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EC number: 213-668-5 | CAS number: 999-97-3
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- 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:
- guideline study with acceptable restrictions
- Remarks:
- 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:
- 1 997
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- 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 substance name:
- 1,1,1,3,3,3-hexamethyldisilazane
- EC Number:
- 213-668-5
- EC Name:
- 1,1,1,3,3,3-hexamethyldisilazane
- Cas Number:
- 999-97-3
- Molecular formula:
- C6H19NSi2
- IUPAC Name:
- bis(trimethylsilyl)amine
- Test material form:
- liquid
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- S. typhimurium, 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: 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: 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: 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 resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- 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
- Remarks:
- at 2500 μg/plate 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 ammonia.
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
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.(μg/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:
- 1,1,1,3,3,3-Hexamethyldisilazane (CAS 999-97-3, EC 213-668-5) has been tested according to OECD Test Guideline 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.
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