Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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
Cross-reference
Reason / purpose:
reference to other study
Remarks:
OECD 471
Reference
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
his-
Species / strain
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.
Controls
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

Results and discussion

Test results
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.