Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (OECD 471): negative

Gene mutation in mammalian cells (HPRT, OECD 476): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Nov to 19 Nov 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Authority (VWA), den Haag, The Netherlands

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon and trp operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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:

all strains: 62, 185, 556, 1667, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: milli-Q water

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

ethylnitrosurea

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 10E8 to 10E9

DURATION
- Preincubation period: 10 -16 h
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 replications in one experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control

Evaluation criteria:

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Statistics:

No statistical analysis was performed.

Key result

Species / strain:

S. typhimurium, other: T 1535, TA 1537, TA 98, 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:

E. coli WP2 uvr A

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

Table 3. Number of revertants counted in bacterial reverse mutation test

		TA 1535		TA 1537		TA98		TA 100		E. Coli
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0 µg/plate		32	16	16	23	31	60	185	217	46	34
		26	10	6	6	37	60	163	205	46	38
		13	31	13	24	26	61	145	226	34	32
	Mean	24	19	12	18	31	60	164	216	42	35
	StDev	10	11	5	10	6	1	20	11	7	3
62 µg/plate		18	28	16	20	32	58	150	159	44	46
		17	17	17	16	32	47	198	180	35	35
		23	14	14	22	20	50	291	157	44	41
	Mean	19	20	16	19	28	52	213	165	41	41
	StDev	3	7	2	3	7	6	72	13	5	6
185 µg/plate		19	24	8	22	29	44	143	169	42	44
		30	20	14	17	38	42	136	162	37	32
		20	23	13	13	38	40	269	161	35	30
	Mean	23	22	12	17	35	42	183	164	38	35
	StDev	6	2	3	5	5	2	75	4	4	8
556 µg/plate		16	16	8	19	35	43	***	133	30	32
		18	18	16	19	29	***	141	130	38	41
		14	13	19	23	22	56	174	121	25	40
	Mean	16	16	22	20	29	50	158	128	31	38
	StDev	2	3	15	2	7	9	23	6	7	5
1667 µg/plate		13	24	13	8	24	40	129	144	34	22
		10	13	5	20	23	55	143	150	34	25
		7	6	8	***	34	54	154	137	43	52
	Mean	10	14	9	14	27	50	142	144	37	33
	StDev	3	9	4	8	6	8	13	7	5	17
5000 µg/plate		12	16	7	14	38	46	127	136	32	38
		29	20	11	16	29	50	136	117	32	25
		19	14	8	28	23	47	129	106	29	35
	Mean	20	17	9	19	30	48	131	120	31	33
	StDev	9	3	2	8	8	2	5	15	2	7
Pos. control		659	558	4339	342	956	1673	674	1494	323	1784
		659	609	3154	264	688	1441	709	1565	307	1613
		620	519	3881	383	1261	1238	641	1719	358	1533
	Mean	646	562	3791	330	968	1451	675	1593	329	1643
	StDev	23	45	598	60	287	218	34	115	26	128

*** plate not counted due to contamination

Conclusions:

Based on the results of the conducted study the test item did not exhibit mutagenic properties in bacterial cells.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jan - 18 May 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted in 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

less then 2x10E6 cells cultured during expression

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

adopted in 1991

GLP compliance:

yes (incl. QA statement)

Remarks:

Staatstoezicht op de Volksgezondheid, Veterinaire Hoofdinspectie, Rijswijk, The Netherlands

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

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:

First experiment: 39, 78, 157, 313, 625, 1250, 2500 and 5000 µg/mL with and without metabolic activation
Second experiment: 313, 625, 1250, 2500 and 5000 µg/mL with and without metabolic activation

Vehicle / solvent:

Ham's F-12 growth medium (without serum)

Untreated negative controls:

yes

Remarks:

culture treated with growth medium

Negative solvent / vehicle controls:

yes

Remarks:

Ham's F-12 growth medium without serum

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 x 10E6

DURATION
- Preincubation period: 20 h
- Exposure duration: 4 h with and without metabolic activation
- Expression time (cells in growth medium): 8 d
- Selection time 8 d
- Fixation time: 16 d

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 3 (the second assay was discontinued due to bacterial infection)

STAINING TECHNIQUE USED: not specified

NUMBER OF CELLS EVALUATED: 10E6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Colonies were counted automatically using an Artek colony counter model 928B.

