Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-05-29 - 2013-09-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Council Regulation No. 440/2008 of 30 May 2008. Official Journal of the European Communities of May 31, 2008, L 142/248 - L142/255. B.13/14. Mutagenicity - Reverse Mutation Test using Bacteria.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 471. "Bacterial Reverse Mutation Test". Adopted: 21st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Health Effects Test Guidelines; United States Environmental Protection Agency; Prevention, Pesticides and Toxic Substances (7101); EPA712-C-98-247, August 1998. OPPTS 870.5100 -Bacterial Reverse Mutation Test.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-diethyl-2,4-diisocyanato-5-methylbenzene
Cas Number:
2162-70-1
Molecular formula:
C13H14N2O2
IUPAC Name:
1,3-diethyl-2,4-diisocyanato-5-methylbenzene
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, light protected
- Solubility and stability of the test substance in the solvent/vehicle: stable for al least 24 hours at room temperature, only used at the same day within the time range of stability data

The batch used was analytically examined prior to study initiation and was approved for use for the test period. A stability test in the solvent did not reveal significant degradation of the active ingredient

Method

Target gene:
his-
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The original strains were obtained from Prof. Bruce Ames (Berkeley, CA, USA).
- Suitability of cells: Histidinc-deficient mutants of Salmonella typhimurium LT2 served as indicators to demonstrate point mutagenic effects. These strains were selected specifically for the Salmonella/ microsome test. Since point mutations can be divided into two basic classes, base-pair substitutions and frameshift mutations, several strains were used which cover both types.
These included the strains Salmonella typhimurium TA1535, TA1537, TA100, TA98 and TA102 (Gatehouse etal. (1994), Levin et al. (1982)). TA 1535 and TA100 bear the base-pair substitution, his G 46, and TA100 additionally contains the plasmid pKM 101. This R factor also contained in TA98 and TA102, codes for an ampicillin resistance and should raise the sensitivity of the strains' TA102 carries the ochre mutation his G 428 on the multicopy plasmid pAQl, which codes in addition for tetracycline resistance. TA1537 and TA98 bear frameshift markers. TA1537 exhibits the +1 mutant, his C 3076, while TA98 bears the +2 type, his D 3052.
Furthermore, the strains have other properties, which should increase their sensitivity. They arc all deep rough, i.e. partly deficient in lipopolysaccharide side chains in their cell walls, enabling larger molecules to penetrate the bacterial cell wall and produce mutations. With the exception of TA102, all strains have reduced capability to repair DNA-damage which increases the likelihood that such damage results in mutations.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague Dawley rat liver S9
Test concentrations with justification for top dose:
0, 10, 25, 50, 160, 500, 1600, 5000 µg/plate
The highest dose tested should cause toxicity or should correspond to the substance's precipitation range (visible precipitates on the plates ) but should not exceed 5 mg/plate or 5 µL/plate, at least six doses were routinely used.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: EDGE, 1,2-Dimethoxyethane
- Justification for choice of solvent/vehicle: A stability test in the solvent did not reveal significant degradation of the active ingredient.
Controls
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
EDGE
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
cumene hydroperoxide
mitomycin C
other: 4-Nitro-1,2-phenylene diamine (4-NPDA); 2-Aminoanthracene (2-AA)
Remarks:
Sodium azide, mitomycin C and cumene hydroperoxide were dissolved in phosphate buffer. The other positive controls were dissolved in DMSO.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 1h (preincubation method)
- Exposure duration: 48 hours (TA102) or 72 hours (TA1535, TA100, TA1537, TA98)

SELECTION AGENT (mutation assays): his minimal agar

NUMBER OF REPLICATIONS: All plates were prepared in triplicate, two independent experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: The toxicity of the substance was assessed by a gross appraisal of background growth on the plates for mutant determination. Moreover, 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 solvent controls.
Rationale for test conditions:
as set out in the guideline
Evaluation criteria:
Assessment Criteria
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA 100, TA1537 and TA98 this increase should be about twice that of solvent controls. For TA102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none in the plate incorporation; started at 5000 µg/plate in the preincubation test

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed, but plates were assessable up to higher ranges (see tables)
Remarks on result:
other: negative for gene mutation in both plate incorporation and preincubation test

Applicant's summary and conclusion

Conclusions:
The study was performed scientifically reasonably according to OECD TG 471 under GLP and is well documented. Positive and negative controls gave the appropriate response. Hence, the results can be considered as sufficiently reliable to assess the mutagenic potential of the test item in bacteria. Under the experimental conditions reported (direct plate incorporation procedure and preincubation modification) the test item did not induce gene mutations by base-pair changes or frame-shifts in the genome of the S. typhimurium strains used, when tested up to a maximum recommended dose of 5000 µg/plate in the absence and presence of S9 mix. Hence, the test item does not need to be considered as mutagenic in bacteria.
Executive summary:

Diethyltolyldiisocyanat, was initially investigated for point mutagenic effects in the plate incorporation test. The test item, dissolved in EGDE, was administered in doses of up to and including 5000 µg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102, according to the OECD guideline 471 under GLP. Doses up to and including 0.05 mg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect, however, ranges up to 5000 µg per plate could be used for assessment purposes. Substance precipitation did not occur.

The test item was investigated in an independent repeat using the preincubation modification. Other experimental conditions remained unchanged. Doses up to and including 0.05 mg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strain-specific bacteriotoxic effect. This range could be used up to 5000 µg per plate for assessment purposes. Substance precipitation occurred at the dose of 5000 µg per plate.

In both experiments, the employed positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonics compared to the corresponding solvent controls.

Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison to the solvent controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well in the preincubation modification, under the experimental conditions applied.

Therefore, the test item 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.