Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test is available where testing was conducted in strains TA98, TA100, TA1535 and TA1537. An in vitro chromosome aberration study in CHO cells is available. An in vitro mammalian mutation study in mouse lymphoma L5178Y cells at the thymidine kinase locus is also available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 June 1992 - 03 July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Reversion to histidine independence
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction supplemented with salts and co-factors (S9-mix)
Test concentrations with justification for top dose:
Preliminary test - a threefold dilution series from 5.0 to 0.062 mg/plate.
Main test (mutagenicity test) - 5.0, 2.5, 1.3, 0.63, 0.31 mg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: initial investigations found that the test substance was readily soluble in water up to at least 16.7 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Used for strains TA100 and TA1535 in the absence of S9- mix. Migrated to IUCLID6: 1.0µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For strains TA98 and TA 1537 in the absence of S-9 mix. Migrated to IUCLID6: 1.0 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthrazene, 1.5µg/plate
Remarks:
For all four strains in the presence of S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: First test series: in agar (plate incorporation);
Second test series: preincubation


DURATION
- Preincubation period: 30 minutes (second test series only)
- Exposure duration: 48 to 72 hours



NUMBER OF REPLICATIONS: Three plates per dose level.


DETERMINATION OF CYTOTOXICITY
- Method: Depression of the background growth.


OTHER EXAMINATIONS:
- Other: Observation of the number of revertant colonies.


OTHER:
Evaluation criteria:
The data obtained were evaluated with respect to the following criteria:
- Dose-response relationship.
- A doubling or more of the revertant level as compared to the control level.
Statistics:
Statistically significant increase in the level of revertants on the test plates as compared to the control plates. The Analysis of Variance test was used
to compare the test and negative control groups. When necessary the Student's t-test was used to compare the negative and positive control groups.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Not toxic up to 5 mg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation recorded. High dose level (5 mg/plate) used 0.3 mL per plate in the preliminary toxicity test rather than 0.1 mL per
plate as usual. In the main test, a 50 mg/mL solution was used for dosing the high level (5 mg/plate) - the solution was not clear, but was judged to
be sufficiently homogeneous for dosing.
- Other confounding effects: None recorded


RANGE-FINDING/SCREENING STUDIES:
A three-fold dilution series (from 5.0 to 0.062 mg/plate) was tested with and without S-9 mix. No toxicity, measured as a colony count reduction,
was observed at any dose with or without S-9 mix.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Two statistically significant increases in revertant counts were found in TA 100 without S-9 mix. Although, the increases were only small and there was no dose-response, it was decided to perform a supplementary test to see if the increases could be confirmed. No indication of any increase was seen in the supplementary test and it was concluded that the small increases were incidental, i.e. due to incidentally low controls.

Conclusions:
Interpretation of results (migrated information):
negative

The test substance Di-TMP was found to be non-mutagenic in the Ames test.
Executive summary:

