Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1) Ames-test, 1990, S. typhimurium strains TA 1535, TA 100, T 1537 and TA 98, according to OECD 471, tested up to precipitation, negative / non mutagenic

2) Ames-test, 2018, E. coli WP2 uvrA, according to OECD 471, tested up to precipitation, negative / non mutagenic

3) Chromosome aberration test, 2018, human lymphocytes, according to OECD 473, tested up to precipitation, negative / non mutagenic

4) Forward gene mutation assay in mammalian cells, mouse lymphoma L5178Y, according to OECD 490, tested up to cytotoxic concentrations, negative / non mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-08-03 - 1989-11-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study, only 4 Salmonella strains tested (TA98, TA100, TA1535 and TA1537) - study providing information on the fifth strain is available
Reason / purpose:
reference to other study
Remarks:
OECD 471
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
yes
Remarks:
only 4 Salmonella strains tested (TA98, TA100, TA1535, TA1537)
Qualifier:
according to
Guideline:
other: EEC Directive 84/449/EEC B.14. Other Effects - Mutagenicity Salmonella typhimurium Reverse Mutation Test
Qualifier:
according to
Guideline:
other: New and Revised Health Effects Test Guidelines October 1984. (U.S.) Environmental Protection Agency Washington, DC (PB 84-233295). HG - Gene Muta - S. typhimurium, October 1984
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine-auxotrophic strains
Metabolic activation:
with and without
Metabolic activation system:
The 9000 g fraction of homogenized mammalian livers: The rat S9 mix comprised 30% S9 fraction, 70% cofactor solution.
Test concentrations with justification for top dose:
Doses up to and including 5000 µg per plate

The following doses of Triisopropyldiisocyanatobenzene were evaluated:

Negative control: 0
Triisopropyldiisocyanatobenzene: 5000, 1000, 200, 40, 8 µg per plate
Positive control:
Na-azide 10 µg per plate (only TA 1535)
NF 0.2 µg per plate (only TA 100)
4-NPDA 10 µg per plate (only TA 1537)
4-NPDA 0.5 µg per plate (only TA 98)
2-AA: 3 µg per plate

Due to the substance's toxicity and precipitation, doses ranging from 62.5 µg to 2000 µg per plate were chosen for the repeat tests.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethylene glycol dimethylether (EGDE) and for positive controls dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The used solvent was chosen out of the following solvents, in the order given: water, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.
Untreated negative controls:
yes
Remarks:
Solvent minus test substance
Negative solvent / vehicle controls:
yes
Remarks:
EGDE (test substance) & DMSO (positive control)
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene
Remarks:
The positive controls sodium azide, nitrofurantoin and 4-nitro-1,2-phenylene diamine were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and experimental conditions:
The original strains were obtained from Prof. Bruce Ames and arrived at the testing laboratory on December 12, 1986.

Mammalian metabolism, which is of great significance in chemical mutagenesis, is simulated in this test by the 9000 g fraction of homogenised mammalian livers. It was made from the livers of at least six adult male Sprague Dawley rats. The S9 mix was freshly prepared (Ames et al., 1973a) and used only on the same day. It was placed in a vessel with a double glass wall until used. The hollow wall was filled with ice to keep the S9 mix permanently cold.
Together with co-factors, this forms the "S9 mix" which represents the metabolic model in this test. S9 mix consists of a cofactor solution* and the corresponding volume of S9 fraction. In all tests, the S9 mix comprised 30% (v/v) S9 fraction.

* 10 mL of cofactor solution are composed as follows:
MgCl2 x 6 H2O: 162.6 mg
KCl: 246.0 mg
Glucose-6-phosphate, disodium salt: 179.1 mg
NADP, disodium salt: 315.0 mg
Phosphate buffer: 100.0 mM

The count was made after the plates had been incubated for 48 hours at 37 ° C. If no immediate count was possible, plates were temporarily stored in a refrigerator.
Evaluation criteria:
The toxicity of the substance was assessed in three ways. The first was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed, it was indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, is noted for a concentration, this "b" represents four plates with background growth. (The same applies to the signs "c", "v", "p", "n" or "-" which may also be used in the tables.) Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titre was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.
The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titre determinations had to demonstrate sufficient bacterial density in the suspension.

