Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-03-21 to 1997-05-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate OECD test guideline and was compliant with GLP, however testing with positive controls was not fully undertaken, strains TA98 and 100 only were tested with positive controls.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
incomplete testing with positive controls with activation
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): hexamethyl disilazane

- Substance type: monoconstituent

- Physical state: colourless liquid

- Analytical purity: 98.8%

- Purity test date: 1996-10-07

- Expiration date of the lot/batch: October 1997

- Storage condition of test material: in the dark, under nitrogen and at room temperature

Method

Species / strain
Species / strain / cell type:
other: TA98, TA100, TA1535, TA1537, TA102
Additional strain / cell type characteristics:
other: histidine deficient
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
See Table 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: not given
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
with DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
strain TA98 without MA

Migrated to IUCLID6: 5.0 ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
with strains TA100 and TA1535 without MA

Migrated to IUCLID6: 2.0 ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
with strain TA1537 without MA

Migrated to IUCLID6: 50 ug/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DSMO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: glutaraldehyde (25.0 ug/plate)
Remarks:
with strain TA102 without MA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
strains TA 100 and TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
no
Remarks:
strains TA1535, TA 1537 and TA102 were not tested with a positive control in the presence of MA.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation


DURATION

- Preincubation period: 1 hour at 37 degC

- Exposure duration: 3 days


NUMBER OF REPLICATIONS: 3 per exposure, 5 with solvent control.

ACTIVATION: S9 mix included 10% Arocolor-induced rat liver S9, and glucose-6-phosphate, NADP, MgCl2, KCl. 0.5 ml was added with 0.1 ml bacterial suspension and 0.1 ml test or control substance to 2.5 ml of agar to give a final concentration of approximately 1.5% S9.

DETERMINATION OF CYTOTOXICITY

- Method: toxicity to background lawn.
Evaluation criteria:
The test article was considered to be mutagenic if a dose related and reproducible increase in the number of revertants was induced at one or more test concentration. An increase in revertant colonies was at least two times the mean negative control counts in strains TA98, TA100 and TA102, or three times in strains TA1535 and TA1537.
Statistics:
Colonies were counted electronically using a Seescan Colony Counter (Seescan plc) and the background lawn inspected for signs of toxicity.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 μg/plate in strain TA102 with MA
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Other confounding effects: in water the test substance hydrolyses into ammoniac.


RANGE-FINDING/SCREENING STUDIES: 8, 40, 200, 1000 and 5000 μg/plate with TA100 without S9 resulted in no evidence of toxicity (results with S9 were not scorable due to contamination). The results were used in the mutagenic assessment and are presented in Table 2.


COMPARISON WITH HISTORICAL CONTROL DATA: Following Experiment 1 treatments of strains TA98 and TA102 in the absence and presence of S-9, Experiment 2 treatments of strain TA98 in the absence and presence of S9 and strain TA100 in the absence of S9, mean revertant colony plate counts for the solvent control treatments were just outside the historical range of this laboratory. The mean solvent control plate counts were however well within other published ranges, and so these strains were considered to have been behaving characteristically, and these data were considered valid.


ADDITIONAL INFORMATION ON CYTOTOXICITY: In Experiment 1 a slight thinning of the background lawn was observed at the highest concentrations with strain TA1537 with S9. In Experiment 2 cytotoxicity was observed in the form of complete mortality of the bacteria at the maximum concentration of 2500 μg/plate with strain TA102 with S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2a. Experiment 1 -Mutagenicity Assay. Number of revertants per plate (average).

Positive control was 2-nitrofluorene without metabolic activation and 2-aminoanthracene with metabolic activation.

 

 Strain

 TA 98

 Conc. (μg/plate)

 - MA

  + MA

 Cytotoxic

(yes/no)

 0*

 34.0

 50.2

 no

 8

 49.7

 43.3

 no

 40

 43.0

 51.7

 no

 200

 46.7

 47.7

 no

 1000

 50.0

 49.0

 no

 5000

 44.0

 47.3

 no

 Positive control

 766.3

 1774.3

 no

* = solvent control with DMSO

 

 

Table 2b. Experiment 1 -Mutagenicity Assay. Number of revertants per plate (average).

Positive control was sodium azide for strains without metabolic activation, the positive control with metabolic activation was 2-aminoanthracene with strain TA100 and none for strain TA1535

 Strain

 TA 100

 TA 1535

 Conc.g/plate)

 -MA

 +MA

 Cytotoxic

(yes/no)

 -MA

 +MA

 Cytotoxic

(yes/no)

 0*

 115.0

 145.4

 no

 17.6

 23.8

 no

 80

 126.0

 162.3

 no

 28.0

 26.3

 no

 40

 106.7

 161.3

 no

 26.0

 26.7

 no

 200

 116.7

 162.3

 no

 21.7

 26.7

 no

 1000

 109.7

 183.7

 no

 19.7

 25.7

 no

 5000

 109.0

 165.7

 no

 22.3

 24.3

 no

 Positive control

 593.0

 1448.0

 no

 387.0

 -

 no

* = solvent control with DMSO

 

 

Table 2c. Experiment 1 - Mutagenicity Assay. Number of revertants per plate (average).

Positive control was 9-aminoacridine without metabolic activation, and no positive control with metabolic activation.