Evaluation criteria:

- a concentration-related increase in mutant frequency,
- a reproducible positive response for at least one of the test substance concentrations (e.g. the mean mutant frequency should be more than 20 mutants per 1,000 ,000 clonable cells (final survival))
- both numerical and biological significance were considered together in the evaluation.

Statistics:

The absolute percentage initial survival and the absolute percentage final survival of the cells were calculated from the mean number of colonies on the Petri dishes and the number of cells seeded per dish.

Two criteria were used to assess the initial survival of the cells, i.e. the mean relative cell yield after the recovery period (expressed as percentage of the yield in the unexposed cell population obtained with the vehicle control), and the mean relative and absolute cloning efficiency of these cells.

The absolute percentage final survival of the cells was used, together with the mean number of mutant colonies recovered, to calculate the mutant frequency. The mutant frequency was expressed as the number of 6-TG resistant mutants per 1,000,000 clonable (final survival) cells.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1.Summary results of the first mutation assay without metabolic activation

Treatment	Dose	Mean relative initial yield (1)	Mean relative initial cloning efficiency (1)	Mean absolute initial cloning efficiency (2)	Mean absolute final cloning efficiency (2)	Mean number of mutant colonies/dish	Mutant frequency per 10E6 clonable cells (3)
	(mg/mL)	(%)	(%)	(%)	(%)
control	+ FCS	108.9	89.5	81.8	96.0	4.1	21.4
solvent	- FCS	100.0	100.0	91.4	95.8	2.0	10.4
test substance	0.039	108.8	80.4	73.5	95.8	0.9	4.7
	0.078	100.9	94.5	86.4	88.1	1.6	9.1
	0.157	96.5	92.0	84.1	93.4	3.7	19.8
	0.313	98.1	99.2	90.7	85.1	1.4	8.2
	0.625	91.5	93.3	85.3	92.5	3.8	20.5
	1.250	106.5	101.9	93. 1	101.7	1.3	6.4
	2.500	111.6	94.7	86.6	93.4	3.8	20.3
	5.000	113.1	94.0	85.9	106.8	2.5	11.7
EMS	0.500	91.4	65.9	60.2	87.2	54.7	313.6

 FCS = Fetal calf serum

(1) relative mean values are given as percentage of the vehicle control - FCS (100%)

(2) absolute mean values are given as percentage of the number of cells seeded for analysis

(3) mutant frequency per 10E6 clonable cells = mean number of mutant colonies/dish divided by mean absolute final cloning efficiency multiplied with 5

 

 

Table 2. Summary results of the first mutation assay with metabolic activation

Treatment	Dose	Mean relative initial yield (1)	Mean relative initial cloning efficiency (1)	Mean absolute initial cloning efficiency (2)	Mean absolute final cloning efficiency (2)	Mean number of mutant colonies/dish	Mutant frequency per 10E6 clonable cells (3)
	(mg/mL)	(%)	(%)	(%)	(%)
control	+ FCS	90.3	117.2	100.7	109.5	2.6	11.9
solvent	- FCS	100.0	100.0	85.9	107.7	2.5	11.6
test substance	0.039	106.1	99.7	85.6	105.9	1.7	8.0
	0.078	100.0	110.1	94.6	11 1.3	1.4	6.3
	0.157	83.7	96.3	82.7	101.6	1.3	6.4
	0.313	92.9	105.2	90 .4	96.6	1.1	5.7
	0.625	89.9	109.1	93.7	89.1	2.5	14.0
	1.250	95.4	113.2	97.2	95.0	0.7	3.7
	2.500	105.1	105.9	91.0	94.7	1.7	9.0
	5.000	94.2	95.9	82.4	98.4	1.7	8.6
DMBA	0.020	68.2	63.7	54.7	85.9	29.9	174.0