The mutagenic potential of Di-TMP was investigated in a Ames test using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. Triplicate cultures of all tester strains were exposed to di-TMP at concentrations up to the limit concentration of 5 mg/plate in the absence and presence of an exogenous metabolic activation system (S9 fraction). No reproducible increase in the number of revertant colonies was seen; di-TMP is therefore considered to be negative in this study. Appropriate control substances confirmed the sensitivity of this assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 May 2015 to 15 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Target gene:
Not applicable (chromosome aberration)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Chinese hamster ovary cells (CHO 10 B4) obtained from the University of Leiden in 1987
- Type and identity of media: Ham's F-10, containing HEPES buffer and supplmented with the antibiotic minocycline. Foetal bovine serum was added for cell growth and treatment in the absence of S9 mix. The medium used for treatment in the presence of S9 mix and for washing cultures before or after treatment was serum-free.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no
- The cells were grown as monolayers and have a generation time of ca 12-14 h. The modal chromosome number was determined to be 21 for these cells.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix: obtained from the livers of Aroclor 1254-induced adult SD rats
Test concentrations with justification for top dose:
Toxicity test: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 µg/mL
Main Test (with S9 mix): 500, 750, 1000, 1250, 1500, 1750, 2000 μg/mL
Main Test (without S9 mix, 6 h treatment): 500, 750, 1000, 1250, 1500, 1750, 2000 μg/mL
Main Test (without S9 mix, 22 h treatment): 250, 500, 750, 1000, 1250, 1500, 1750, 2000 μg/mL
Vehicle / solvent:
Ham's F-10 medium: The test item was soluble in Ham’s F-10 medium with vortex mixing and warming (37°C).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
All experiments included vehicle control cultures and these cultures were subjected to the same experimental manipulations as the treated cultures, both in the presence and absence of S9 mix.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Cells were trypsinised from stock flasks at passage numbers 8 (Toxicity Test) and 12 (Main Test), and resuspended in fresh culture medium at a density of 0.1 x 10E6. The cell suspensions (5 mL) were dispensed into 25 cm² tissue culture flasks. Test cultures were established from the stock flask 20-24 h before testing. All experimental procedures, up to harvesting, were conducted using aseptic technique and under amber light. Tests were conducted both in the presence and absence of S9 mix. All treatments in the toxicity test were performed on single cultures and in the main test were performed on duplicate cell cultures. Cultures to be treated in the presence of S9 mix were washed with serum-free medium before treatment. Immediately before dosing, exposure medium was prepared in sterile containers.

Cultures were established 20-24 h before exposure. The treatment period in the presence of S9 mix was 0-6 h (6-22 h recovery period), and in the absence of S9 mix was 0-22 h (no recovery period). Colcemid was added at 22-24 h (main test only) and cells were harvested at 24 h. After treatment, cells were washed twice with serum-free medium. Full growth medium was added for the recovery period and colcemid treatment (main test only). The volume of medium for the recovery period was 5 mL. Living cultures were examined for evidence of changes to cell morphology, once at the end of the treatment period and again before harvesting of cultures.

Toxicity test: At harvest time, the monolayer cells were trypsinised and counted. This provided a quantitative measure of toxicity.

Main test: Colcemid was added to all cultures at a final concentration of 0.1 μg/mL. Culturing the cells in medium containing colcemid for 2 h accumulated cells in metaphase (the stage of cell division at which chromosomes can be examined using light microscopy). Mitotic cells were harvested by gently tapping flasks to release these cells from the monolayer. Cells were sedimented by centrifugation and treated with hypotonic solution (trisodium citrate; 1%, w/v), for 15 min at room temperature. The cells were then fixed (after sedimentation as before) using freshly prepared fixative (4 mL; methanol:glacial acetic acid; 3:1, v/v). After sedimentation two further fixative changes were conducted. Monolayer cells were trypsinised, counted and discarded. This provided a quantitative measure of toxicity. Three slides per culture were prepared. All slides were marked with the study number and assigned a unique coded number from a computer-generated sequence. Slides were prepared by dropping the cell suspension onto clean grease-free slides. The slides were allowed to air dry overnight and were then stained with Giemsa (5%, v/v), then made permanent by mounting coverslips with DPX mountant.

