Undecyl glucoside

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:

04 Jan - 22 Feb 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study. Due to cytotoxicity more marked with pre-incubation, of the six tested doses, only four instead of 5 doses were analysable in the second assay in tester strain TA100 (+S9) as recommended by OECD guideline 471.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

Groupe interministeriel des produits chimiques, Paris, France

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Undecyl glucoside

EC Number:

308-766-0

EC Name:

Undecyl glucoside

Cas Number:

98283-67-1

Molecular formula:

(C6H10O5)nC11H24O, n - number of D-glucopyranose units

IUPAC Name:

(2R,3S,4S,5R)-2-(hydroxymethyl)-6-(undecyloxy)oxane-3,4,5-triol

Test material form:

other: solid

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 1535, TA 1537, TA 98 and TA 100 carry a mutation of the uvr B gene and the deep rough mutation (rfa), TA100 and TA98 contain the R-factor plasmid (pkM101). TA 102 carries the deep rough mutation (rfa) and contains the R-factor plasmid (pAQ1)

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:

Preliminary toxicity test:
- 50, 150, 500, 1500, 5000 µg/plate (with and without metabolic activation)
Experiment I:
- 5, 15, 50, 150, 500, 1000 µg/plate (without metabolic activation; TA 1535, TA 1537, TA 100 and TA 102)
- 5, 15, 50, 150, 500, 1500 µg/plate (without metabolic activation, TA 98)
- 15, 50, 150, 500, 1500, 3000 µg/plate (with metabolic activation, all tester strains)
Experiment II:
- 5, 15, 50, 150, 500, 1000 µg/plate (without metabolic activation, TA 1535)
- 1.5, 5, 15, 50, 150, 500 µg/plate (without metabolic activation, TA 1537)
- 5, 15, 50, 150, 500, 750 µg/plate (without metabolic activation, TA 98, TA 100 and TA 102)
- 5, 15, 50, 150, 500, 1500 µg/plate (with metabolic activation, TA 98, TA 102 and TA 1535, pre-incubation method)
- 5, 15, 50, 150, 500, 1000 µg/plate (with metabolic activation, TA 100 and TA 1537, pre-incubation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: The vehicle was chosen due to the solubility properties of the test item.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9-mix: sodium azide (TA 1535, TA 100), 9-amino-acridine (TA 1537), 2-nitro fluorene (TA 98), mitomycin c (TA 102); +S9-mix: 2-anthramine (TA 1535, TA 1537, TA 98, TA 100), benzo[a]pyrene (TA 102)

Remarks:

sodium azide (1 µg/plate), 9-amino-acridine (50 µg/plate), 2-nitro fluorene (2 µg/plate), mitomycin c (0.125 µg/plate), 2-anthramine (2/1 µg/plate without/with pre-incubation), benzo[a]pyrene (2 µg/plate without/with pre-incubation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; pre-incubation (experiment II with metabolic activation)

DURATION
- Pre-incubation period: 60 minutes

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: In order to choose the range of doses for the test, the toxic activity of the test item was determined. The toxicity assay was carried out in all the strains to be tested under the same conditions as the mutagenicity test with and without metabolic activation but using only one plate per dose instead of 3. The plates were incubated for approximately 44 hours at ca. 37°C, and the revertants were counted. The maximum dose in the preliminary toxicity assay was the maximum dose according to OECD Guideline 471 e.g. 5000 μg/plate. Toxicity was checked by microscopic examination of the background growth.

Evaluation criteria:

A test system is considered as mutagenic if
- a test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 3 times the value for the solvent control, is considered positive in the assay (TA 1535 and TA 1537)
- a test item causing a positive response proportional to the dose for at least 3 doses with, for the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay (TA 98, TA 100 and TA 102)
In some borderline cases, an additional criterion to be considered is the comparison between the number of revertants induced by the test item and the laboratory historical control data. Indeed, an increase in each individual value that is above the highest value of corresponding historical control data can help supporting a conclusion such as “equivocal” or “weak” mutagen. If a test item causes a positive response during a single assay and that result cannot be reproduced in at least 1 independent assay, the initial positive result may be considered as not significant. If, in all experimental conditions examined, none of the above criteria are fulfilled, a test item is considered clearly negative and unable to induce mutations in this test system.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated. In addition, data was analysed by means of Dunnett's method allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp I: -S9: 1000 µg/plate: TA1535, TA1537, TA100; 1500 µg/plate: TA98; +S9: 1500 µg/plate: TA100; 3000 µg/plate: TA1535, TA1537, TA98; Exp II: -S9: 750 µg/plate: TA100; 1000 µg/plate: TA1535; +S9: 500 µg/plate: TA1535, TA1537, TA100; 1500 µg/plate: TA98