 

 Strain

TA 1537

 Conc. (μg/plate)

 - MA

 + MA

 Cytotoxic

(yes/no)

 0*

 9.4

 12.6

 no

 80

 16.7

 14.7

 no

 40

 15.0

 6.3

 no

 200

 11.3

 16.3

 no

 1000

 12.3

 15.0

 no

 5000

 15.7

 14.3

 no

 Positive control

 419.0

 -

 no

* = solvent control with DMSO

 

 

Table 2d. Experiment 1 - Mutagenicity Assay. Number of revertantsper plate (average).

Positive contrrol was glutaraldehyde without metabolic activation, and there was no positive control with metabolic activation.

 

 Strain

TA 102

 Conc. (μg/plate)

 - MA

  + MA

 Cytotoxic

(yes/no)

 0*

 404.8

 454.8

 no

 80

 429.3

 506.0

 no

 40

 452.0

 444.3

 no

 200

 430.0

 475.3

 no

 1000

 410.3

 472.0

 no

 5000

 326.0

 538.7

 no

 Positive control

 575.7

 -

 no

* = solvent control with DMSO

 

 

Table 3a. Experiment 2 -Mutagenicity Assay. Number of revertants per plate (average).

Positive control was 2-nitrofluorene without metabolic activation and 2-aminoanthracene with metabolic activation.

 

 Strain

 TA 98

 Concentration

 - MA

  + MA

 Cytotoxi

(yes/no)

 0*

 29.8

 39.2

 no

 1

 36.3

 39.3

 no

 2

 29.7

 36.7

 no

 3

 38.3

 31.7

 no

 4

 32.0

 35.0

 no

 5

 40.3

 28.7

 yes

 Positive control

 776.0

 1412.7

 no

* = solvent control with DMSO

Concentrations: 0, 1, 2, 3, 4 & 5 without metabolic activation were respectively = 0, 312.5, 625, 1250, 2500 and 5000 μg/plate; and with metabolic activation respectively = 0, 156.25, 312.5, 625, 1250, 2500 μg/plate.

 

 

Table 3b. Experiment 2 -Mutagenicity Assay. Number of revertantsper plate (average).

Positive control was sodium azide for both strains without metabolic activation, the positive control with metabolic activation was 2-aminoanthracene with strain TA100 and none for strain TA1535

 Strain

 TA 100

 TA 1535

 Concentration

 -MA

 +MA

 Cytotoxic

(yes/no)

 -MA

 +MA

 Cytotoxic

(yes/no)

 0*

126.6

 133.0

 no

 22.0

 18.0

 no

 1

126.5

 136.0

 no

 20.3

 30.0

 no

 2

121.7

 138.3

 no

 19.3

 26.0

 no

 3

125.0

 129.0

 no

 22.7

 21.3

 no

 4

139.0

 145.3

 no

 30.5

 26.0

 no

 5

121.0

 134.7

 no

 20.7

 25.0

 no

 Positive control

717.0

1061.0

 no

 549.3

 -

 no

* = solvent control with DMSO

Concentrations: 0, 1, 2, 3, 4 & 5 without metabolic activation were respectively = 0, 312.5, 625, 1250, 2500 and 5000 μg/plate; and with metabolic activation respectively = 0, 156.25, 312.5, 625, 1250, 2500 μg/plate.

 

 

Table 3c. Experiment 2 - Mutagenicity Assay. Number of revertants per plate (average).

Positive control was 9-aminoacridine without metabolic activation, and no positive control with metabolic activation.

 

 Strain

TA 1537

 Concentration

 - MA

 + MA

 Cytotoxic

(yes/no)

 0*

 12.6

 12.4

 no

 1

 10.3

 11.0

 no

 2

 11.7

 11.7

 no

 3

 10.7

 12.0

 no

 4

 10.0

 16.0

 no

 5

 11.7

 9.0

 no

 Positive control

 853.0

 -

 no

* = solvent control with DMSO

Concentrations: 0, 1, 2, 3, 4 & 5 without metabolic activation were respectively = 0, 312.5, 625, 1250, 2500 and 5000 μg/plate; and with metabolic activation respectively = 0, 156.25, 312.5, 625, 1250, 2500 μg/plate.

 

 

Table 3d. Experiment 2 -Mutagenicity Assay. Number of revertantsper plate (average).

Positive control was glutaraldehyde without metabolic activation, and there was no positive control with metabolic activation.

 

 Strain

TA 102

 Concentration

 - MA

  + MA

 Cytotoxic

(yes/no)

 0*

 336.8

 370.8

 no

 1

 315.7

 354.7

 no

 2

325.0

 362.7

 no

 3

 298.0

 344.7

 no

 4

 330.3

 351.7

 no

 5

 287.3

  -

 yes

 Positive control

 619.0

 -

 no

* = solvent control with DMSO

Concentrations: 0, 1, 2, 3, 4 & 5 without metabolic activation were respectively = 0, 312.5, 625, 1250, 2500 and 5000 μg/plate; and with metabolic activation respectively = 0, 156.25, 312.5, 625, 1250, 2500 μg/plate.

 

Applicant's summary and conclusion

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

HMDZ has been tested according to OECD TG 471 and in compliance with GLP. No test substance induced increase in the number of revertants in S. typhimurium strains TA 98, TA100, TA 1535, TA 1537 and TA 102 was observed. The results obtained with and without metabolic activation in the initial plate incorporation test and in the repeat pre-incubation assay were in agreement. Appropriate solvent and positive controls were included and gave expected results. The test material was therefore considered to be negative for mutagenicity to bacteria under the conditions of this test.