1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

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:

09 - 23 May 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E.coli and/or TA 102 tester strain were not included in the study; cytotoxicity/precipitation was not determined.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

EC Number:

226-159-8

EC Name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

Cas Number:

5306-85-4

Molecular formula:

C8H14O4

IUPAC Name:

1,4:3,6-dianhydro-2,5-di-O-methyl-D-glucitol

Test material form:

other: liquid

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: All TA strains carry a mutation of the uvr B gene coding for the DNA excision repair system (uvrB) and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101) to detect weak mutagens.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: All TA strains carry a mutation of the uvr B gene coding for the DNA excision repair system (uvrB) and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101) to detect weak mutagens.

Metabolic activation:

with and without

Metabolic activation system:

cofactor 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
- 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without metabolic activation)
Experiment II
- 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without metabolic activation)
Experiment III
- 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Acridine Mutagen ICR191 (AM), 2-Aminoanthracene (2AA), Daunorubicin HCl (DR), 4-Nitro-o-phenylene diamine (4NPD), N-Methyl-N´-nitro-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3 plates for each test concentration, 5 plates for negative controls, 2 plates for each positive control

Evaluation criteria:

A positive response in an experiment is achieved when a two-fold or greater increase in the mean number of revertant colonies per test plate (over and above that observed for the solvent control plates) is obtained at at least one dose level. A second criterion for a positive result is the observation of a statistically significant dose-related increase in the number of revertants. In either case, the observed effect should be reproducible.

Statistics:

Mean values (colony count/plate) and standard deviation were calculated. An assessment of the statistical significance was carried out using a one-tailed Student´s t-test. The corresponding probability for each dose level was determined from a t-table using the appropriate degrees of freedom.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: No information is given, if a range-finding study was performed. In addition, cytotoxicity was neither examined in a previous study nor during the course of the study.

COMPARISON WITH HISTORICAL CONTROL DATA: No information is available for historical control data.

Any other information on results incl. tables

Table 1: Test Results - Experiment I (Plate Incorporation)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type		Frameshift type
		TA 1535	TA 100	TA 1537	TA 1538	TA 98
–	0	19.0 ± 2.7	114.0 ± 16.2	10.0 ± 4.0	5.0 ± 1.9	15.2 ± 4.1
–	1.6	21.0 ± 1.7	101.7 ± 16.3	14.0 ± 0.0	6.7 ± 3.1	14.7 ± 3.5
–	8	20.7 ± 1.2	109.0 ± 3.6	12.0 ± 0.0	5.0 ± 2.0	16.7 ± 2.1
–	40	18.7 ± 1.5	96.0 ± 15.6	10.0 ± 1.4	6.7 ± 1.2	16.3 ± 3.5
–	200	18.7 ± 4.0	120.0 ± 16.1	C	5.7 ± 3.8	17.3 ± 4.6
–	1000	20.7 ± 2.1	108.7 ± 13.6	C	7.0 ± 2.6	17.3 ± 1.5
–	5000	26.0 ± 6.9	114.3 ± 12.7	18.3 ± 3.1	6.0 ± 1.0	17.0 ± 4.0
Positive controls, –S9	Name	MNNG	MNNG	AM	4NPD	DR
	Concentrations (μg/plate)	1	1	1	1	0.2
	Mean No. of colonies/plate (average of 2 ± SD)	59.0 ± 26.9	215.0 ± 2.8	90.0 ± 0.0	49.5 ± 0.7	298.0 ± 24.0
	Concentrations (μg/plate)	2	2	2	2	0.5
	Mean No. of colonies/plate (average of 2 ± SD)	854.0 ± 69.3	825.0 ± 21.2	125. 0 ± 12.7	100.5 ± 19.1	700.5 ± 3.5
	Concentrations (μg/plate)	5	5	-	5	1
	Mean No. of colonies/plate (average of 2 ± SD)	2285.0 ± 257.4	2160.5 ± 85.6	-	167.0 ± 25.5	1019.5 ± 64.3
+	0	14.0 ± 2.2	127.2 ± 18.9	15.4 ± 3.2	8.4 ± 3.8	16.0 ± 5.1
+	1.6	17.0 ± 3.6	132.3 ± 17.2	15.0 ± 2.0	7.0 ± 2.6	17.7 ± 5.7
+	8	15.3 ± 4.0	134.3 ± 7.4	15.0 ± 0.0	7.3 ± 1.5	20.7 ± 3.5
+	40	15.7 ± 4.6	132.3 ± 15.3	13.3 ± 3.5	7.3 ± 1.2	15.0 ± 3.6
+	200	22.3 ± 4.7	123.3 ± 11.6	9.0 ± 4.4	7.0 ± 1.0	16.0 ± 1.7
+	1000	16.3 ± 1.5	119.7 ± 8.5	18.5 ± 4.9	8.0 ± 1.7	15.3 ± 0.6
+	5000	18.3 ± 2.1	130.7 ± 1.2	8.0 ± 0.0	9.0 ± 4.0	13.0 ± 2.0
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	0.5	0.2	0.5	0.2	0.2
	Mean No. of colonies/plate (average of 2 ± SD)	57.5 ± 2.1	282.0 ± 17.0	33.0 ± 0.0	4.5 ± 2.1	132.5 ± 19.1
	Concentrations (μg/plate)	1	0.5	1	0.5	0.5
	Mean No. of colonies/plate (average of 2 ± SD)	78.0 ± 5.7	479.0 ± 137.2	66.5 ± 16.3	4.0 ± 0.0	353.5 ± 12.0
	Concentrations (μg/plate)	2	1	2	1	1
	Mean No. of colonies/plate (average of 2 ± SD)	125.5 ± 26.2	953.0 ± 7.1	184.0 ± 1.4	6.0 ± 1.4	730.5 ± 91.2