Toxicity Screen: Slides were examined for evidence of metaphase cells and signs of cellular necrosis.
Chromosomal Aberrations: Based on the toxicity (i.e. cell counts and slide/culture observations) 3-4 concentration levels were selected for assessment of chromosomal aberrations. From 2-3 slides per culture, up to 75 metaphase cells per slide, a total of 150 metaphase cells per culture (ie 300 metaphase cells per concentration), were examined where possible. Slides were scored in order of coded number. The observations were performed using a microscope at a magnification of x 1250. The number of chromosomes in each metaphase cell and all abnormalities, using the nomenclature of Gebhart (1970), was recorded. The positions on the slides of any structurally aberrant cells were recorded using the Vernier scale on the microscope stage.
Evaluation criteria:
Toxicity: A dose level of test item was considered to be toxic to the CHO cells if the RICC of both cultures was 45% ± 5% of the vehicles (i.e. 55% ± 5% cytotoxcity). If the RICC was greater than 45% ± 5%, then dose levels were considered toxic if consistent evidence of changes to cell morphology, either in the living cultures or on the slides, was observed.
Clastogenicity: The results for test item and positive control-treated cultures were evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures were within the 95% limits for the historical negative control data. The response at a single dose was classified as significant if the percentage of aberrant cells was consistently greater than the 99% limits for the historical negative control data or was greater than double the frequency of an elevated vehicle control culture. An experimental condition was positive if the response in at least one acceptable dose level was significant by the criteria described above. Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive.
Statistics:
Not performed.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 to 2000 µg/L with S9; at 1250 to 2000 µg/L without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity test: In the presence of S9 mix, toxicity was observed in the cultures treated with Di-Trimethylolpropane at 1000 and 2000 μg/mL, with greatly reduced cell counts (RICC value below 40% of the vehicle control culture) in the culture treated with 2000 μg/mL and 1000 μg/mL was deemed toxic to the cells from culture observations. In the absence of S9 mix (both 6 h and 22 h treatment), greatly reduced cell counts (RICC value below 40% of the vehicle controls) were observed in the cultures treated with Di-Trimethylolpropane at 2000 μg/mL.

Main test: In the presence of S9 mix, toxicity was observed in the cultures treated with Di-Trimethylolpropane at 1000-2000 μg/mL, with RICC values below 40% of the vehicle control cultures in the cultures treated with 1500-2000 μg/mL. The RICC values observed in the duplicate cultures treated with Di-Trimethylolpropane at 1250 μg/mL were in the 45% ± 5% range and 1000 μg/mL was deemed toxic to the cells from slide observations only.
In the absence of S9 mix (6 h treatment), no toxicity was noted in any of the cultures treated with Di-Trimethylolpropane. In the longer treatment (22 h), toxicity was noted in the cultures treated with Di-Trimethylolpropane at 1250-2000 μg/mL, with RICC values below 40% of the vehicle control cultures in the cultures treated with 1500-2000 μg/mL. The RICC values observed in the duplicate cultures treated with Di-Trimethylolpropane at 1250 μg/mL were in the 45% ± 5% range.
In the main assay, cultures treated with the following dose levels of Di-Trimethylolpropane were selected for assessment of chromosomal aberrations: Presence of S9 Mix, 6 h treatment: 500, 750, 1000, and 1250 μg/mL; Absence of S9 Mix, 6 h treatment: 1500, 1750 and 2000 μg/mL; Absence of S9 Mix, 22 h treatment: 500, 1000 and 1250 μg/mL
Remarks on result:
other: strain/cell type: CHO 10 B4
Remarks:
Migrated from field 'Test system'.

The experiments in this study were deemed to be valid because they fulfilled the following criteria: there was no evidence of contamination; cells in vehicle control cultures grew normally; the results of vehicle and positive control cultures were acceptable; there were 3 acceptable dose levels of the test item for assessment. The levels of structural and numerical aberrations in the vehicle control cultures were within the 95% limits of the historical negative control data. All the cultures treated with Di-Trimethylolpropane showed levels of aberrations within the 95% limits of the historical negative control data.

