Pyrolytic sugar

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:

2020-2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,6-anhydro-β-D-glucose

EC Number:

207-855-0

EC Name:

1,6-anhydro-β-D-glucose

Cas Number:

498-07-7

Molecular formula:

C6H10O5

IUPAC Name:

(1R,2S,3S,4R,5R)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol

Constituent 2

Reference substance name:

Glycollaldehyde

EC Number:

205-484-9

EC Name:

Glycollaldehyde

Cas Number:

141-46-8

Molecular formula:

C2H4O2

IUPAC Name:

2-hydroxyacetaldehyde

Constituent 3

Reference substance name:

Organic acids

IUPAC Name:

Organic acids

Constituent 4

Reference substance name:

Ketones

IUPAC Name:

Ketones

Constituent 5

Reference substance name:

Phenols

IUPAC Name:

Phenols

Constituent 6

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Constituent 7

Reference substance name:

Oligomers of sugars and anhydrosugars

IUPAC Name:

Oligomers of sugars and anhydrosugars

Test material form:

liquid: viscous

Method

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:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH, Gießen
- method of preparation of S9 mix: Produced from the livers of male Sprague-Dawley rats which were treated with Phenobarbital/5,6-Benzoflavone
- concentration or volume of S9 mix and S9 in the final culture medium: S9-mix - Phosphate buffer 22.5 mL; 0.1M NADP-solution 1.0 mL; 1M G6P-solution 0.125 mL; Salt solution 0.5 mL; Rat liver S9 1.0 mL.

Test concentrations with justification for top dose:

Experiment 1: 5, 1.5, 0.5, 0.15, 0.05 µL/plate
Experiment 1b: 5, 1.5, 0.5, 0.15, 0.05, 0.015, 0.005 µL/plate
- TA 102, was not tested in Experiment 1.
- For TA100 (+/-S9) experiment 1 was invalid, because the values of spontaneous re-vertants of the solvent controls demin. water and DMSO did not meet the historical control data range.

Top dose of 5 µL/plate as recommended on the OECD Guideline

Two valid experiments were performed; the initial experiment had to be repeated with additional lower concentrations due to an insufficient number of analyzable non-toxic concentrations as indicated in the guideline.
For TA100 (+/-S9) experiment 1 was invalid, because the values of spontaneous revertants of the solvent controls demin. water and DMSO did not meet the historical control data range.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-Amino-anthracene 4-Nitro-1,2-phenylenediamine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of replicates: 3 replicates per bacteria strain with (+S9) and 3 replicates per bacteria strain without metabolic activation(-S9)
- Number of independent experiments: 2

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48h

Rationale for test conditions:

The test was conducted in compliance with the following guideline(s):
- OECD Guidelines for the Testing of Chemicals Part 471, adopted 26. Jun. 2020 “Bacterial Reverse Mutation Test“
- Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”

Evaluation criteria:

A result is considered as positive if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the concentrations occurs in at least one tested strain with or without metabolic activation.
A biologically relevant increase is described as follows:
-  if in the bacteria strainsS. typhimuriumTA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
-  if in the bacteria strainsS. typhimuriumTA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

A substance is not mutagenic if it does not meet the criteria above.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.

Statistics:

No statistical analysis was conducted for this test

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At concentration 5 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At concentration 5 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At concentration 5 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At concentration 5 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At concentration 5 µL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Mean Revertants Experiment 1

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	12	14	iv	iv	-	-	6	7	7	4
	sd	1.0	1.5	iv	iv	-	-	1.0	2.1	0.0	1.5
DMSO	Mean	9	16	iv	iv	-	-	9	6	5	5
	sd	1.0	2.6	iv	iv	-	-	2.1	1.0	2.0	1.7
Positive Controls*	Mean	576	128	iv	iv	-	-	309	297	59	17
	sd	16.0	7.6	iv	iv	-	-	14.0	8.3	1.0	6.0
	f(I)	64.00	8.00	iv	iv	-	-	51.50	49.50	11.80	3.40
5 µL/plate	Mean	0	0	iv	iv	-	-	0	0	0	0
	sd	0.0	0.0	iv	iv	-	-	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	iv	iv	-	-	0.00	0.00	0.00	0.00
1.5 µL/plate	Mean	15	24	iv	iv	-	-	18	23	7	5
	sd	2.5	1.5	iv	iv	-	-	4.9	1.0	1.2	1.5
	f(I)	1.67	1.50	iv	iv	-	-	2.00	3.83	1.40	1.00
0.5 µL/plate	Mean	12	19	iv	iv	-	-	14	13	4	3
	sd	1.2	2.0	iv	iv	-	-	3.8	3.6	2.5	0.6
	f(I)	1.33	1.19	iv	iv	-	-	1.56	2.17	0.80	0.60
0.15 µL/plate	Mean	13	21	iv	iv	-	-	15	9	5	4
	sd	0.6	1.5	iv	iv	-	-	3.6	3.5	1.5	3.0
	f(I)	1.44	1.31	iv	iv	-	-	1.67	1.50	1.00	0.80
0.05 µL/plate	Mean	14	26	iv	iv	-	-	9	11	3	3
	sd	1.2	4.0	iv	iv	-	-	2.6	1.5	1.5	1.5
	f(I)	1.56	1.63	iv	iv	-	-	1.00	1.83	0.60	0.60