2AA: 2-aminoanthracene

MNNG: N-methyl-N´-nitro-N-nitrosoguanidine

DR: Daunorubicin

AM: Acridine mutagen

4NPD: 4-nitro-o-phenylenediamine

C: Contaminated

Table 2: Test Results - Experiment II (Plate Incorporation)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type		Frameshift type
		TA 1535	TA 100	TA 1537	TA 1538	TA 98
–	0	13.0 ± 4.0	103.6 ± 20.4	10.5 ± 2.1	8.6 ± 3.4	12.8 ± 3.6
–	1.6	13.7 ± 4.6	107.0 ± 16.5	7.0 ± 1.0	12.0 ± 1.0	14.0 ± 3.0
–	8	12.3 ± 4.9	118.7 ± 9.1	6.5 ± 0.7	8.0 ± 1.0	11.7 ± 2.9
–	40	11.3 ± 3.5	121.3 ± 13.7	8.7 ± 0.6	7.7 ± 2.1	13.3 ± 1.5
–	200	14.0 ± 2.6	122.3 ± 4.5	5.0 ± 0.0	7.3 ± 3.1	11.3 ± 0.6
–	1000	11.7 ± 2.1	124.0 ± 26.6	6.7 ± 2.1	6.0 ± 1.7	14.3 ± 3.5
–	5000	12.0 ± 1.0	118.7 ± 16.1	6.5 ± 0.7	9.7 ± 3.2	13.7 ± 4.2
Positive controls, –S9	Name	MNNG	MNNG	AM	4NPD	DR
	Concentrations (μg/plate)	1	1	0.5	1	0.2
	Mean No. of colonies/plate (average of 2 ± SD)	133.0 ± 62.2	328.0 ± 89.1	49.0 ± 2.8	52.0 ± 15.6	323.5 ± 68.6
	Concentrations (μg/plate)	2	2	1	2	0.5
	Mean No. of colonies/plate (average of 2 ± SD)	901.5 ± 252.4	1326.5 ± 26.2	70.0 ± 2.8	88.0 ± 2.8	770.0 ± 87.7
	Concentrations (μg/plate)	5	5	2	5	1
	Mean No. of colonies/plate (average of 2 ± SD)	1672.0 ± 32.5	1920.0 ± 83.4	127.0 ± 14.1	200.0 ± 46.7	809.5 ± 67.2
+	0	18.8 ± 5.1	125.4 ± 11.8	11.0 ± 5.0	10.2 ± 1.1	17.0 ± 1.9
+	1.6	18.7 ± 2.1	128.7 ± 4.5	11.3 ± 1.2	12.3 ± 5.7	18.0 ± 1.7
+	8	20.7 ± 3.1	142.0 ± 9.5	8.3 ± 4.2	13.0 ± 0.0	19.7 ± 4.7
+	40	15.0 ± 1.7	128.0 ± 21.0	10.0 ± 0.0	9.3 ± 1.5	17.0 ± 3.0
+	200	18.7 ± 3.1	124.3 ± 11.1	9.3 ± 1.5	9.0 ± 1.0	12.0 ± 1.0
+	1000	15.0 ± 1.0	127.7 ± 4.2	11.7 ± 2.5	9.3 ± 0.6	17.3 ± 3.8
+	5000	22.0 ± 4.0	135.3 ± 9.5	8.5 ± 2.1	10.7 ± 3.2	15.3 ± 3.5
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	0.5	0.2	1	0.2	0.2
	Mean No. of colonies/plate (average of 2 ± SD)	48.0 ± 8.5	242.0 ± 35.4	51.0 ± 7.1	78.5 ± 2.1	78.0 ± 4.2
	Concentrations (μg/plate)	1	0.5	2	0.5	0.5
	Mean No. of colonies/plate (average of 2 ± SD)	70.5 ± 12.0	442.0 ± 60.8	271.5 ± 112.4	330.5 ± 103.9	183.5 ± 17.7
	Concentrations (μg/plate)	2	1	-	1	1
	Mean No. of colonies/plate (average of 2 ± SD)	114.5 ± 0.7	749.0 ± 282.8	-	487.5 ± 224.2	510.5 ± 38.9

2AA: 2-aminoanthracene

MNNG: N-methyl-N´-nitro-N-nitrosoguanidine

DR: Daunorubicin

AM: Acridine mutagen

4NPD: 4-nitro-o-phenylenediamine

Table 3: Test Results - Experiment III (Plate Incorporation)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Frameshift type
		TA 1537 (-S9-Mix)	TA 1538 (+ S9-Mix)
–/+	0	10.8 ± 1.9	7.0 ± 1.2
–/+	1.6	9.0 ± 1.7	9.0 ± 3.0
–/+	8	10.0 ± 4.6	6.7 ± 1.5
–/+	40	11.3 ± 2.1	9.7 ± 3.2
–/+	200	8.0 ± 4.4	8.0 ± 1.7
–/+	1000	12.7 ± 1.5	10.0 ± 2.6
–/+	5000	8.7 ± 4.0	11.0 ± 4.0
Positive controls, –S9	Name	AM	2AA
	Concentrations (μg/plate)	0.5	0.2
	Mean No. of colonies/plate (average of 2 ± SD)	67.0 ± 9.9	70.5 ± 2.1
	Concentrations (μg/plate)	1	0.5
	Mean No. of colonies/plate (average of 2 ± SD)	82.5 ± 6.4	141.0 ± 8.5
	Concentrations (μg/plate)	2	1
	Mean No. of colonies/plate (average of 2 ± SD)	123.0 ± 2.8	402.0 ± 1.4

AM: Acridine mutagen

2AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) tested with and without metabolic activation.