Aberration data: Main Test, With S9 Mix, 6 h Treatment, 24 h Harvest

Group

Structural Aberrations

Aberration Frequency

Aberrant Cell Frequency

Numerical Aberrations

Chromatid

Chromosome

Complex

Multiple

Other

Lesions/Cell

Including Gaps

Excluding Gaps

% Cells With

G

B

F

G

B

F

E

D

R

Judge

%   Judge

%   Judge

AE

ER

PP

Negative Control1

0

2

0

0

0

0

1

0

0

0

0

0.03

-

1

-

1

-

0

1

0

0

0

0

0

0

0

1

0

0

0

0

0.01

-

1

-

1

-

0

0

0

Di-TMP 500 µg/mL

0

0

0

0

0

1

0

0

0

0

0

0.01

-

1

-

1

-

0

1

0

0

2

0

0

0

0

2

0

0

0

0

0.04

-

2

-

2

-

1

0

0

Di-TMP 750 µg/mL

0

0

0

0

0

0

3

0

0

0

0

0.04

-

2

-

2

-

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0.00

-

0

-

0

-

0

0

0

Di-TMP 1000 µg/mL

0

1

0

0

0

0

0

0

0

0

0

0.01

-

1

-

1

-

0

1

0

0

0

0

0

0

0

1

0

0

0

0

0.01

-

1

-

1

-

0

0

0

Di-TMP 1250 µg/mL

0

5

0

0

0

0

0

0

0

0

0

0.03

-

2

-

2

-

1

1

0

0

3

0

0

0

0

1

0

0

0

0

0.03

-

2

-

2

-

0

0

1

Positive Control2

0

14

0

0

0

0

14

0

0

0

0

0.28

+

12

+

12

+

1

0

0

0

17

0

0

0

0

17

0

0

0

0

0.34

+

15

+

15

+

3

0

1

1Ham’s F-10 medium

2Cyclophosphamide

Aberration data: Main Test, Without S9 Mix, 6 h Treatment, 24 h Harvest

Group

Structural Aberrations

Aberration Frequency

Aberrant Cell Frequency

Numerical Aberrations

Chromatid

Chromosome

Complex

Multiple

Other

Lesions/Cell

Including Gaps

Excluding Gaps

% Cells With

G

B

F

G

B

F

E

D

R

Judge

%   Judge

%   Judge

AE

ER

PP

Negative Control1

1

0

0

0

0

0

0

0

0

0

0

0.01

-

1

-

0

-

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0.01

-

1

-

1

-

1

0

0

Di-TMP 1500 µg/mL

2

1

1

0

0

0

0

0

0

0

0

0.03

1

3

-

1

-

0

3

1

0

0

0

0

0

0

0

0

0

0

0

0.00

-

0

-

0

-

0

6

1

Di-TMP 1750 µg/mL

2

2

0

0

0

0

0

0

0

0

0

0.03

-

2

-

1

-

0

3

1

0

0

0

0

0

0

0

0

0

0

0

0.00

-

0

-

0

-

0

2

1

Di-TMP 2000 µg/mL

1

0

0

0

0

0

0

0

0

0

0

0.01

-

1

-

0

-

0

1

0

0

1

0

0

1

1

0

0

0

0

0

0.02

-

2

-

2

-

0

1

1

Positive Control2

3

11

4

0

2

5

3

0

0

0

0

0.21

+

15

+

13

+

3

0

1

2

16

2

1

4

3

12

0

0

0

0

0.35

+

18

+

17

+

4

0

1

1Ham’s F-10 medium

2Methyl methanesulphonate

Key: G = Gap; B = Break; F = Fragment; E = Exchange; D = Dicentric; R = Ring; + = positive; - = negative; AE = Aneuploid; ER = Endoreduplicated; P = Polyploidy

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Di-TMP was not clastogenic to Chinese hamster ovary cells in vitro, when tested both in the presence and absence of metabolic activation (S9).
Executive summary:

