2,4,8,10-Tetraoxaspiro[5.5]undecane-3,9-diethanol, .beta.3,.beta.3,.beta.9,.beta.9-tetramethyl-

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:

Testing was conducted between 17 May 2006 and 16 June 2006.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test was performed according to guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 2

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:

-

EC Number:

485-230-3

EC Name:

-

Cas Number:

1455-42-1

Molecular formula:

Hill formula: C15H28O6 CAS formula: C15H28O6

IUPAC Name:

2-[9-(1-hydroxy-2-methylpropan-2-yl)-2,4,8,10-tetraoxaspiro[5.5]undecan-3-yl]-2-methylpropan-1-ol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor’s identification: SPG
Description: White powder
Batch number: 50908-02
Date received: 05 May 2006
Storage conditions: Room temperature in the dark.

Method

Target gene:

S. typhimurium: Histidine
E. coli: Tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S 9 mix; pretreated

Test concentrations with justification for top dose:

Preliminary toxicity test: Concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Mutation test experiments 1 and 2: Concentrations tested were 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

Solvent: Dimethyl sulphoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

Preliminary toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing an aliquot of bacterial culture, 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0. 5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel Bonner Minimal agar (30 mL/plate). Ten concentrations of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test material and 2 mL molten trace histidine or tryptophan supplemented, top agar, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation test – Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots of one of the bacterial cultures were dispensed into sets of test tubes followed by2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL or S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed into the surface of Vogel Bonner Minimal agar plates (one tube per plate). This procedure was repeated in triplicate for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a colony counter.

Mutation test – Experiment 2
The second experiment was performed as above, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as experiment 1 (50 to 5000 µg/plate).

Acceptance criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell wall mutation and pKM101 plasmid R-factor.
All tester strain cultures should be in the approximate range of 1 to 9.9E09 bacteria per mL.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistically methods, as recommended by the UKEMS can also be used as an aid to evaluation, however statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(> 5000 µg/plate)

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(> 5000 µg/plate)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(> 5000 µg/plate)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(> 5000 µg/plate)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(> 5000 µg/plate)

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test results:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

Mutation test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not provided in the report. The S9 mix and the amino-acid supplemented top agar used in both experiments were shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale, particulate precipitate was observed at 5000 µg/plate which did not prevent the scoring of revertant colonies.
No significant increases in the frequency or revertant colonies were recorded for any of the strains of bacteria at any dose level, with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary toxicity test

The number of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA 100	110	99	87	102	104	106	98	113	107	104	89P
+	TA 100	89	88	106	75	88	74	81	78	60	62	73P
-	WP3uvrA-	21	21	23	31	24	13	30	24	13	18	23P
+	WP3uvrA-	26	24	46	26	31	25	36	30	27	21	21P

Where P = Precipitate

Spontaneous mutation rates (concurrent negative controls)

Experiment 1:

Number of revertants (mean number of colonies per plate)
Base pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA-		TA98		TA1537
98	(107)	13	(11)	22	(27)	20	(18)	19	(10)
123		19		40		14		7
99		24		19		21		3

 

Experiment 2:

Number of revertants (mean number of colonies per plate)
Base pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA-		TA98		TA1537
67	(81)	15	(19)	22	(25)	29	(19)	7	(11)
96		19		26		14		14
81		24		26		14		11

Test results – Experiment 1 – Without metabolic activation

With or without S9-mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
-	0	102	(91) 11.5#	10	(11) 1.7	18	(21) 2.9	13	(19) 8.7	15	(19) 4.5
		79		10		23		15		19
		91		13		23		29		24
-	50	84	(88) 4.0	14	(13) 2.1	21	(21) 0.0	25	(25) 2.5	14	(12) 3.5
		89		11		21		27		8
		92		15		21		22		14
-	150	88	(89) 1.0	9	(10) 2.3	29	(21) 7.6	14	(20) 5.3	19	(13) 5.3
		90		13		14		24		9
		89		9		19		22		11
-	500	104	(91) 11.7	10	(12) 2.5	18	(20) 1.5	20	(22) 4.0	12	(14) 2.1
		89		12		21		27		16
		81		15		20		20		13
-	1500	80	(81) 3.1	15	(13) 2.5	20	(21) 0.6	19	(21) 7.2	15	(13) 3.5
		78		13		21		15		9
		84		10		21		29		15
-	5000	85P	(85) 0.6	19P	(13) 4.9	25P	(20)	22P	(19) 7.0	11P	(9) 2.0
		85P		11P		15P		24P		9P
		86P		10P		20P		11P		7P
Positive controls S9-mix -	Name	EENG		EENG		EENG5.0		4NQO		9AA
	Concentration (µ/plate)	3		5		2		0.2
	Number of colonies per plate	558	(553) 4.2	676	(528) 166.2	749	(841) 90.1	172	(175) 47.1	2716	(2543) 229.0
		550		559		846		130		2283
		552		348		929		224		2629

 

Test results – Experiment 1 – With metabolic activation

With or without S9-mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base pair substitution type						Frameshift type
		TA100		TA1535		WP2uvrA-		TA98		TA1537
+	0	86	(86) 1.0#	13	(14) 1.2	25	(30) 7.2	33	(31) 4.4	9	(10) 0.6
		87		15		26		34		10
		85		13		38		26		10
+	50	80	(81) 0.6	11	(11) 0.6	31	(26) 4.7	30	(18) 10.1	7	(8) 1.2
		81		12		14		12		9
		81		11		22		13		9
+	150	77	(82) 8.4	16	(13) 2.5	41	(29) 10.1	26	(27) 6.6	8	(10) 2.6
		78		11		23		21		13
		92		13		24		34		9
+	500	79	(88) 10.8	11	(10) 1.0	26	(26) 4.0	32	(29) 3.6	12	(10) 2.1
		100		10		22		30		9
		85		9		30		25		8
+	1500	84	(87) 2.9	13	(12) 1.7	23	(25) 2.0	21	(23) 2.0	12	(8) 4.0
		89		13		25		25		4
		89		10		27		23		8
+	5000	76P	(84) 9.7	10P	(10) 1.5	26P	(28) 4.0	29P	(28) 2.3	10P	(9) 2.1
		95P		12P		26P		25P		7P
		82P		9P		33P		29P		11P
Positive controls S9-mix +	Name	2AA		2AA		2AA		BP		2AA
	Concentration (µ/plate)	1		2		10		5		2
	Number of colonies per plate	1828	(1869) 53.7	230	(234) 6.1	300	(370) 62.0	213	(227) 12.7	591	(657) 106.6
		1850		231		394		238		600
		1930		241		417		229		780

P = Precipitate

# = Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of the test both with and without metabolic activation.

Executive summary:

Introduction.

The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals no 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

 

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % Liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test formulations.

 

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale, particulate precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Conclusion.

The test material was considered to be non-mutagenic under the conditions of the test.