2-methyl-m-phenylene diisocyanate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2, 6 Toluene diisocynate is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: Similar to OECD 471

Principles of method if other than guideline:

Salmonella/microsome test was performed on 2, 6 Toluene diisocyanate chemical to know its mutagenic effect.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: Salmonella typhimurium LT2 mutants TA 1535, TA 100, TA 1537 and TA 98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was obtained from Aroclor 1254 500 mgrkg in corn oil, single intraperitoneal injection, 5 days prior to sacrifice. induced male Sprague–Daw- ley rats

Test concentrations with justification for top dose:

0,150,300,600,1200,2400 and 4800µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethyleneglycoldimethylether (EGDE)
- Justification for choice of solvent/vehicle: No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene with S9 metabolic activation system

Remarks:

No data on control chemical available

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data available
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate. Three plates were used for each concentration and each strain.

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY The toxicity of the substance was assessed as a gross appraisal of background growth on mutant plates and/or as a marked and dose-dependent reduction in the mutant count.

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: No data available

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 98, this increase should be about twice that of negative controls, whereas for TA 1537 at least a threefold increase is required.

Statistics:

No data available.

Species / strain:

S. typhimurium, other: TA 1535, TA 100, TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

upto 150 µg/plate for 10% S9 mix and upto 300 µg/plate for 30% S9 mix

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: Yes, observed
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table: Result of 2,6 TDI dissolved in EDGE and metabolic activation

Strain S9, µg/plate	TA1537		TA98
	10% S9	30% S9	10% S9	30% S9
0	12	14	47	45
150	13	21	67	57
300	13	16	87*	61
600	15	15p	130*	73*
1200	7p	10p	166*p	97*p
2400	7p	9p	128*p	82*p
4800	P	P	P	P
2-Aminianthracene	360*	66*	1538*	618*

*: Mutagenic effect

p: precipitation

Conclusions:

Interpretation of results (migrated information):
negative With and without Salmonella typhimurium TA 1535, TA 100, TA 1537
negative without metabolic activation Salmonella typhimurium strain TA98
negative with metabolic activation Salmonella typhimurium strain TA98; upto 150 µg/plate for 10% S9 mix and upto 300 µg/plate for 30% S9 mix

2, 6 Toluene diisocyanate is negative for the strains TA 1535, TA1537 and TA100 with and without S9 activation system and negative for Salmonella typhimurium strain TA98 upto 150 µg/plate for 10% S9 mix and upto 300 µg/plate for 30% S9 mix. Hence 2, 6 Toluene diisocyanate is not likely to classify as a gene mutant in vitro.

Executive summary:

Salmonella/microsome test was performed on 2, 6 Toluene diisocyanate to determine its mutagenic effect in the bacterial tester strains TA1535, TA1537, TA100 and TA98 of Salmonella typhimurium LT2 mutants. Plate incorporation method was performed and the reversion count was made after 48hrs incubation period.

2, 6 Toluene diisocyanate is negative for the strains TA 1535, TA1537 and TA100 with and without S9 activation system and negative for Salmonella typhimurium strain TA98 upto 150 µg/plate for 10% S9 mix and upto 300 µg/plate for 30% S9 mix. Hence 2, 6 Toluene diisocyanate is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Gene mutation in vitro:

Various peer reviewed publications were viewed to determine the mutagenic nature of 2, 6 Toluene diisocynate. The summary is as mentioned below:

Salmonella/microsome test was performed by Seel et al (1999) on 2, 6 Toluene diisocyanate (CAS no 91 -08 -7) to determine its mutagenic effect in the bacterial tester strains TA1535, TA1537, TA100 and TA98 of Salmonella typhimurium LT2 mutants. Plate incorporation method was performed and the reversion count was made after 48hrs incubation period. 2, 6 Toluene diisocyanate is negative for the strains TA 1535, TA1537 and TA100 with and without S9 activation system and negative for Salmonella typhimurium strain TA98 upto 150 µg/plate for 10% S9 mix and upto 300 µg/plate for 30% S9 mix. Hence 2, 6 Toluene diisocyanate is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration test was performed by Gulati et al (1989) to determine the mutagenic nature of 2, 6 Toluene diisocynate. Approximately 24 hr prior to cell treatment, 1.2 x 10⁶cells were seeded per 75 cm²flask. A culture was established for each dose both with and without metabolic activitation. For assays without metabolic activation, the cells were treated for about 10 hr. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off. In the test protocol for assays with metabolic activation, the cells were harvested approximately 11 hr after removal of the S9 fraction. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. The standard time for obtaining second-division metaphase cells was 26 hr. Harvest time was extended in increments of 5hrs with colcemid present for the last 2 hr. Treatment of cell cultures with concentrations of 600 - 1,000µg/mL 2, 6 toluene diisocyanate, under conditions of delayed harvest to compensate for chemical-induced cell cycle delay, produced a significant, dose-related increase in ABS. A pronounced increase occurred at the highest dose tested, which also caused severe toxicity. 2, 6 Toluene diisocynate induced chromosomal abberrations in CHO cell line in the absence if S9 metabolic activation system and failed to induce mutation in the presence of S9.

In the same study by Gulati et al (1989), Sister chromatid exchange assay was performed to determine the mutagenic nature of 2, 6 Toluene diisocynate. Approximately 24 hr prior to cell treatment, 1 x 10⁶cells were seeded per 75 cm²flask. A culture was established for each dose both with and without metabolic activitation. For assays without metabolic activation, the medium was replaced with fresh medium immediately before chemical treatment. Cells were treated with test or control substances for 2 hr to allow interaction with cells before the addition of bromodeoxyuridine (BrdUrd). BrdUrd was then added (final concentration 10 pM), and incubation was continued for an additional 24 hr. The medium was removed, and fresh medium containing 10 pM BrdUrd and colcemid was added and incubation was continued for 2-3 hr. For assays with metabolic activation, the cells were rinsed twice with phosphate buffered saline (PBS), after which culture medium without FBS was added. Cells were incubated for 2 hr in the presence of the test or control substances and the S9 reaction mixture. FBS was omitted to avoid the binding of serum proteins to short-lived, highly reactive intermediates. After the 2 hr exposure period, cells were washed twice with PBS, and then complete medium containing 10% FBS and 10 pM BrdUrd was added. Cells were incubated for an additional 26hr,with colcemid present for the final 2- 3 hr of incubation. Two to three hours after addition of colcemid, cells were harvested by mitotic shake-off. Prior to harvesting, the percent confluency in each flask was estimated using a widefield microscope. Harvested cells were treated for about 3 min at room temperature with hypotonic KCI (75 mM), washed with fixative (3: 1 methano1: glacial acetic acid, v/v), dropped onto slides, and air dried. Staining for the detection of SCE was accomplished by a modified fluorescence plus Giemsa (FPG) technique. Fifty second division metaphase cells were scored per dose for the incidence of SCE. The number of chromosomes in each cell was also recorded. Any cell that had fewer than 19 or more than 23 chromosomes was excluded. All slides except for the high-dose positive controls were coded. Induction of SCE occurred within a concentration range 50-300µg/ml, but the level of response did not always correlate with dose, possibly because of precipitation of the test chemical and the resulting variable decreases in cell culture exposures. 2, 6 Toluene diisocynate induced sister chromatid exchange in CHO cell line in the absence if S9 metabolic activation system and failed to induce SCEs in the presence of S9.

Based on the data summarized, 2, 6 Toluene diisocynate is not mutagenic in vitro. Mutagenic nature is observed for CHO cell line without mammalian metabolic activation system in the studies reviewed. Human concern arises with positive data with mammalian metabolic activation system. Since 2, 6 Toluene diisocynate gives negative results with mammalian metabolic activation system, thus, it can be considered as negative. Hence 2, 6 Toluene diisocynate is not likely to classify as a gene mutant in vitro.

Justification for selection of genetic toxicity endpoint

Data is from peer reviewed publication

Justification for classification or non-classification

2, 6 Toluene diisocynate is not likely to classify as a gene mutant in vitro.