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
No further data on statistics available
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:
cytotoxicity
Remarks:
weak bacteriotoxic effects at 40 µg per plate and above. Substance precipitation occurred at 500 µg per plate and above.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Remarks:
No "untreated" negative control was set for EGDE, since sufficient evidence was available in the literature and from experience indicating that this solvent had no influence on the spontaneous mutant counts of the bacterial strains used.
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 500 µg per plate, the substance started to precipitate. Therefore doses of 1000 µg per plate and above could only be used to a limited extent for assessment purposes.
- Other confounding effects: There was no indication of a bacteriotoxic effect of Triisopropyldiisocyanatobenzene at 8 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses had a weak, strain-specific bacteriotoxic effect.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the repeat tests.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1, 2-phenylene diamine and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Table 1: Summary of the Results With Triisopropyldiisocyanatobenzene in the Salmonella/Microsome Test

 S9 mix  TA 1535 TA 100 TA 1537 TA 98 
 without  negative (-ve)  (-ve)  (-ve)    (-ve)
 with   (-ve)  (-ve)   (-ve)   (-ve)

Table 2: Summary of tabulated data without S-9 Mix

Summary of Mean Values Without S9 Mix From Tables 1-8
Table and group µg/plate Strain
TA 1535 TA 100  TA 1537 TA98
1-4 0 16 114 8 20
8 15 105 8 21
40 17 91 8 20
200 15 98 7 23
1000 17 85 - 23
5000 -- 107 - --
Na-a2id 799      
NF   370    
4-NPDA     41 62
5-8 0 19 98 9 23
62.5 13 80 7 22
125 12 76 7 22
250 12 76 10 22
500 13 57 6 21
1000 14 61 5 23
2000 14 74 20
Na-acid 782      
NF   376    
4-NPDA     38 126

Table 3: Summary of tabulated data with S-9 Mix

Summary of Mean Values With S9 Mix From Tables 1-8
Table and group µg/plate Strain
TA 1535 TA 100  TA 1537 TA98

1 -4    

30 % S-9

0 19 146 13 36
8 22 126 11 35
40 21 133 8 33
200 20 101 10 28
1000 23 123 - 36
5000 --- - --
2-AA 121 639 60 395

5-8 

30 % S-9

0 15 86 7 33
62.5 16 89 5 26
125 16 92 8 24
250 14 88 8 26
500 14 101 9 28
1000 15 106 7 20
2000 -- 83 - --
2-AA 100 539 60 435
Conclusions:
The study was performed according to the OECD Guideline 471 with deviations (only 4 Salmonella strains tested) and considered to be of good quality (reliability Klimisch 2). The vehicle and the positive control substances fulfilled validity criteria of the test system. The Salmonella/microsome test, employing doses up to 5000 µg per plate, showed Triisopropyldiisocyanatobenzene to produce only weak bacteriotoxic effects at 40 µg per plate and above. Substance precipitation occurred at 500 µg per plate and above. The test material did not induce significant increases in the frequency of revertant colonies in the bacterial strains TA98, TA100, TA1535 and TA1537. No indications of mutagenic effects of Triisopropyldiisocyanatobenzene could be found at assessable doses up to 5000 µg per plate in any of the Salmonella typhimurium strains used.
Executive summary:

Triisopropyldiisocyanatobenzene was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, T 1537 and TA 98.

The study was performed according to the OECD Guideline 471 with deviations (only 4 Salmonella strains tested) and considered to be of good quality (reliability Klimisch 2). 8 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect. Substance precipitation occurred at the dose 500 µg per plate and above. Therefore this range could only be used to a limited extent up to 5000 µg per plate for assessment purposes.

Evidence of mutagenic activity of triisopropyldiisocyanatobenzene was not seen. No biologically relevant increase in the mutant count, in comparison with the negative control was observed.

