5-methyl-2-(1-methylbutyl)-5-propyl-1,3-dioxane

Genetic toxicity: in vitro

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:

1981-05-01 to 1982-10-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study was not conducted under GLP but performed according to existing test prodecures (Ames et al. 1977).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The study was conducted according to Ames et al. (1978) and followed the main principles of the later OECD TG 471 (first adopted 26 May 1983).

GLP compliance:

no

Remarks:

GLP in EU started in 1981

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Troenan

IUPAC Name:

Troenan

Test material form:

other: not reported

Details on test material:

- Name of test material (as cited in study report): Troenan

Method

Target gene:

gene for synthesis of Histidin in strains of Salmonella typhimurium

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: Mutations in Histidin synthesis: his G46 in TA 1535 and TA 100, his C 3076 in TA 1537, his D3052 in TA 98; additional mutations: rfa (LPS), uvrB (repair), R- in TA 1535 and TA 1537, R- in TA 100 and TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: Mutations in Histidin synthesis:his D3052 in TA 1538; additional mutations: rfa (LPS), uvrB (repair), R- in TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

4, 20, 100, 500 and 2500 µg/L

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not reported

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminoantracen

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: bacteria suspension in 10 mL standard I nutrition bouillon, cell concnetration 10^9
2 mL Top Agar (including 10 mL sterile Histidin (0.5 mM)-Biotin (0.5 mM)-solution per 100 mL Top Agar), 0.1 mL bacteria suspension, 0.1 mL test substance solution were transferred into sterile vessels and thoroughl y mixed.
5 test concentrations without S9-mix: addition of 0.5 mL sterile water
5 test concentrations with S9-mix: addition of 0.5 mL S9-mix
After mixing dirstibuten of the soft fluid into Agar plates
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: not reported

DETERMINATION OF CYTOTOXICITY: relative total growth

Evaluation criteria:

Assessment of a positive result:
- background of not reverted bacteria with no growth reduction in comparison to the negative controls
- spontaneous mutation rates of the used bacteria strains within the charactictic range of spontaneous mutations
range of spontaneoas mutations without S-9 mix:
TA 1535: mean = 6, extreme values: 1-15 (Ames at al.: mean=20, extreme values 5-50)
TA 100: mean = 87, extreme values: 35-201 (Ames at al.: mean=160, extreme values 60-220)
TA 1537: mean = 7, extreme values: 1-24 (Ames at al.: mean=7, extreme values 3-25)
TA 1538: mean = 14, extreme values: 6-27 (Ames at al.: mean=25, extreme values 5-40)
TA 98: mean = 20, extreme values: 5-39 (Ames at al.: mean=40, extreme values 15-75)
range of spontaneoas mutations with S-9 mix:
TA 1535: mean = 8, extreme values: 1-25
TA 100: mean = 105, extreme values: 54-252
TA 1537: mean = 6, extreme values: 1-23
TA 1538: mean = 18, extreme values: 3-48
TA 98: mean = 27, extreme values: 7-76
- positive controls with mutation rates of at least 2.0 (TA 100) and 3.0 (other strains)
- an effect is considered if the test substance causes an increase of spontaneous mutations relative to the negative controls of at least 2.0 (TA 100) or 3.0 (other strains)

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Number of revertants in cultures without S-9 mix

Treatment	Conc.	TA1535		TA100		TA1537		TA1538		TA98
(without S-9 mix)	(µg/plate)	mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
negative control	---	4.5	2.3	100.5	15.1	4.2	1.5	9.3	3.8	13	3.6
positive controls
sodium azide	1	276.7	107.9	489.3	231.9	---	---	---	---	---	---
F-factor	---	61.5	---	4.9	---	---	---	---	---	---	---
4-nitrol-o-phenylendiamin	---	---	---	---	---	109.7	40	1084	56	472	205
F-factor	---	---	---	---	---	26.1	---	116.6	---	36.6	---
2-aminoanthracene	5	3	1	106.3	14.7	6.7	1.6	16.7	9.9	18.3	0.6
test substance
Troenan	2500	2	0	40	14.1	---a	---a	---a	---a	12	4.2
	500	2	1.4	62.5	9.2	---a	---a	7.5	3.5	6	1.4
	100	3	0	61	9.9	3.5	0.7	10	0	11.5	4.9
	20	1.5	0.7	70.5	3.5	3.5	0.7	7.5	2.1	12.5	3.5
	4	2	1.4	66	5.7	4	1.4	11	2.8	7	2.8

F-factor: increase of revertants relative to the negative control

a. Partial or complete inhibition of bacterial growth

Table 2 Number of revertants in cultures with S-9 mix

Treatment	Conc.	TA1535		TA100		TA1537		TA1538		TA98
(with S-9 mix)	(µg/plate)	mean	SD	mean	SD	mean	SD	mean	SD	mean	SD
negative control	---	9.2	4.3	95.3	21.1	11.3	21.6	18.4	5.5	23.7	7.7
positive controls
sodium azide	1	26.3	3.1	85.7	30.2	---	---	---	---	---	---
F-factor	---	2.9	---	---	---	---	---	---	---	---	---
4-nitrol-o-phenylendiamin	---	---	---	---	---	41	11.5	985	127	709	10
F-factor	---	---	---	---	---	3.6	---	53.6	---	29.9	---
2-aminoanthracene	5	55	9.5	1079	341	591	120	440	74	1404	366
F-factor	---	6	---	11.3	---	52.4	---	23.9	---	59.2	---
test substance
Troenan	2500	4	0	109	1.1	---a	---a	19	8.5	23	0
	500	5	0	92.5	31.8	---a	---a	18	7.1	27	2.8
	100	7	2.8	82.5	23.3	6	0	21	4.2	28	0
	20	2	0	90	35.3	5.5	0.7	16.5	4.9	23.5	0.7
	4	10	0	84.5	2.1	6	2.8	29	1.4	24	5.7

F-factor: increase of revertants relative to the negative control

a. Partial or complete inhibition of bacterial growth

Applicant's summary and conclusion

Conclusions:

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

In a reliable and conclusive study according to Ames et al. (1977), the test substance Troenan is considered to be not mutagenic under the conditions of the test.

Executive summary:

The test substance Troenan was tested for mutagenic activity in the bacterial tester strains Samonella typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98. The test was conducted on agar plates in the absence or presence pf postmitochondrial supernatant fluids from the liver of male rates treated with Aroclor 1254 (S-9 mix). Solutions of the test substance were freshly made up in DMSO before use. The following test concentrations were tested: 4. 20, 100, 500 and 2500 µg Troenan per plate.Troenan did not induce reverse mutations in the absence or presence of S-9 mix in the tester strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98. Therefore, Troenan did not show mutagernic activity in vitro.