1-O-β-(2 ,3-di-O-alka(e)noyl-6 -O-acetyl-D-mannopyranosyl)-D-erythritol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay: Negative (Non-mutagenic); OECD 471; T. Morinaga. (2018)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 March - 30 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted under GLP in accordance with the international guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

S9 mix

Test concentrations with justification for top dose:

Doses selected for the mutagenicity assay was based on data generated in preliminary study (Doses of 0.305, 1.22,4.88, 19.5, 78.1, 313, 1250 and 5000 μg per plate) Based on the outcome of the range finder study in which bacterial growth inhibition was observed at the lowest dose was used as high dose, the doses for the main test were as follows; In the absence of S9 mix:
0, 9.77, 19.5, 39.1, 78.1, 156 & 313 μg per plate for TA100 & TA1537, 39.1, 78.1, 156, 313, 625 & 1250 μg per plate for TA98 & TA1535 and 156, 313, 625, 1250 & 5000 μg per plate for WP2 uvrA. In the present of S9 mix:
39.1, 78.1,156, 313, 625 & 1250 μg per plate for TA100 & TA1535 and 156, 313, 625, 1250 & 5000 μg per plate for TA98, TA1535 & WP2 uvrA.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate
- Number of independent experiments: Preincubation method

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Yes
- Test substance added in medium; preincubation: 45 mL of each tester strain was directly inoculated into 20 mL 0f 2.5% nutrient broth (Nutrient broth No. 2, Oxoid). The resulting mixture was subject to gyratory culture at 50rpm for 10h at 37°C. After incubation, the optical density of the bacteria suspension was measured to confirm that the viable bacterial count was not less than 1.0x10*9/mL using conversion table.
For tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or the negative control solution. For the test with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of 0.1 M Na-phosphate buffer. This was followed by pre-incubation with shaking at 37°C for the 20 mins, then 2.0mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate. And 0.1 mL of positive control solution was carried out equally.
For sterility test, 0.1 mL of test solution of the maximum concentration and 0.5 mL of S9 mix were added to each tube followed by 2.0 mL agar then transferred to surface of minimal glucose agar plate.
These preparations took place over UV absorbent filter.
As top agar, 0.5 mM biotin-0.5 mM L-histidine solution and 0.5 mM l-tryptophan solution were added to the safe agar solution (0.6% agar, 0.5% NaCl) by volume of 1/10 for TA and E.Coli strains respectively.
All plates were incubated at 37°C for 48 hrs, the number of revertant colonies were counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 mins
- Exposure duration/duration of treatment: 48 hours at 37°C
- Harvest time after the end of treatment (sampling/recovery times): not stated

Rationale for test conditions:

The study was based on the in vitro (pre-incubation) technique described by Ames et al (1975), Maron and Ames (1983), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence is used to complement the Salmonella strains.

Evaluation criteria:

To ensure the validity of the study, the following conditions should be met:
1) Colonies on the plate should be countable without contamination of the test plate or other unexpected events.
2) The number of revertant colonies in the negative control and positive control groups should be within the range of the background data (mean +- 3 standard deviations) of the Shiga laboratory, NISSEI BILIS Co., Ltd. Moreover, the number of revertant colonies in each positive control group should be at least twice higher in each negative control.
3) The number of doses without bacterial growth inhibition should be four or more.
4) No contamination should be observed in the sterility test.

For the number of revertant colonies on each plate, the mean values (rounded to integers) in each dose group are obtained. The case where all the following criteria are met, the results are considered to be positive;
1) The number of revertant colonies in the test article group is equal to or greater than twice the mean negative control value.
2) Dose-dependency is observed.
3) Reproducibility was obtained by the dose-finding study and main study.