The positive control sodium azide, nitrofurantoin, 4 -nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, Triisopropyldiisocyanatobenzene was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-05 - 2018-03-13 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Upon dossier evaluation, testing of a fifth strain was requested by ECHA in addition to the already available OECD 471 study. So although only one strain was tested which does not meet the requirements of the OECD 471 TG, it was conducted otherwise according to the guideline and provides consistent results in addtion to the available study, so reliability Klimisch 1 was assigned.
Reason / purpose:
reference to other study
Remarks:
OECD 471
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for the Testing of Chemicals (1997). Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471
Deviations:
yes
Remarks:
Only one strain tested. However, as this study was conducted in addtion to the available OECD 471, this deviation is not only uncritical but even intended.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EC Commission Regulation No. 440/2008. Method B.13/14: Mutagenicity - Reverse mutation test using bacteria. OJ L 142/248
Deviations:
yes
Remarks:
Only one strain tested. However, as this study was conducted in addtion to the available OECD 471, this deviation is not only uncritical but even intended.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
US EPA Health Effects Test Guidelines (1998). OPPTS 870.5100 Bacterial reverse mutation test. EPA 712-C-98-247
Deviations:
yes
Remarks:
Only one strain tested. However, as this study was conducted in addtion to the available OECD 471, this deviation is not only uncritical but even intended.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
trp-/-
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Remarks:
Escherichia coli WP2 uvrA (pKM101) trpE ochre
Details on mammalian cell type (if applicable):
This strain was used to detect base change mutations.
The strain was obtained from Moltox Inc. and the batch of the strain was stored at -90
to -70°C as aliquots of nutrient broth culture. Dimethyl sulfoxide (DMSO) was added to the
cultures at 8% v/v as a cryopreservative. The batch of the frozen strain was tested for amino
acid requirement, deficiency in DNA excision repair system (uvrA mutation) and the
pKM101 plasmid that confers resistance to antibiotics. The responses of the strain to a series
of reference mutagens were also assessed.
For use in tests, an aliquot of frozen culture was added to 25 mL of nutrient broth and
incubated, with shaking, at 34 to 39°C for 10 hours. This culture was intended to provide a
viable cell density of at least 10E9 per mL, which was confirmed by performing viability
counts, in which aliquots (0.1 mL) of a 10E-6 dilution of the 10-hour cultures were spread on
the surface of plates of nutrient agar. After incubation at 34 to 39°C for 24 hours, the total
number of resultant colonies was counted.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/5,6-benzoflavoneinduced rat liver S9
Test concentrations with justification for top dose:
The highest concentration of TRIDI tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows.
Tested concentration: 0, 5 (test 1 only), 15, 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethylene glycol dimethyl ether (EGDE) was used as the vehicle for this study.
- Justification for choice of solvent/vehicle: To be in line with the vehicle chosen in the already available OECD 471 study
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
Remarks:
0.1 ml EDGE
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, first test); preincubation (second test)

DURATION
- Preincubation period: 30 min (preincubation test only)
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): trp minimal agar

NUMBER OF REPLICATIONS: Three Petri dishes were used for each treatment, two independent experiments were performed.

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test item may be detected by a reduction in mean revertant colony numbers to ≤50% of the concurrent vehicle control count, by a sparse or absent background bacterial lawn, or both.
Rationale for test conditions:
As indicated by the guideline.
Evaluation criteria:
Analysis of Data
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Criteria for Assessing Mutagenic Potential
If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
which are also precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
please refer to 'Overall remarks'
Conclusions:
The study was performed under GLP according to OECD 471, so in general the study was well performed and the results are reliable. The study was conducted on E. coli WP2 uvrA (pKM101) only in order to complete the already available OECD 471 study with four S. typhimurium strains. It was concluded that TRIDI showed no evidence of mutagenic activity in this bacterial system under the test conditions employed. So, the results are consistently negative.
Executive summary:

In this in vitro assessment of the mutagenic potential of TRIDI according to OECD 471 under GLP, tryptophan-dependent mutants of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to TRIDI diluted in ethylene glycol dimethyl ether (EGDE). EGDE was used as the vehicle control and untreated controls were also included.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

Concentrations of TRIDI up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca. half-log10 dilutions of the highest concentration.

No signs of toxicity towards the tester strain were observed in either mutation test following exposure to TRIDI. Precipitate was observed on all plates containing TRIDI at 1500 and 5000 µg/plate in the absence of S9 mix and at 5000 µg/plate in the presence of S9 mix in both tests.

No evidence of mutagenic activity was seen at any concentration of TRIDI in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle and untreated controls were within or close to the current historical control range for the laboratory.