The clastogenic potential of Di-Trimethylpropane was evaluated in a chromosomal aberration assay. The assay was performed in Chinese hamster ovary cell cultures according to OECD guideline 473. The study was conducted incorporating a preliminary toxicity test (in single cultures) followed by a main test (in duplicate cultures). Ham’s F-10 medium was the vehicle and cyclophosphamide and mitomycin C were the positive controls used in the main test. All tests were conducted in the presence and absence of S9 mix (exogenous source of metabolic activation). Cultures, established 20-24 h before testing, were treated for 6 h in the presence and 6 h and 22 h in the absence of S9 mix. Cultures were harvested at 24 h post treatment. Di-Trimethylolpropane was toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix. It was tested up to the maximum permitted concentration of 2000 μg/mL. In the toxicity test, toxicity was noted at 1000 and 2000 μg/mL in the presence of S9 mix and at 2000 μg/mL in the absence of S9 mix. In the Main Test, toxicity was noted in cultures treated with Di-Trimethylolpropane at 1000-2000 μg/mL (presence of S9 mix) and in cultures treated at 1250-2000 μg/mL (absence of S9 mix, 22 h treatment). No toxicity was noted in the cultures treated with Di-Trimethylolpropane in the shorter treatment (6 h) in the absence of S9 mix. There was no evidence that Di-Trimethylolpropane induced structural chromosomal aberrations in either the presence or absence of S9 mix. In conclusion, Di-Trimethylolpropane was not clastogenic when tested with Chinese hamster ovary cell in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 March 2016 to 23 January 2017
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)
GLP compliance:
yes
Type of assay:
other: Mammalian gene mutation study
Specific details on test material used for the study:
I
Target gene:
tk gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced S9
Test concentrations with justification for top dose:
15.4, 30.9, 61.8, 123.6, 247.3, 494.5, 989, 1483.5 and 1978 µg/mL (preliminary toxicity test; highest concentration slightly less than 2000 µg/mL due to an error in not applying the correction factor). In the main study 250, 375, 500, 750, 1000, 1500 and 2000 µg/mL
Vehicle / solvent:
RPMI 1640 media with sodium bicarbonate, supplemented with peniciliin (100 units/mL), streptomycin (100 µg/mL) and Poloxamer 188 (0.05% v/v), referred to as R0P.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
In the preliminary experiment, the exposure of the cells was similar to that described for the mutation assays with the
exception that only one culture was prepared for each treatment.
Relative suspension growth was used to measure toxicity in the range-finding experiment. The cell population densities
were recorded over 2 days (following treatment) using a Beckman Coulter Z1 particlecounter. The suspension growth was calculated as (Day1 count/Concentration on Day 0) x (Day 2 count /final concentration on Day 1). The total suspension growths were expressed as percentages of the vehicle control group (= relative suspension growth, or RSG). The toxicity test was performed using the standard 4 h exposure period in the absence and presence of S9 mix.
Observations of pH change (colour change in the indicator in RPMI medium) were made and if any change was noted, pH measurements were made. Osmolality measurements were made and there was no substantial change in any of the dose levels compared to the vehicle control.

In the Main Mutation Assay, on the day of the test (Day 0), samples of cell culture (5 mL) were dispensed to sterile tubes. Freshly prepared S9 mix or R0P (1 mL) was added to each tube followed by the test formulation (4.0 mL) or vehicle (R0P). Positive control cultures received the appropriate solution (0.1 mL) and R0P (3.9 mL). The final reaction mixture in all cultures contained 10 mL of cells, at a population density estimated to be 6.0 x 10^5 cells/mL, in R5P medium (R0P with 5% heat-inactivated horse serum).
All tubes were placed on a 10 r.p.m. rotating drum, inside an incubator set to maintain a temperature of 37°C, for 4 h. After this, the cells were gently sedimented by centrifugation at 200 g for 5 min and resuspended in 20 mL R10P medium (R0P with 10% heat inactivated horse serum). This step was repeated to give a cell density estimated to be 3 x 10^5/mL.
The cells were returned to the rotating drum and allowed to express their genetic lesions for 2 days. Cell numbers were adjusted, after counting, to an estimated 3 x 10^5 cells/mL on Day 1.

On Day 2 cell counts were determined. The cell counts over the 2 days of the experiments provided a measure of suspension growth. This in turn provided a measure of RSG. This was used when choosing dose levels to carry through to final assessment, as no other measures of toxicity were known at the time the decision was required.
In each assay, the treated cultures from 5 concentrations of Di-Trimethylolpropane giving satisfactory cell survival were selected for final assessment.
The cultures were then assessed for expression of genetic damage. This was determined by performing parallel cloning assays for cloning efficiency and mutant selection.For the cloning efficiency assay, each culture was diluted into cloning medium to give an estimated 8 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 1.6 cells per well.
For the mutant selection assay, TFT stock solution was added to cloning medium to give a final concentration of 3 μg/mL. Into this medium, the cell cultures were diluted to give an estimated 1 x 10^4 cells/mL. Two 96-well dishes were filled with 200 μL cell culture per well, so giving an estimated 2000 cells per well.
All dishes were placed in an incubator set to maintain a humid atmosphere of 5% CO2:95% air (v/v) at 37°C until the colonies were fully developed (at least 9 days for cloning efficiency assay, at least 12 days for mutant selection assay).

