleated cell (MNC) frequency has been described in literature indicating that females have higher MNC frequency than males. Since our historical data refer mainly to male individuals, this result was not considered critical.

Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed in both Main Assays following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. The study was accepted as valid.

In the absence of S9 metabolism, no statistically significant increase in the incidence of micronucleated cells over the concurrent negative control was observed at any dose level, nor concentration related increase of cells bearing micronuclei was seen using the short term treatment or the continuous treatment. In the presence of S9 metabolism, a statistically significant increase (p<0.01) of micronucleated cells over the concurrent negative control value was observed in Main Assay I at the highest concentration scored which yielded marked cytotoxicity (67%). A statistically significant linear trend was also indicated. However, a high heterogeneity was observed between the two replicate cultures at the two highest concentrations analysed, therefore an extended analysis to increase the sample size was performed for negative control and for cultures treated at 667 and 444 µg/mL. When small departures from control values are
observed, clarification/confirmation of the results can be obtained by extending the assessment of slides for micronucleated cells, hence one thousand binucleated cells per culture were scored for the presence of micronuclei using slides which had not been examined previously. Data generated in this additional analysis were combined with the data previously generated. Once again, a statistically significant increase of cells bearing micronuclei was observed only at 667 µg/mL, where the cytotoxicity level exceeded the recommended range and a remarkable difference in the incidence of micronucleated cells between replicate cultures was noted. These results have been attributed to an excessive toxic effect. Since there is considerable evidence that excessive toxicity can give rise to 'misleading' positive results that occur only under cytotoxic conditions and not at lower concentrations, Main Assay II was performed using a narrowed dose range.

No statistically significant increase in the incidence of micronucleated cells over the concurrent control was seen at any dose level, although an adequate level of cytotoxicity (53%) was achieved at the top concentration scored. No concentration related increase of cells bearing micronuclei was observed. On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce micronuclei in human lymphocytes after in vitro treatment.

Any other information on results incl. tables

Summary table (Main phase 1, 3 hour treatment)

Treatment

Dose level (µg/mL)

Presence of S9 metabolism

Absence of S9 Metabolism

% Mn cells

Sig.

%Cytotoxicity

Dose level (µg/mL)

% Mn cells

Sig.

%Cytotoxicity

Solvent

0

0.33

-

-

-

0.3

-

-

Test item

296

0.3

NS

20

132

0.25

NS

26

Test item

444

0.60

NS

30

198

0.35

NS

31

Test item

667

0.98

***

67

296

0.45

NS

47

Cyclophosphamide

20

3.30

***

45

-

-

-

-

Note: results for combined scoring of 2000 cells.

 

Summary table (Main phase 2, 3 hour with S9 mix and continuous (31 hour) treatment without S9 mix)

Treatment

Dose level (µg/mL)

Presence of S9 metabolism

Absence of S9 Metabolism

% Mn cells

Sig.

%Cytotoxicity

Dose level (µg/mL)

% Mn cells

Sig.

%Cytotoxicity

Solvent

-

0.7

-

-

-

0.95

-

-

Test item

303

1.15

NS

28

7.9

0.5

NS

19

Test item

348

1.25

NS

42

11.9

0.9

NS

33

Test item

400

0.85

NS

53

17.8

0.6

NS

50

Cyclophosphamide

15

3.85

***

50

-

-

-

-

Colchicine

-

-

-

-

0.04

4.5

***

99

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions employed, the substance does not induce micronuclei in human lymphocytes after in vitro treatment.

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