 

FCS = Fetal calf serum

(1) relative mean values are given as percentage of the vehicle control - FCS (100%)

(2) absolute mean values are given as percentage of the number of cells seeded for analysis

(3) mutant frequency per 10E6 clonable cells = mean number of mutant colonies/dish divided by mean absolute final cloning efficiency multiplied with 5

 

 

Table 3. Summary results of the second mutation assay without metabolic activation

Treatment	Dose	Mean relative initial yield (1)	Mean relative initial cloning efficiency (1)	Mean absolute initial cloning efficiency (2)	Mean absolute final cloning efficiency (2)	Mean number of mutant colonies/dish	Mutant frequency per 10E6 clonable cells (3)
	(mg/mL)	(%)	(%)	(%)	(%)
control	+ FCS	119.6	87.8	89.4	86.7	2.6	15.0
solvent	- FCS	100.0	100.0	101.8	86.0	3.8	22.1
test substance	0.313	108.1	86.1	87.6	81.8	0.5	3.1
	0.625	106.0	103.0	104.9	84.7	0.9	5.3
	1.250	107.6	100.6	102.4	88.2	0.9	5.1
	2.500	105.4	95.1	96.8	86.6	1.4	8.1
	5.000	104.0	98.0	99.8	92.2	3.0	16.3
EMS	0.500	106.7	63.8	64.9	72.8	36.2	248.6

 

FCS = Fetal calf serum

(1) relative mean values are given as percentage of the vehicle control - FCS (100%)

(2) absolute mean values are given as percentage of the number of cells seeded for analysis

(3) mutant frequency per 10E6 clonable cells = mean number of mutant colonies/dish divided by mean absolute final cloning efficiency multiplied with 5

 

Table 4. Summary results of the second mutation assay with metabolic activation

Treatment	Dose	Mean relative initial yield (1)	Mean relative initial cloning efficiency (1)	Mean absolute initial cloning efficiency (2)	Mean absolute final cloning efficiency (2)	Mean number of mutant colonies/dish	Mutant frequency per 10E6 clonable cells (3)
	(mg/mL)	(%)	(%)	(%)	(%)
control	+ FCS	95.2	98.8	101.5	82.6	4.3	26.0
sovent	- FCS	100.0	100.0	102.7	99.2	2.7	13.6
test substance	0.313	113.9	90.4	92.8	93.4	4.1	21.9
	0.625	96.6	78.3	80.4	87.7	4.5	25.7
	1.250	110.6	101.9	104.7	92.0	1.3	7.1
	2.500	113.1	94.5	97.1	101.3	6.5	32.1
	5.000	113.6	93.6	96.1	99.1	2.1	10.6
DMBA	0.020	57.3	32.0	32.9	81.6	50.4	308.4

 

FCS = Fetal calf serum

(1) relative mean values are given as percentage of the vehicle control - FCS (100%)

(2) absolute mean values are given as percentage of the number of cells seeded for analysis

(3) mutant frequency per 10E6 clonable cells = mean number of mutant colonies/dish divided by mean absolute final cloning efficiency multiplied with 5

 

Conclusions:

Based on the results of the conducted study the test item did not exhibit mutagenic properties in mammalian cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test in somatic cells, rat (OECD 474): negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Oct 2000 to 1 Feb 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

guideline study but no final report available (draft report available)

Reason / purpose for cross-reference:

reference to other study

Remarks:

reference to concurrent repeated dose toxicity study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

1000 instead of 4000 erythrocytes counted

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 1992

Principles of method if other than guideline:

A concurrent control group of 10 rats/sex was kept on basal diet supplemented with 10% pregelatinized wheat starch.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspectorate for Health Protection, Commodities and Veterinary Public Health, Ministry of Health, Welfare and Sport, The Haque, The Netherlands