It was concluded that TRIDI showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Feb 2018 to 02 May 2018 (experimental phase)
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)
Version / remarks:
OECD Guideline for the Testing of Chemicals. (2016) In Vitro Mammalian Chromosome Aberration Test, Test Guideline 473
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
EC Commission Regulation No. 2017/735. Method B.10: Mutagenicity – In Vitro Mammalian Chromosome Aberration Test. OJ L 112/23.
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5375 In Vitro Mammalian Chromosome Aberration Test. EPA 712-C-98-223
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Correction factor for formulation: None, tested as supplied
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: human donors
- Suitability of cells: Suitable. Indicated in the guideline. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen.
- Sex, age and number of blood donors if applicable: Human blood was collected from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled (in equal volumes from each donor) and diluted with HML media.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: HML media.
RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.
- Properly maintained: yes
Cultures were established from the pooled sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 34 to 39°C, and the cells re-suspended (twice daily) by gentle inversion.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: phenobarbital and 5,6-benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
Following treatment, precipitate (visible by eye) was present at final concentrations of 50 μg/mL, when compared with the vehicle control. Therefore 50 μg/mL was the maximum concentration assessed for mitotic index.
TRIDI caused a reduction in the mitotic index to 43 % of the vehicle control value at 30 μg/mL. At higher tested concentrations overt toxicity was observed. The concentrations selected for metaphase analysis were 10, 20 and 30 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethylene glycol dimethyl ether (EGDE)
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test item in ethylene glycol dimethyl ether (EGDE) was assessed. TRIDI was found to be soluble at 200 mg/mL in EGDE.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
1% EDGE
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h or 21h
- Expression time (cells in growth medium): 18h or none

SPINDLE INHIBITOR (cytogenetic assays): 0.1 μg/mL colcemid for 2h

STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8)

NUMBER OF REPLICATIONS: 3 independent experiments, duplicate cultures were prepared for all cultures and two slides were prepared per culture

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Harvesting and Fixation: Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemi to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl) for a 10 minute period at between 34 to 39°C. The suspensions were centrifuged at 500g for 5 minutes and the cell pellets fixed by addition of ice-cold fixative (methanol:glacial acetic acid (3:1 v/v)). Following further centrifugation the supernatant was removed and replaced with fixative; this was repeated until the fixative was clear.
Slide Preparation: The fixed pellets were re-suspended, then centrifuged at 500g for 5 minutes and re-suspended in a small volume of fixative. A few drops of the cell suspensions were dropped onto precleaned microscope slides and allowed to air dry. One slide was prepared per culture. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainders of the cell suspensions in fixative were stored at 2 to 8°C until slide analysis was completed.

NUMBER OF CELLS EVALUATED: 1000 cells in each culture

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): The proportion of mitotic cells per 1000 cells in each culture was recorded. From each culture 150 metaphase figures were examined, however, this number was reduced in cultures showing a high level of aberrant cells, where 15 cells with structural aberrations (excluding gaps) were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Rationale for test conditions:
As indicated in the guideline.
Evaluation criteria:
Assessment Criteria:
Providing that all of the acceptance criteria have been met, the test item 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 are outside the distribution of the historical vehicle control data (above the upper 95% confidence limit).
If all of these criteria were met, the test item 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 vehicle control.
There was no concentration-related increase when evaluated with an appropriate trend test.
All results are inside the distribution of the historical vehicle control data (within the 95% confidence limits).
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.
Statistics:
Statistical analysis was performed via SAFEStat (SAS statistical applications for end users) Chromosome Aberrations application which was developed in SAS (SAS INSTITUTE 2002).
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / Effects of osmolality:
The osmolality and pH of TRIDI in medium were measured by analysing samples of HML media, dosed at 1% (v/v), with either the vehicle (EGDE) or a TRIDI formulation at 200 mg/mL (to give a final concentration of 2000 μg/mL). For medium dosed with TRIDI at 2000 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 2000 μg/mL as this is the standard limit concentration within this test system as recommended in the current OECD Guideline 473 (2016).
- Evaporation from medium: none
- Precipitation: Precipitate was observed by eye at the end of treatment at 62.5 µg/mL