The plates were scored using a dissecting microscope fitted to a light box with dark field illumination. The number of empty wells in each plate in the cloning efficiency assay was counted. When scoring the mutant selection assay, separate counts were made of the numbers of wells containing large type and small type colonies. Large colonies are defined as covering greater than ¼ of the floor of the well, while small colonies cover less than ¼ of the well (Moore et al (2000)). In addition, there are morphological differences. Large colonies tend to be similar to those found on the cloning efficiency plates, being generally flat. Small colonies tend to look dense in comparison. Any wells containing both colony
types were scored as a large type. The total number of empty wells is required for the calculation of mutant fraction, so each well could only be scored once.

The recommended endpoint for assessing cytotoxicity in mouse lymphoma mutation tests is Relative Total Growth (RTG) (Moore et al (2002)). RTG combines the total suspension growth above with the cloning efficiency (CE) of the non-mutants at the end of the expression period, again expressing individual values as percentages of the vehicle control mean. The CE is calculated from the zero term of the Poisson distribution using the formula:
Cloning efficiency = (-In (number of empty wells/total number of wells))/number of cells per well
Total growth - suspension growth x cloning efficiency
Relative total growth (%) = (individual total growth/mean vehicle control total growth) x 100
The number of empty wells from the non-mutant cloning efficiency assay and the number of empty wells from the TFT-resistance assay were used to calculate the mutant fraction, as below. The CE of both cell types was calculated as in Section 7.1.2. The mutant fraction per viable cell was calculated as below:
Mutant fraction per viable cell = (CE in medium containing TFT/CE in non - selective medium) = (CE of mutants/CE of non-mutants).
Each mutant fraction was expressed per 10^6 viable cells.
Rationale for test conditions:
The highest concentration of test item should be limited by solubility or toxicity, or, in the absence of these, should be the maximum practicable concentration of test item, based on the recommendations in the OECD Guideline, i.e. 2000 μg/mL or 10 mM, whichever is lower.
Evaluation criteria:
A test item would be considered positive if the increase in MF above the concurrent background (IMF) exceeds 126 x10^ -6 and the increase is concentration related.
An experiment may also be classed as positive in the absence of a linear trend if there is mitigating evidence. This may be, for example, the presence of a similar level of toxicity at all concentrations assessed. In such a case, the confirmatory experiment would be expected to assess concentrations covering different levels of toxicity, to establish a linear trend.
Additional comparisons that can aid interpretation of results include:
Comparison of the IMF with the historical maximum for difference between vehicle controls (No-effect Maximum).
Comparison of the mutant fraction of a treated group with the historical range of vehicle control values.
In the absence of any significant findings or other criteria for a positive response, a test item was defined as non mutagenic provided data have been obtained in both the absence and the presence of S9 mix that accompany one or more of the following:
A suitable maximum concentration has been achieved.
The highest practical concentration limited by the solubility or pH of the test item
RTG in the range 10-20%
It is acknowledged (Moore et al (2002)) that there are some circumstances under which a chemical may be determined to be non mutagenic when there is no treatment showing an RTG value between 10 and 20%. These situations are as follows:
There is no evidence of mutagenic activity in a series of data points within 100% to 20% RTG and there is at least one data point between 20% and 25% RTG.
There is no evidence of mutagenic activity in a series of data points between 100% to 25% RTG and there is also a data point between 10% and 1% RTG.
Statistics:
None
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the preliminary test, no toxicity was observed in either the absence or presence of S9 mix following 4 h exposure to Di-Trimethylolpropane up to concentrations of 1978 μg/mL. No change to osmotic pressure was noted. There was a colour change to the culture medium in culture treated with 1483.5 and 1978 μg/mL, therefore pH measurements of these cultures were conducted. The culture medium was within 1 pH unit of corresponding vehicle control treated cultures, therefore no further measurements were required.