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp I: -S9: 1000 µg/plate: TA102; +S9: 3000 µg/plate: TA102; Exp II: -S9: 750 µg/plate: TA102; +S9: 150 µg/plate: TA102

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully soluble in the vehicle (sterile water).
- Precipitation: No precipitation was observed in the main mutagenicity assay (experiment I and II) neither in the presence nor in the absence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES
Cytotoxicity of the test item was assessed in a preliminary experiment resulting in:
- an important toxicity with the presence of microcolonies at the two highest tested doses of 5000 and 1500μg/plate without metabolic activation in strains TA1535, TA1537, TA100 and TA102
- an important toxicity with the presence of microcolonies at the highest tested dose of 5000μg/plate in all strains with metabolic activation and in strain TA98 without metabolic activation
- a moderate toxicity at the tested dose of 1500μg/plate in strain TA98 without metabolic activation and in strain TA100 with metabolic activation
- a slight toxicity at the tested dose of 500μg/plate in strains TA1535 ad TA1537 without metabolic activation and at the tested dose of 1500μg/plate in strains TA1535, TA1537, TA98 and TA102 with metabolic activation.
Therefore, the maximum doses retained for the first mutagenicity assay in the absence of metabolic activation were 1000 µg/plate (TA1535, TA1537, TA100 and TA102) and 1500μg/plate (TA 98) and 3000 µg/plate in all strains in the presence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA
The results were within the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Cytotoxicity was recorded at 1000 µg/plate (TA 1535, TA 1537, TA 100, TA 102) and 1500 µg/plate (TA 98) in the absence of metabolic activation and at 1500 µg/plate (TA 100) and 3000 µg/plate (TA 1535, TA 1537, TA 98, TA 102) in the presence of metabolic activation (experiment I). In the second experiment cytotoxicity was present at 750 µg/plate (TA 100, TA 102) and at 1000 µg/plate /TA 1535) in the absence of metabolic activation and at 150 µg/plate (TA 102), 500 µg/plate (TA 1535, TA 1537, TA 100) and 1500 µg/plate (TA 98) in the presence of metabolic activation.

Any other information on results incl. tables

Table 1: Results of the preliminary cytotoxicity test

Toxicity Assay	Dose in µg/plate	TA 1535		TA 1537		TA 98		TA 100		TA 102
		T	P	T	P	T	P	T	P	T	P
Test item without S9-mix	0 50 150 500 1500 5000	- - - + +++ +++	- - - - - -	- - - + +++ +++	- - - - - -	- - - - ++ +++	- - - - - -	- - - - +++ +++	- - - - - -	- - - - +++ +++	- - - - - -
Top Dose in first mutagenicity assay		1000		1000		1500		1000		1000
Test item with S9-mix	0 50 150 500 1500 5000	- - - - + +++	- - - - - -	- - - - + +++	- - - - - -	- - - - + +++	- - - - - -	- - - - ++ +++	- - - - - -	- - - - + +++	- - - - - -
Top dose in first mutagenicity assay		3000		3000		3000		3000		3000

T: toxicity (- non toxic: + slightly toxic: ++ moderately toxic: +++ strongly toxic)

P: precipitation (- absence: + slight precipitate: ++ moderate precipitate: +++ important precipitate)

Table 2: Results of mutagenicity assay (experiment I)

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 100	TA1535	TA 102	TA98	TA1537
–	0	93.8 ± 6.1	14.0 ± 2.8	107.7 ± 23.9	16.2 ± 1.9	6.8 ± 3.4
–	5	85.7 ± 7.1	14.3 ± 3.5	105.3 ± 15.5	10.3 ± 1.2	6.0 ± 3.0
–	15	97.3 ± 3.5	17.3 ± 5.9	112.7 ± 9.2	14.3 ± 2.1	4.7 ± 0.6
–	50	95.0 ± 11.3	16. 7 ± 1.5	127.3 ± 8.1	15.7 ± 4.2	4.0 ± 1.0
–	150	85.3 ± 6.5	13.0 ± 2.6	114.7 ± 1.2	10.7 ± 6.4	3.7 ± 3.8
–	500	82.7 ± 29.1	15.3 ± 4.5	100.7 ± 32.9	12.0 ± 4.0	4.0 ± 2.6
–	1000	18.7 ± 1.5	9.3 ± 2.1	50.0 ± 2.0	-	**
–	1500	-	-	-	4.3 ± 2.1	-
Positive controls, –S9	Name	NaN	NaN	MM-c	2-NF	9-AA
	Concentrations (μg/plate)	1	1	0.125	2	50
	Mean No. of colonies/plate (average of 3 ± SD)	624.0 ± 97.3	477.3 ± 258.8	480.0 ± 142.2	230.7 ± 31.9	450.7 ± 117.1
+	0	110.5 ± 16.1	12.0 ± 3.7	179.3 ± 27.1	21.7 ± 4.1	6.7 ± 1.9
+	15	102.7 ± 5.5	8.7 ± 2.9	210.0 ± 28.8	24.0 ± 4.0	6.0 ± 1.0
+	50	104.0 ± 5.0	12.3 ± 2.5	193.3 ± 17.5	19.3 ± 8.1	6.7 ± 3.2
+	150	84.3 ± 2.5	9.0 ± 2.0	155.3 ± 25.0	19.0 ± 5.2	8.0 ± 2.6
+	500	94.7 ± 5.7	7.7 ± 0.6	189.3 ± 12.2	27.0 ± 6.6	5.0 ± 4.6
+	1500	31.3 ± 4.9	11.0 ± 2.6	126.0 ± 30.0	24.0 ± 7.9	3.0 ± 2.6
+	3000	**	**	22.7 ± 6.7	**	**
Positive controls, +S9	Name	2-AA	2-AA	BaP	2-AA	2-AA
	Concentrations (μg/plate)	2	2	2	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	1738.7 ± 268.8	418.7 ± 66.2	912.0 ± 224.0	1888.0 ± 279.0	154.7 ± 24.1