RANGE-FINDING/SCREENING STUDIES:
In all exposure conditions the highest concentration tested was 2000 µg/mL and precipitate was observed by eye at the end of treatment at 62.5 µg/mL as assessed in concurrently treated HML media-only cultures. Cultures at 125 µg/mL and greater were discarded.
In the absence of S9 mix following 3-hour treatment, TRIDI caused a reduction in the mitotic index to 35% of the vehicle control value at 62.5 µg/mL, the highest tested concentration analyzed for mitotic index.
In the presence of S9 mix following 3-hour treatment, TRIDI caused a reduction in the mitotic index to 54% of the vehicle control value at 31.25 µg/mL. At higher tested concentrations overt toxicity was observed.
In the absence of S9 mix following 21-hour treatment, TRIDI caused a reduction in the mitotic index to 37% of the vehicle control value at 62.5 µg/mL, the highest tested concentration analyzed for mitotic index.
The concentrations used in the main test were based upon these data.
Conclusions:
The study was conducted under GLP according to OECD guideline 473 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response, doses were chosen as indicated in the guideline. Hence, the results can be considered as reliable to assess the potential of TRIDI to induce chromosome aberrations in mammalian cells. The test item TRIDI has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system in human lymphocytes, under the experimental conditions described. Therefore, TRIDI is considered to be non-genotoxic in this in vitro chromosome aberration test, when tested up to the highest required concentration.
Executive summary:

A study was performed according to OECD TG 473 under GLP to assess the ability of TRIDI to cause structural chromosome aberrations in human lymphocytes cultured in vitro.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test item both in the absence and presence of exogenous metabolic activation (S9 mix). Vehicle and positive control cultures were also included where appropriate. 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 21 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. A single main test was performed for the 3-hour exposure in the absence of S9 mix, two main tests for the 3-hour exposure in the presence of S9 mix and three main tests for the 21-hour exposure in the absence of S9 mix. The mitotic index was assessed for all cultures to determine cytotoxicity. Based on the data from the preliminary toxicity test, test item concentrations were selected for the main test.

In the main test, justification for the highest analyzed concentration was determined by cytotoxicity.

The following test item concentrations were selected for metaphase analysis:

In the absence of S9 mix, 3-hour treatment: 10, 20 and 30 µg/mL.

In the presence of S9 mix, 3-hour treatment: 10, 30 and 32.5 µg/mL.

In the absence of S9 mix, 21-hour treatment: 1, 17.5 and 30 µg/mL.

Under the conditions described above, TRIDI caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations (excluding gaps), at any analyzed concentration, when compared with the vehicle control. There was no evidence of a linear dose-concentration relationship. The mean proportion of cells with chromosomal aberrations for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits.

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

It is concluded that TRIDI has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Jun 2018 to 16 Jul 2018 (experimental phase)
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)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 490: "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene", 2016.
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5300 In vitro mammalian cell gene mutation test. EPA 712-C-98-221.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
EC Commission Regulation No. 440/2008. Method B.17: Mutagenicity – In vitro mammalian cell gene mutation test. OJ L 142/262.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
tk+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: L5178Y mouse lymphoma (3.7.2c) cells (Clive and Spector, 1975), were obtained from American Type Culture Collection (ATCC), Virginia.
- Suitability of cells: The cells are heterozygous at the thymidine kinase (tk) locus (TK+/-), and forward mutation (TK-/-) is detected by the ability of these cells to divide and form colonies in the presence of trifluorothymidine (TFT), a toxic analogue of thymidine. This test system detects a wide range of genetic damage in viable cells capable of forming colonies. The test methodology was based on established procedures (Amacher et al., 1979; Amacher et al., 1980; Clive et al., 1979; Cole et al., 1990; Moore et al., 1985).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
The following media, obtained from a suitable supplier, were used:
R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamicin.
R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R10p: medium was used for cell culture unless otherwise specified.
Selective medium consisted of R10p containing 4 µg/mL trifluorothymidine (TFT).
Cells were kept in a humidified incubator at 34 to 39°C in an atmosphere of 5% CO2 in air
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes. Cell stocks are periodically checked for freedom from mycoplasma contamination.
- Periodically 'cleansed' against high spontaneous background: Yes. Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196 to -150°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: phenobarbital and 5,6-benzoflavone induced male Sprague-Dawley rat liver S9Type and composition of metabolic activation system:
- method of preparation of S9 mix: S9 mix contains: S9 fraction (5% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in R0. The co‑factors were prepared, neutralised with 1N NaOH and filter sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: The final volume of the cultures was 10 mL and the final concentration of the S9 fraction was 2% v/v, if present
Test concentrations with justification for top dose:
Concentrations used in the main test were based upon the results of the preliminary test (See results section):
-S9 mix (3 hours): 5, 10, 12.5, 15, 20, 25 and 30 µg/mL
+S9 mix (3 hours): 5, 10, 15, 17.5, 20, 22.5, 25, 27.5 and 30 µg/mL
-S9 mix (24 hours): 5, 10, 15, 17.5, 20, 22.5, 25, 27.5 and 30 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: EGDE (Ethylene glycol dimethyl ether)
- Justification for choice of solvent/vehicle: The test item was found to be soluble at 200 mg/mL in Ethylene glycol dimethyl ether (EGDE). It is not considered to have any adverse effects in this study.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
1% EGDE
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Cultures contained a total of 12 x 10E6 cells in a final volume of 10 mL (3h exposure). Duplicate 10 mL cultures containing 3 x 10E6 cells were treated for 24 hours.