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI)WU BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 135 - 191 g (males) and 109 - 135 g (females)
- Fasting period before study: no
- Housing: individually in suspended stainless steel cages (h x l x w = 18 x 32 x 18 cm), fitted with wire-mesh floor and front
- Diet: powdered diet: Rat & Mouse No. 3 Breeding Diet, RM3; SDS, Special Diets Services, Witham, England (ad libitum)
- Water: tap water for human consumption (ad libitum)
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
Each batch of diet is analysed by the supplier for nutrients and contaminants (the CoAs of the used batches are attached to the report). Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the CRO.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 50-70
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Oct 2000 To: 26 Jan 2001

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency):
approximately every three weeks (19 Oct 2000, 07 Nov 2000, 07 and 29 Dec 2000)
- Mixing appropriate amounts with: powdered
diet (Rat & Mouse No.3 Breeding Diet, RM3; SDS, Special Diets Services, Witham, England)
- Storage temperature of food: in the freezer (≤ -18 °C) in plastic bags containing portions sufficient to cover the need for 3 - 4 days

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days/week

Post exposure period:

not applicable

Dose / conc.:

10 other: %

Remarks:

100 000 ppm (corresponding to ca. 7000 and 8400 mg/kg bw/day in males and females, respectively)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes

Positive control(s):

Mitomycin C (administered once in week 14, 24 h before collection of bone marrow cells)
- Route of administration: intraperitonreally
- Dose: 1.5 mg/kg bw
- Other: positive control rats were kept on purified control diet from the start of the 13-week treatment period

Tissues and cell types examined:

femural bone marrow cells, numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 1000 erythrocytes/animal

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dietary level of 10% was selected as a high level which was previously found to be tolerable without obvious signs of toxicity.

TREATMENT AND SAMPLING TIMES:
test item animals and control animals: week 14, day 92/93/94
positive control animals: week 14, day 91

DETAILS OF SLIDE PREPARATION: At necropsy, bone marrow cells from one of the femurs was immediately collected into foetal calf serum and processed into glass drawn smear according to the method described by Schmidt (1976). Two bone marrow smears per animal were made and air­ dried, fixed in methanol and stained with May-Grünwald Giemsa.

METHOD OF ANALYSIS: Slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 1000 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 1000 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE/animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

The study is considered valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range.

A response is considered positive if the mean number of MPE/2000 PE is statistically significant higher compared to the vehicle control.

A test substance is considered to cause chromosomal damage and/or damage to the mitotic apparatus if a clear dose-related increase in the mean number of MPE/2000 PE compared to the vehicle control is observed, and/or if a single positive dose is observed.

A test substance is considered negative in the micronucleus test if it produces no positive response at any dose level or time point.

The test substance or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test substance reduces the mean number of PE/E, or causes systemic toxicity.

Both statistical significance and biological relevance will be considered in the evaluation.

Statistics:

Data on MPE and PE was subjected to a one way ANOVA with factor group (controls and test item groups) for each sex. If the ANOVA yielded a significant effect (p < 0.05), the ANOVA was followed by pooled error variance t-tests or if variances were inhomogenous, separate variance t-tests. These t-tests were applied to the negative control versus tets item treatments per sex. In addition, the positive control group and the negative control group for each sex, were compared using pooled error variance t-tests or if variances were inhomogenous, separate variance t-tests. All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The incidences of MPE per 2000 PE of the positive control group differed significantly from the vehicle control group (p < 0.001) in both sexes, demonstrating the sensitivity of the system.

The incidences of PE per 1000 E of the positive control females differed significantly from the vehicle control group (p < 0.01) demonstrating the sensitivity of the system.

