Reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-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:

28 Jun - 09 Aug 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Verkehr Baden-Württemberg, Stuttgart, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

EC Number:

945-924-3

Molecular formula:

C13H18O2

IUPAC Name:

reaction mass of rel-(2S,4R,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane and rel-(2S,4S,6S)-2,4,6-trimethyl-4-phenyl-1,3-dioxane

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I:
15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment II:
15, 50, 150, 500 and 1500 µg/plate without metabolic activation
15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

Statistics:

The X²-test (Mohn and Ellenberger, 1977) was used.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDY: Based on an initial toxicity test, the test substance was tested in concentrations of 15 to 5000 µg/plate.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1

EXPERIMENT 1
S9-Mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	35 ± 8	99 ± 10	260 ± 17	33 ± 6	9 ± 2
Solvent control (DMSO)	36 ± 9	113 ± 6	259 ± 15	35 ± 4	12 ± 4
15	-	112 ± 25	-	-	-
50	33 ± 3	100 ± 13	239 ± 13	41 ± 4	9 ± 3
150	41 ± 6	111 ± 10	243 ± 12	45 ± 3	11 ± 6
500	31 ± 4	114 ± 10	244 ± 9	41 ± 5	9 ± 2
1500	30 ± 6	140 ± 12	263 ± 11	40 ± 6	13 ± 2
5000	14 ± 5 (T)	91 ± 19 (T)	109 ± 12 (T)	29 ± 5 (T)	2 ± 1 (T)
NaN3 (0.7)	-	465 ± 41	-	657 ± 71	-
2-NF (2.5)	269 ± 29	-	-	-	-
9-AA (50)	-	-	-	-	364 ± 29
Mitomycin C (0.15)	-	-	581 ± 12	-	-
S9-Mix	With
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	40 ± 4	114 ± 4	280 ± 11	20 ± 3	14 ± 3
Solvent control (DMSO)	42 ± 7	114 ± 5	254 ± 26	23 ± 3	13 ± 3
15	-	120 ± 10	-	-	-
50	38 ± 5	103 ± 11	293 ± 16	16 ± 3	19 ± 3
150	36 ± 6	115 ± 8	298 ± 6	22 ± 5	16 ± 3
500	40 ± 4	107 ± 5	305 ± 13	19 ± 3	13 ± 3
1500	47 ± 10	117 ± 10	285 ± 5	20 ± 3	10 ± 2
5000	41 ± 7	70 ± 8 (T)	245 ± 27 (T)	18 ± 2 (T)	4 ± 3 (T)
2-AA (0.7)	1189 ± 70	1092 ± 92	-	-	-
2-AA (1.5)	-	-	725 ± 22	625 ± 38	508 ± 20
NC = Negative Control T = bacteriotoxic 2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

Table 2: Test Results of Experiment 2

EXPERIMENT 2
S9-Mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	47 ± 6	118 ± 10	269 ± 11	30 ± 8	16 ± 4
Solvent control (DMSO)	44 ± 12	108 ± 9	262 ± 15	35 ± 4	15 ± 5
15	47 ± 3	133 ± 10	283 ± 12	35 ± 3	14 ± 1
50	33 ± 9	121 ± 13	274 ± 19	29 ± 7	15 ± 5
150	52 ± 5	140 ± 19	265 ± 24	34 ± 8	18 ± 3
500	43 ± 5	132 ± 14	275 ± 8	39 ± 3	12 ± 4
1500	42 ± 11	149 ± 5	262 ± 13	43 ± 3	16 ± 2
NaN3 (0.7)	-	515 ± 9	-	624 ± 39	-
2-NF (2.5)	222 ± 21	-	-	-	-
9-AA (50)	-	-	-	-	212 ± 19
Mitomycin C (0.15)	-	-	820 ± 53	-	-
S9-Mix	With
Test item (µg/plate)	TA 98	TA 100	TA 102	TA 1535	TA 1537
NC	48 ± 4	107 ± 7	307 ± 29	22 ± 4	15 ± 4
Solvent control (DMSO)	38 ± 7	91 ± 13	298 ± 9	22 ± 5	19 ± 5
15	-	124 ± 9	322 ± 21	18 ± 4	16 ± 5
50	37 ± 10	124 ± 9	330 ± 7	18 ± 3	17 ± 3
150	40 ± 6	120 ± 9	328 ± 13	21 ± 8	11 ± 3
500	39 ± 3	110 ± 11	296 ± 20	16 ± 3	17 ± 4
1500	45 ± 2	116 ± 8	347 ± 6	17 ± 2	17 ± 5
5000	27 ± 4 (T)	-	-	-	-
2-AA (0.7)	613 ± 33	903 ± 136	-	-	-
2-AA (1.5)	-	-	1106 ± 92	619 ± 51	259 ± 25
NC = Negative Control T = bacteriotoxic 2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

The test substance induced a slight increase in the mutation frequency in the tester strain TA 1535 and TA 100 in the absence of a metabolic activation system in both experiments. In the presence of S9 mix a slight increase in the frequency of revertants of strain TA102 was observed. However, the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X²-test did not reveal a significant effect at any of the test points.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA 100, TA 1535, TA 102, TA 98 and TA 1537) tested with and without metabolic activation up to 5000 µg/plate.