DURATION
- Preincubation period: none
- Exposure duration: 3 or 24 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10 to 12 days

SELECTION AGENT (mutation assays): 4 µg/mL trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was measured in terms of relative suspension growth (RSG) and relative total growth (RTG).
Rationale for test conditions:
As indicated in the guideline.
Evaluation criteria:
Due to limitations of this free-text field, see "Any other information on materials and methods"
Statistics:
Statistical analysis was performed by Fluctuation application SAFEStat (SAS statistical applications for end users). Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
precipitation occurred at concentrations which were too cytotoxic
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: occurred, but testing concentrations were limited by cytotoxicity
- Other confounding effects: none known
- Data on osmolality and data on pH: The osmolality and pH of TRIDI in medium were measured by analysing samples of R10p media, dosed at 1% (v/v), with either the vehicle (EGDE) or a TRIDI formulation at 200 mg/mL (to give a final concentration of 2000 μg/mL). For medium dosed with TRIDI at 2000 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 2000 μg/mL as this is the standard limit concentration within this test system as recommended in the current OECD Guideline 490 (2016).


RANGE-FINDING/SCREENING STUDIES:
Precipitate (observed by eye at the end of treatment) was observed at concentrations of 250 µg/mL and greater in the absence and presence of S9 mix, following a 3-hour exposure. 250 μg/mL was, therefore, the highest concentration carried forward for assessment of toxicity. Exposure to TRIDI at concentrations from 3.91 to 250µg/mL in the absence and presence of S9 mix (3-hour exposure) resulted in relative suspension growth (RSG) values
from 101 to 0% and from 108 to 1% respectively. Following a continuous exposure for 24 hours, precipitation (assessed by eye at the end of treatment) was observed at concentrations of 125 µg/mL and greater. 125 μg/mL was,
therefore, the highest concentration carried forward for assessment of toxicity. Exposure to concentrations from 3.91 to 125 µg/mL resulted in RSG values from 109 to 1%. Concentrations used in the main test were based upon these data.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see attachment
Conclusions:
The study was conducted under GLP according to OECD guideline 490 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response, concentrations were properly chosen based on cytotoxicity. Hence, the results can be considered as reliable to assess the potential of the test item to induce mutations in mammalian cells. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. It can be concluded that TRIDI did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
Executive summary:

TRIDI was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

TRIDI was found to be soluble at 200 mg/mL in Ethylene glycol dimethyl ether (EGDE). A final concentration of 2000 mg/mL, dosed at 1% v/v was used as the maximum concentration in the preliminary toxicity test. This is the standard limit concentration within this test system as recommended in the current OECD Guideline 490 (2016).

Precipitate was observed by eye at the end of treatment following a 3-hour exposure to TRIDI in the absence and presence of S9 mix at concentrations 250 µg/mL and above. Relative suspension growth (RSG) was reduced from 101 to 0% and from 108 to 1% at concentrations from 3.91 to 250 µg/mL in the absence and presence of S9 mix respectively. Precipitate was observed by eye at the end of treatment following a 24-hour exposure in the absence of S9 mix at concentrations 125 µg/mL and above. RSG was reduced from 109 to 1% at concentrations from 3.91 to 125 µg/mL. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix were 15 and 17.5 µg/mL respectively. In both the absence and presence of S9 mix the mean RTG was reduced to 19%.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 20 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The mean RTG was reduced to 8%.