Table 1. Micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE)

	Group mean ± Standard Dev. numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE)
	starch supplemented control diet	Test item 10%	Mitomycin C (1.5 mg/kg bw)
males	3.0 ± 1.7	1.4 ± 0.5	27.6 ± 4.0***
females	3.0 ± 1.2	3.2 ± 0.8	29.0 ± 6.6***

***p<0.001: two-sided asymptotic t-test, treated versus control

Table 2. Polychromatic erythrocytes (PE) per 1000 erythrocytes (PE)

	Group mean ± Standard Dev. numbers of polychromatic erythrocytes (PE) per 1000 erythrocytes (E)
	starch supplemented control diet	Test item 10%	Mitomycin C (1.5 mg/kg bw)
males	435.0 ± 136.7	387.0 ± 46.8	373.0 ± 26.0
females	465.2 ± 41.6	450.6 ± 39.0	284.8 ± 71.9**

**p<0.01: two-sided asymptotic t-test, treated versus control

Conclusions:

Interpretation of results: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro genetic toxicity

A reliable bacterial gene mutation assay (Ames test) was performed with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol (CAS 64519-82-0) according to OECD 471 and in compliance with GLP (Wijngaard, 2008). The S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were tested using the plate incorporation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 3 replicates in one experiment up to the limit concentration of 5000 µg/plate (vehicle: demineralized water). Neither cytotoxicity nor precipitation of the test item was recorded either in the presence and absence of metabolic activation up to and including 5000 µg/plate. Appropriate positive and solvent controls were included into the test and showed the expected results. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system.

Two furtherbacterial gene mutation assays (Ames tests) were performed with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol according or similar to OECD 471 (Herbold, 1978; Blijleven, 1995) using the S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. In the experiments the test item was tested up to the limit concentration of 5000 µg/plate (vehicle: demineralized water) without causing cytotoxicity or an increase in revertant numbers in the absence or presence of metabolic activation.

Thus, under the conditions of the tests, Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

 

An in vitro Mammalian Cell Gene Mutation Test was performed with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol in Chinese hamster ovary (CHO) cells according to OECD 476 and under GLP (van Delft, 1995). In the first experiment, the cells were treated with the test item at concentrations of 39, 78, 157, 313, 625, 1250, 2500 and 5000 µg/mL µg/mL for 4 hours in the presence or absence of metabolic activation (Aroclor 1254-induced rat liver S9-mix). In the second experiment, test concentrations of 313, 625, 1250, 2500 and 5000 µg/mL were applied with and without metabolic activation for 4 hours. The vehicle (growth medium) controls had acceptable mutant frequency values that were within the normal range for the CHO cell line at the HPRT locus. The positive controls ethylmethanesulfonate (-S9-mix) and dimethylbenzanthracene (+S9-mix) induced marked increases in the mutant frequency indicating the satisfactory performance of the test. No cytotoxicity was observed at any dose level. No significant increase in the mutation frequency at the HPRT locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. Thus, the test substance was considered to be non-mutagenic in CHO cells under the conditions of the test.

 

In vivo genetic toxicity

An in vivo erythrocyte micronucleus test with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol was performed according to OECD 474 and in compliance with GLP (de Vogel, 2001). The assay was part of a 90-day dietary repeated dose toxicity study (Jonker and Lina, 2001). The test item was administered orally via the feed to 5 Wistar rats of each sex. A concurrent control group of 5 rats/sex was kept on basal diet supplemented with 10% pregelatinized wheat starch. The test item was administered in the feed at a level of 10% (100 000 ppm, dry matter) for 13 consecutive weeks. This level was equivalent to an overall test item intake of approximately 7000 and 8400 mg/kg bw/day in males and females, respectively. The positive control substance (mitomycin C, 1.5 mg/kg bw, i.p.) was administered once to animals previously kept on standard diet. Bone marrow cells were freshly isolated at necropsy following at least 13 weeks of administration of the test item and 24 hours post-application of mitomycin C. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes or polychromatic erythrocytes compared to the control values were seen in either sex. The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen. Based on the results of the study the test item is negative for the induction of micronuclei under the conditions of the test.

Justification for classification or non-classification

Based on the available data, there is no indication that Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol has any mutagenic or clastogenic potential in vitro and in vivo. The available data on genetic toxicity are therefore conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008.