Statistics:

Not stated

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium 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

Key result

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: 
- Data on pH:Not specified
- Data on osmolality:Not specified
- Possibility of evaporation from medium:Not specified
- Water solubility:Not specified
- Precipitation and time of the determination: not observed.
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no


RANGE-FINDING/SCREENING STUDIES (if applicable): Yes

Grrowth inhibition was observed at 313 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 in E. coli WP2 uvr A without metabolic activation. In presence of of metabolic activation growth inhibition observed at 1250 μg per plate or more in TA100 and 1535 and at 5000 in TA1537. No precipitation was also observed with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Both within historical control range

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: No increase in revertant were observed.
- Statistical analysis; not stated
- Any other criteria: not stated

Ames test:
- Signs of toxicity: Toxicity was observed beginning at 650 μg per plate in all test conditions
- Individual plate counts: yes

Remarks on result:

other: No mutagenicity

Table 1. Result of the Dose-finding study

Experimental Period: March 2 – 5^th2009
Metabolic activation	Dose of test item μg per plate	Number of revertant colonies/plate
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
S9 mix (-)	Negative control (DMSO)	100	9	22	11	7
	0.305	92	11	19	14	11
	1.22	105	9	22	14	8
	4.88	96	8	20	17	7
	19.5	117	7	18	14	12
	78.1	106	10	16	13	8
	313	78*	8	24	19	2*
	1250	65*	4*	20	18*	3*
	5000	58*	5*	14*	14*	2*
S9 mix (+)	Negative control (DMSO)	102	7	21	22	16
	0.305	109	9	26	31	21
	1.22	105	11	19	26	11
	4.88	98	8	17	18	11
	19.5	96	11	25	27	14
	78.1	102	6	28	21	13
	313	97*	8	26	23	9
	1250	68*	6*	24	24	9
	5000	44*	8*	20	23	7*
Positive control (-S9)	Name	ENNG	ENNG	ENNG	2-NF	9-AAc
	μg per plate	3	5	2	1	80
	No. of revertant colonies/plate	620	845	801	461	267
Positive control (+S9)	Name	2-AAn	2-AAn	2-AAn	2-AAn	2-AAn
	μg per plate	1	2	10	0.5	2
	No. of revertant colonies/plate	810	309	285	542	229

2-AAn: 2-aminoanthracnen

9-AAc: 9-aminoacridine

2-NF: 2-nitroflurene

ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine

*: Bacterial growth inhibition observed

Table 2. Result of the main study.

Experimental Period: March 10 – 13^th2009
Metabolic activation	Dose of test item μg per plate	Number of revertant colonies/plate
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2uvrA	TA98	TA1537
S9 Mix (-)	Negative control (DMSO)	100	11	24	21	8
	9.77	115				10
	19.1	114				9
	39.1	103	9		14	8
	78.1	110	9		19	7
	156	74*	10	26	16	5*
	313	67*	10	24	16	3*
	625		8	26	17
	1250		7*	22	18*
	2500			26
	5000			18*
S9 Mix (+)	Negative control (DMSO	104	9	27	25	17
	39.1	106	9
	78.1	113	8
	156	112	12	20	28	14
	313	101	11	21	32	18
	625	78*	6*	34	30	15
	1250	63*	4*	20	31	12
	2500			29	33	6*
	5000			20	28	8*
Positive control (-S9)	Name	ENNG	ENNG	ENNG	2-NF	9-AAc
	μg per plate	3	5	2	1	80
	No. of revertant colonies/plate	655	827	914	432	391
Positive control (+S9)	Name	2-AAn	2-AAn	2-AAn	2-AAn	2-AAn
	μg per plate	1	2	10	0.5	2
	No. of revertant colonies/plate	793	279	419	480	189

2-AAn: 2-aminoanthracnen

9-AAc: 9-aminoacridine

2-NF: 2-nitroflurene

ENNG: N-ethyl-N’-nitro-N-nitrosoguanidine

*: Bacterial growth inhibition observed

Conclusions:

Based on the condition of the study, the test item did not induce mutagenicity in bacterial reverse mutation assay with or without metabolic activation.

Executive summary:

OECD 471 ( 2011): The test item was tested to evaluate its mutagenic potential using the pre-incubation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Doses selected for the mutagenicity assay was based on data generated in preliminary study (Doses of 0.305, 1.22,4.88, 19.5, 78.1, 313, 1250 and 5000 μg per plate). Based on the outcome of the range finder study in which bacterial growth inhibition was observed the lowest dose inducing bacterial growth inhibition among the doses with growth inhibition was settled as the highest dose in the main study. The remaining five doses were gradually decreased at the common ratio of two, and a total of six does were set. For the tester strains in which bacterial growth inhibition was not observed, 5000 μg per plate was set as the highest dose in accordance with the guideline. The remaining five doses were gradually decreased at the common ratio of two, and a total of six does up to 156 μg per plate . Therefore,  the doses for the main test were as follows;

In the absence of S9 mix:

0, 9.77, 19,5, 39.1, 78.1, 156 & 313 μg per plate for TA100 & TA1537, 39.1, 78.1, 156, 313, 625 & 1250 μg per plate for TA98 & TA1535 and 156, 313, 625, 1250, 2500 & 5000 μg per plate for WP2 uvrA.  