It was concluded that TRIDI did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria (OECD 471)

Herbold 1990

Triisopropyldiisocyanatobenzene was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, T 1537 and TA 98.

The study was performed according to the OECD Guideline 471 with deviations (only 4 Salmonella strains tested) and considered to be of good quality (reliability Klimisch 2). 8 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a weak, strain-specific bacteriotoxic effect. Substance precipitation occurred at the dose 500 µg per plate and above. Therefore this range could only be used to a limited extent up to 5000 µg per plate for assessment purposes.

Evidence of mutagenic activity of triisopropyldiisocyanatobenzene was not seen. No biologically relevant increase in the mutant count, in comparison with the negative control was observed.

The positive control sodium azide, nitrofurantoin, 4 -nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, Triisopropyldiisocyanatobenzene was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Envigo 2018

In this in vitro assessment of the mutagenic potential of TRIDI according to OECD 471 under GLP, tryptophan-dependent mutants of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to TRIDI diluted in ethylene glycol dimethyl ether (EGDE). EGDE was used as the vehicle control and untreated controls were also included.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

Concentrations of TRIDI up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca. half-log10 dilutions of the highest concentration.

No signs of toxicity towards the tester strain were observed in either mutation test following exposure to TRIDI. Precipitate was observed on all plates containing TRIDI at 1500 and 5000 µg/plate in the absence of S9 mix and at 5000 µg/plate in the presence of S9 mix in both tests.

No evidence of mutagenic activity was seen at any concentration of TRIDI in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle and untreated controls were within or close to the current historical control range for the laboratory.

It was concluded that TRIDI showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Gene mutation in mammalian cells (OECD 490)

TRIDI was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

TRIDI was found to be soluble at 200 mg/mL in Ethylene glycol dimethyl ether (EGDE). A final concentration of 2000 mg/mL, dosed at 1% v/v was used as the maximum concentration in the preliminary toxicity test. This is the standard limit concentration within this test system as recommended in the current OECD Guideline 490 (2016).

Precipitate was observed by eye at the end of treatment following a 3-hour exposure to TRIDI in the absence and presence of S9 mix at concentrations 250 µg/mL and above. Relative suspension growth (RSG) was reduced from 101 to 0% and from 108 to 1% at concentrations from 3.91 to 250 µg/mL in the absence and presence of S9 mix respectively. Precipitate was observed by eye at the end of treatment following a 24-hour exposure in the absence of S9 mix at concentrations 125 µg/mL and above. RSG was reduced from 109 to 1% at concentrations from 3.91 to 125 µg/mL. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix were 15 and 17.5 µg/mL respectively. In both the absence and presence of S9 mix the mean RTG was reduced to 19%.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 20 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The mean RTG was reduced to 8%.

It was concluded that TRIDI did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Chromosomal aberration (OECD 473)

A study was performed according to OECD TG 473 under GLP to assess the ability of TRIDI to cause structural chromosome aberrations in human lymphocytes cultured in vitro.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test item both in the absence and presence of exogenous metabolic activation (S9 mix). Vehicle and positive control cultures were also included where appropriate. 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 21 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. A single main test was performed for the 3-hour exposure in the absence of S9 mix, two main tests for the 3-hour exposure in the presence of S9 mix and three main tests for the 21-hour exposure in the absence of S9 mix. The mitotic index was assessed for all cultures to determine cytotoxicity. Based on the data from the preliminary toxicity test, test item concentrations were selected for the main test.

In the main test, justification for the highest analyzed concentration was determined by cytotoxicity.

The following test item concentrations were selected for metaphase analysis:

In the absence of S9 mix, 3-hour treatment: 10, 20 and 30 µg/mL.

In the presence of S9 mix, 3-hour treatment: 10, 30 and 32.5 µg/mL.

In the absence of S9 mix, 21-hour treatment: 1, 17.5 and 30 µg/mL.

Under the conditions described above, TRIDI caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations (excluding gaps), at any analyzed concentration, when compared with the vehicle control. There was no evidence of a linear dose-concentration relationship. The mean proportion of cells with chromosomal aberrations for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits.

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

It is concluded that TRIDI has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Justification for classification or non-classification

According to the Regulation (EC) No 1272/2008 the test material does not meet the criteria for classification and will not require labelling as a mutagen.