sd = standard deviation±

* Different positive controls were used, see table under 'Any other information on materials and methods incl. tables' section

s.g.= strong growth, too strong for counting of revertants

n.c. = not calculable

f(I) = increase factor, calculation [mean revertants divided by mean spontaneous revertants]

iv = invalid

- = not tested

Mean Revertants Experiment 1b

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	19	22	73	82	260	247	5	6	3	6
	sd	3.1	1.5	7.0	5.7	18.3	12.2	1.5	2.5	2.5	0.6
DMSO	Mean	14	16	74	76	247	243	6	5	3	3
	sd	1.5	3.0	13.6	13.7	15.1	22.0	1.5	1.5	1.5	2.1
0.9% NaCl	Mean	-	-	-	-	245	-	-	-	-	-
	sd	-	-	-	-	8.3	-	-	-	-	-
Positive Controls*	Mean	601	149	573	2003	581	737	356	145	33	88
	sd	28.1	4.2	31.1	164.2	24.4	31.1	22.3	10.0	14.2	7.2
	f(I)	42.93	9.31	7.85	26.36	2.37	3.03	71.20	29.00	11.00	29.33
5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1.5 µL/plate	Mean	16	18	185	187	239	256	13	19	0	4
	sd	3.6	4.9	4.2	5.3	4.6	14.4	9.2	6.1	0.6	2.5
	f(I)	1.14	1.13	2.50	2.46	0.97	1.05	2.17	3.80	0.00	1.33
0.5 µL/plate	Mean	16	24	124	125	231	251	10	12	3	3
	sd	3.2	5.7	13.0	7.0	4.6	18.0	3.6	0.6	2.5	1.5
	f(I)	1.14	1.50	1.68	1.64	0.94	1.03	1.67	2.40	1.00	1.00
0.15 µL/plate	Mean	11	19	101	91	240	239	8	11	4	4
	sd	2.3	5.6	12.5	4.0	17.4	11.5	2.5	4.0	2.1	2.1
	f(I)	0.79	1.19	1.36	1.20	0.97	0.98	1.33	2.20	1.33	1.33
0.05 µL/plate	Mean	13	22	79	83	229	236	10	7	3	5
	sd	2.9	1.5	2.5	8.2	14.0	16.0	3.5	2.0	1.5	0.0
	f(I)	0.93	1.38	1.07	1.09	0.93	0.97	1.67	1.40	1.00	1.67
0.015 µL/plate	Mean	13	20	80	72	237	225	8	3	2	4
	sd	1.2	3.5	10.7	5.6	12.2	6.1	2.5	1.2	2.3	3.2
	f(I)	0.93	1.25	1.08	0.95	0.96	0.93	1.33	0.60	0.67	1.33
0.005 µL/plate	Mean	11	17	85	76	236	247	7	7	2	7
	sd	0.6	2.6	5.9	2.1	4.0	8.3	0.6	4.6	2.1	1.5
	f(I)	0.79	1.06	1.15	1.00	0.96	1.02	1.17	1.40	0.67	2.33

sd = standard deviation±

* Different positive controls were used, see table under 'Any other information on materials and methods incl. tables' section

s.g.= strong growth, too strong for counting of revertants

n.c. = not calculable

f(I) = increase factor, calculation [mean revertants divided by mean spontaneous revertants]

- = not tested

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium strains TA100 in the presence and absence of metabolic activation and TA1535 in the presence metabolic activation under the experimental conditions in this study.

Executive summary:

The test item was tested in the Bacterial reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). The study procedures described in this report were based on the most recent Guideline OECD 471 (2020) and EU Method B.13/14 (2008). The test was performed with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation. Three valid experiments were performed; the initial experiment had to be repeated with additional lower concentrations due to an insufficient number of analyzable non-toxic concentrations as indicated in the guideline. It was concluded that Pyrolytic Sugar is mutagenic in the Salmonella typhimuriumstrains TA100 in the presence and absence of metabolic activation and TA1535 in the presence of metabolic activation.