**: not analyzable due to toxicity

NaN: sodium azide

MM-c: mitomycin c

2 -NF: 2 -nitrofluorene

9 -AA: 9 -aminoacridine

2 -AA: 2 -anthramine

BaP: benz[a]pyrene

-: concentration not assessed

Table 3: Results of mutagenicity assay (experiment II)

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 100	TA1535	TA 102	TA98	TA1537
–	0	114.3 ± 19.7	12.5 ± 3.2	165.3 ± 20.1	14.8 ± 2.0	5.7 ± 1.6
	1.5	-	-	-	-	9.3 ± 2.1
–	5	109.7 ± 10.1	12.0 ± 3.5	196.0 ± 7.2	18.0 ± 5.2	9.0 ± 2.6
–	15	104.0 ± 6.0	11.0 ± 2.6	188.7 ± 26.6	14.0 ± 1.0	10.0 ± 5.0
–	50	91.0 ± 8.5	14.3 ± 6.4	173.3 ± 3.1	12.0 ± 1.0	7.7 ± 3.8
–	150	107.7 ± 12.2	15.3 ± 4.2	139.3 ± 8.1	14.0 ± 1.7	4.7 ± 0.6
–	500	93.3 ± 11.9	10.3 ± 4.0	141.3 ± 16.0	15.3 ± 4.2	6.7 ± 0.6
	750	65.0 ± 2.8	-	95.3 ± 21.6	13.3 ± 0.6	-
–	1000	-	8.0 ± 2.6	-	-	-
Positive controls, –S9	Name	NaN	NaN	MM-c	2-NF	9-AA
	Concentrations (μg/plate)	1	1	0.125	2	50
	Mean No. of colonies/plate (average of 3 ± SD)	544.0 ± 48.0	421.3 ± 21.2	490.7 ± 33.3	244.7 ± 36.7	580.0 ± 102.1
+	0	71.7 ± 5.9	11.2 ± 3.3	202.0 ± 29.4	20.2 ± 6.5	5.7 ± 1.2
+	5	74.0 ± 12.1	9.7 ± 2.1	178.0 ± 32.7	23.7 ± 7.5	5.7 ± 0.6
+	15	78.3 ± 6.7	10.0 ± 2.0	164.7 ± 9.5	21.3 ± 5.0	9.7 ± 2.3
+	50	81.7 ± 7.2	9.7 ± 1.5	146.0 ± 9.2	22.3 ± 2.5	8.3 ± 8.4
+	150	94.0 ± 6.9	10.3 ± 1.5	116.0 ± 5.3	18.3 ± 4.7	6.7 ± 3.1
+	500	**	6.0 ± 2.0	117.3 ± 11.4	16.3 ± 6.7	2.7 ± 1.5
	1000	**	-	-	-	**
+	1500	-	**	72.0 ± 22.6	**	-
Positive controls, +S9	Name	2-AA	2-AA	BaP	2-AA	2-AA
	Concentrations (μg/plate)	1	1	2	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1280.7 ± 287.0	232.7 ± 20.2	536.0 ± 60.4	1152.0 ± 180.3	83.3 ± 12.7

**: not analyzable due to toxicity

NaN: sodium azide

MM-c: mitomycin c

2 -NF: 2 -nitrofluorene

9 -AA: 9 -aminoacridine

2 -AA: 2 -anthramine

BaP: benz[a]pyrene

-: concentration not assessed

Applicant's summary and conclusion

Conclusions:

A bacterial gene mutation assay (Ames test) was performed with the test material according to OECD TG 471 and in compliance with GLP. The test material was considered not to be mutagenic in the presence and absence of metabolic activation in the tester strains used.