Di-Trimethylolpropane was assessed for mutagenic activity at concentrations of: 500, 750, 1000, 1500 and 2000 μg/mL in the absence and presence of S9 mix.
All mean IMF values were below the minimum 126 mutants per million required to indicate a biologically relevant increase, and therefore the test item was considered not to be demonstrating mutagenic activity.
The RTG at the highest assessed concentration (2000 μg/mL) was 69% in absence of S9 mix and 56% in the presence of S9 mix. Di-Trimethylolpropane was not mutagenic in the absence or in the presence of S9.

Main mutation assay: 4 hour treatment in the absence of S9

Concentration (µg/mL)

Relative total growth (%)

Mutant fraction (x 10-6)

Induced mutant fraction (x10-6)

Ratio of small to large colonies

0
(Vehicle control)

100

85

N/A

1.16

250

NP

NP

NP

NP

375

NP

NP

NP

NP

500

84

104

19

0.78

750

79

92

7

1.33

1000

79

91

6

1.06

1500

69

120

35

0.90

2000

69

180

95

1.39

Positive control – EMS 250 µg/mL

66

682

597

0.40

Positive control – MMS 10 µg/mL

31

1674

1589

0.94

NP: Not plated
N/A: Not applicable
EMS: Ethyl methanesulphonate
MMS: Methyl methanesulphonate

 

Main mutation assay: 4 hour treatment in the presence of S9

Concentration (µg/mL)

Relative total growth (%)

Mutant fraction (x 10-6)

Induced mutant fraction (x10-6)

Ratio of small to large colonies

0
(Vehicle control)

100

78

N/A

1.52

250

NP

NP

NP

NP

375

NP

NP

NP

NP

500

99

86

8

0.78

750

79

97

19

1.25

1000

88

73

-

0.99

1500

78

86

8

1.02

2000

56

132

54

0.78

Positive control – 3-MC 2.5 µg/mL

46

1507

1429

0.72

Positive control – 3-MC 10 µg/mL

19

2034

1956

0.82

NP: Not plated
N/A: Not applicable
3-MC: 3-methylcholanthrene

Conclusions:
Di-Trimethylolpropane was not mutagenic in mouse lymphoma L5178Y cells, in either the absence or presence of S9 mix.
Executive summary:

An in vitro mammalian mutation study was performed with Di-Trimethylolpropane using mouse lymphoma L5178Y cells according to OECD test guideline 490. Testing was conducted in the absence and in the presence of S-9 for 4 hours at concentrations up to 2000 µg/mL in the main experiment. Relative total growth was used to indicate the level of toxicity. Slight toxicity was noted in the 4-hour treatments, though testing was conducted up to a regulatory maximum of 2000 µg/mL. The induced mutant frequency did not exceed the global evaluation factor (126 mutants per million). It can be concluded, therefore, that there is no evidence for the mutagenicity of Di-Trimethylolpropane based on the results of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No evidence of mutagenicity was seen in an Ames test with di-TMP (Jensen, 1992). Di- TMP was also found to be negative in a study of clastogenicity in mammalian cells in vitro (Murie, 2015) and an in vitro mammalian mutation study (Innes, 2017).


Justification for selection of genetic toxicity endpoint
A weight of evidence approach is taken to this endpoint, based on negative responses reported in studies in vitro. No single study is selected as key.

Short description of key information:
No evidence of mutagenicity was seen in an Ames test with di-TMP (Jensen, 1992). Di-TMP was also found to be negative in a study of clastogenicity in mammalian cells in vitro (Murie, 2015) and an in vitro mammalian mutation study (Innes, 2017).


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data do not indicate that Di-Trimethylolpropane is mutagenic: no classification according to Regulation (EC) No 1272/2008 is therefore required.