In the presence of S9 mix:

39.1, 78.1,156, 313, 625 & 1250 μg per plate for TA100 & TA1535 and 156, 313, 625, 1250, 2500 & 5000 μg per plate for TA98, TA1537 & WP2 uvrA.  

All criteria for a valid study were met as described in the protocol. There was no increase in the number of revertant colonies observed in both preliminary and main study. From the preliminary study, growth inhibition was observed at 313 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 in E. coli WP2 uvr A without metabolic activation. In presence of metabolic activation growth inhibition observed at 1250 μg per plate or more in TA100 and 1535 and at 5000 in TA1537. No precipitation was also observed with and without metabolic activation.

From the main study, growth inhibition was observed at 156 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 μg per plate in E. coli WP2 uvr A without metabolic activation. In presence of metabolic activation growth inhibition observed at 625 μg per plate or more in TA100 and TA1535 and at 2500 μg per plate in TA1537. No precipitation was also observed with and without metabolic activation.

No dose dependent increases were observed in all tester strains with or without metabolic activation. The test item was negative for mutagenicity in bacterial reverse mutation assay with or without metabolic activation.

Under the conditions of this study, test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The test item is not mutagenic and therefore no mode of action is required.

Additional information

OECD 471 ( 2011): The test item was tested to evaluate its mutagenic potential using the pre-incubation method by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

Doses selected for the mutagenicity assay was based on data generated in preliminary study (Doses of 0.305, 1.22,4.88, 19.5, 78.1, 313, 1250 and 5000 μg per plate). Based on the outcome of the range finder study in which bacterial growth inhibition was observed the lowest dose inducing bacterial growth inhibition among the doses with growth inhibition was settled as the highest dose in the main study. The remaining five doses were gradually decreased at the common ratio of two, and a total of six does were set. For the tester strains in which bacterial growth inhibition was not observed, 5000 μg per plate was set as the highest dose in accordance with the guideline. The remaining five doses were gradually decreased at the common ratio of two, and a total of six does up to 156 μg per plate . Therefore,  the doses for the main test were as follows;

In the absence of S9 mix:

0, 9.77, 19,5, 39.1, 78.1, 156 & 313 μg per plate for TA100 & TA1537, 39.1, 78.1, 156, 313, 625 & 1250 μg per plate for TA98 & TA1535 and 156, 313, 625, 1250, 2500 & 5000 μg per plate for WP2 uvrA.  

In the present of S9 mix:

39.1, 78.1,156, 313, 625 & 1250 μg per plate for TA100 & TA1535 and 156, 313, 625, 1250, 2500 & 5000 μg per plate for TA98, TA1537 & WP2 uvrA.  

All criteria for a valid study were met as described in the protocol. There was no increase in the number of revertant colonies observed in both preliminary and main study. From the preliminary study, growth inhibition was observed at 313 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 in E. coli WP2 uvr A without metabolic activation. In presence of metabolic activation growth inhibition observed at 1250 μg per plate or more in TA100 and 1535 and at 5000 in TA1537. No precipitation was also observed with and without metabolic activation.

From the main study, growth inhibition was observed at 156 μg per plate or more in all TA100 & 1537 strains, 1250 μg per plate in TA1535, TA98 and 5000 μg per plate in E. coli WP2 uvr A without metabolic activation. In presence of metabolic activation growth inhibition observed at 625 μg per plate or more in TA100 and TA1535 and at 2500 μg per plate in TA1537. No precipitation was also observed with and without metabolic activation.

No dose dependent increases were observed in all tester strains with or without metabolic activation. The test item was negative for mutagenicity in bacterial reverse mutation assay with or without metabolic activation.

Under the conditions of this study, test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Justification for classification or non-classification

The test item did not meet the criteria for classification for mutagenicity in accordance with Globally Harmonized Classification System or the Regulation (EC) No. 1272/2008; relating to the Classification, Labelling and Packaging of Substances and Mixtures.