[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 August 2015 to 15 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: See below.

Version / remarks:

Official notice of MHLW, METI and MOE (31 March 2011)
YAKUSHOKUHATSU 0331 No 7
SEIKYOKU No 5
KANPOKIHATSU No 110331009.
Official Notice of J MOL (16 September 1999).

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Test guideline 4

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

1

Reference substance name:

-

EC Number:

482-480-5

EC Name:

-

Cas Number:

1187571-02-3

Molecular formula:

(R2C5H4NO2)(0-3)(R3C3H5O3)(0-3)H(0-2)B

IUPAC Name:

Coconut oil reaction products with boric acid and diethanolamine

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and
5,6-benzoflavone to stimulate mixed-function oxidases in the liver

Test concentrations with justification for top dose:

Concentrations with high ionic strength and osmolality may cause chromosomal aberrations
(Galloway et al. 1987). Therefore, the osmolality of the test substance in medium was tested
at concentrations of 1800, 3000 and 5000 micro g/mL. Concentrations of 3000 and 5000 micro g/mL caused a change of greater than 50 mOsm/kg when compared with the vehicle control.
No fluctuations in pH of the medium were observed at 1800, 3000 and 5000 micro g/mL of more
than 1.0 unit compared with the vehicle control. As changes of greater than 50 mOsm/kg
were observed at higher concentrations, the maximum final concentration tested in the
preliminary toxicity test was 1800 micro g/mL.

In the main test, justification for the highest analysed concentration was determined by
cytotoxicity for all treatment groups.

Vehicle / solvent:

Prior to commencing testing, the solubility of the test substance in vehicle compatible with this test system was assessed. It was found to be soluble at 500 mg/mL in ethanol.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Preliminary test:
A culture of CHL cells in a 75 cm2 flask was harvested as follows: the supernatant medium was removed and 8 mL Accutase™ (cell detachment solution) was added. The cells were then examined microscopically, and if “rounded”, they were dislodged and a sample taken for counting.
The cells were then re-suspended in an appropriate volume of culture medium to give 2 x 104
cells/mL. Aliquots (5 mL) of the cell suspension were added to 25 cm2 tissue culture flasks and were incubated at 34-39 deg.C in an atmosphere containing 5% carbon dioxide.

Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment
with the test substance for each test condition. No positive control cultures were prepared.

After approximately 24 hours, the culture medium was removed from each culture and replaced with fresh medium, allowing for the volume of S9 mix where appropriate.

The test substance was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control.

At the end of the 3-hour treatment period, cultures were examined for the presence of precipitate. Cultures were washed in saline and fresh medium was added to the flasks which were then incubated (for approximately 12 hours) until the scheduled harvest time.

At the end of the 15-hour treatment period, cultures were examined for the presence of precipitate.

Main test:
The procedure for the main test was the same as that for the preliminary test, with the following exceptions; positive control cultures were included for all tests, duplicate cultures were prepared for all cultures and two slides were prepared for selected cultures.

3-hour treatment in the absence of S9 mix - The test substance was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Mitomycin C, at final concentrations of 0.1 and 0.2 µg/mL, was added to duplicate cultures.

Following 3-hour treatment, medium was removed from the flasks and discarded. Cells were
washed in saline before fresh medium was added to the cells. They were then incubated for a
further 12 hours. The cultures were then harvested and slides prepared.

3-hour treatment in the presence of S9 mix - For treatments in the presence of S9 mix, 1 mL of S9 mix was added to give a concentration of 1% v/v in the final test medium.

RThe test substance was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Cyclophosphamide at final concentrations of 5 and 10 µg/mL was added to duplicate cultures.

Following 3-hour treatment, medium was removed from the flasks and discarded. Cells were
washed in saline before fresh medium was added to the cells. They were then incubated for a
further 12 hours. The cultures were then harvested and slides prepared.

15-hour treatment in the absence of S9 mix - The test substance was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Mitomycin C, at final concentrations of 0.1 and 0.2 µg/mL, was added to duplicate cultures.

Following the end of the treatment period the cultures were harvested and slides prepared.

Evaluation criteria:

Providing that all of the acceptance criteria have been met, the test substance was considered
to be clearly positive if, in any of the experimental conditions examined:

At least one of the test concentrations exhibited a statistically significant
increase compared with the concurrent vehicle control.

The increase was dose-related when evaluated with an appropriate trend
test.

Any of the results were outside the distribution of the historical negative
control data.

If all of these criteria were met, the test substance was considered able to induce chromosome
breaks and/or gain or loss in the test system.

Providing that all of the acceptance criteria have been met, a negative response was claimed
if, in all of the experimental conditions examined:

None of the test concentrations exhibited a statistically significant increase
compared with the concurrent negative control.

There was no concentration-related increase when evaluated with an
appropriate trend test.

All results were inside the distribution of the historical negative control
data.

If all of these criteria are met, the test substance was considered unable to induce
chromosome breaks and/or gain or loss in the test system.

The Study Director used scientific judgement to classify data that did not fall into either of
the above categories.

Results and discussion

Test results

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence and presence of S9 mix following 3 and 15 hours in the absence of S9 mix and
for 3 hours in the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control.

No statistically significant increases in the proportion of polyploid or endoreduplicated
metaphase cells were observed during metaphase analysis, under any treatment condition,
when compared to the vehicle control.

Both positive control compounds caused statistically significant increases in the proportion of
aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Applicant's summary and conclusion

Conclusions:

In an OECD 473 study the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations or of causing an increase in numerical aberrations in the form of polyploidy (or endoreduplication), in this in vitro cytogenetic test system, under the experimental conditions described.

Executive summary:

A study was performed to assess the ability of the test substance to induce chromosomal aberrations in Chinese Hamster Lung (CHL) cells cultured in vitro.

CHL cells were grown and sub-cultured in tissue culture medium at 34-39 deg.C in an atmosphere containing 5% carbon dioxide. They were then incubated with the test substance in both the absence and presence of exogenous metabolic activation (S9 mix). Vehicle and positive control cultures were also included (where applicable). Two hours before the end of the incubation period, cell division was arrested using Colcemid® , the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

The study consisted of a preliminary toxicity test and a main test. In both types of tests the cells were treated for 3 and 15 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. The Relative Increase in Cell Counts (RICC) was assessed for all cultures to determine cytotoxicity.

In the main test, justification for the highest analysed concentration was determined by cytotoxicity for all treatment groups.

The following test substance concentrations were selected for metaphase analysis:

In the absence of S9 mix, 3-hour treatment: 25, 87.5 and 100 µg/mL
In the presence of S9 mix, 3-hour treatment: 50, 200 and 225 µg/mL
In the absence of S9 mix, 15-hour treatment: 6.25, 12.5 and 25 µg/mL

In the absence and presence of S9 mix following 3 and 15 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the
vehicle control.

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared to the vehicle control.

Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

 

In an OECD 473 study the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations or of causing an increase in numerical aberrations in the form of polyploidy (or endoreduplication), in this in vitro cytogenetic test system, under the experimental conditions described.