(1-ethoxyethoxy)cyclododecane

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:

01-02-2000 to 21-02-2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable deviations.

Remarks:

The efficacy of the S9-mix was only validated using 2-Anthramine (2-Aminoanthracene CAS 613-13-8) and benzopyrene or dimethylbenzanthracene was not used in each test. This is reflected in the reliability score.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The efficacy of the S9-mix was only validated using 2-Anthramine (2-Aminoanthracene CAS 613-13-8) and benzopyrene or dimethylbenzanthracene was not used in each test. This is reflected in the reliability score.

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Notification No. 700 of the Environmental Agency, No. 1039 of the Ministry of Health and Welfare and No. 1014 of Ministry of International Trade and Industry, 09 December 1986

Deviations:

yes

Remarks:

see above

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see above

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

yes

Remarks:

see above

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 1999 ; signature: December 1999

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

446-240-3

EC Name:

-

Cas Number:

389083-83-4

Molecular formula:

C16H32O2

IUPAC Name:

(1-ethoxyethoxy)cyclododecane

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: At room temperature, protected from light and under nitrogen gas
- Other: colourless

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test (TA98, TA100 and Wp2urvA): 0, 10, 100, 500, 1000, 2500 and 5000 µg/plate with and without S9 mix
Experiment 1 (plate incorporation method): All strains: 0, 312.5, 625, 1250, 2500, 5000 µg/plate with and without S9 mix
Experiment 2 (preincubation method): All strains: 0, 312.5, 625, 1250, 2500, 5000 µg/plate with and without S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (batch number and supplier listed in full study report).
- Justification for choice of solvent/vehicle: Ethanol was selected as the vehicle. Test item was dissolved in the vehicle at a concentration of 100 mg/ml for the preliminary toxicity test and for both mutagenicity experiments. The preparations were made immediately before use.

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

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preliminary Toxicity Test: in medium; in agar (plate incorporation), Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Preliminary Toxicity Test: To assess the toxicity of the test substance to the bacteria, at least six dose-levels (one plate/dose-level) will be tested in the TA98, TA100 and WP2uvrA strains, with and without S9 mix. Test item solution (0.05 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
Experiment 1. Test item solution (0.05 mL), S9 mix (0.5 mL) when required and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant amino acid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
Experiment 2. Test item solution (0.05 mL), S9 mix (0.5 mL) and bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours incubation at 37 ºC the revertants were scored using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Evidence leading to positive result:
1. Reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level
2. Evidence of dose-relationship, in any strain
Reference to historical data and/or other considerations such as biological relevance would be taken into consideration during the evaluation of data

Applicant assessment indicates: The test item is considered non-mutagenic (negative) in the test system if the above criteria are not met. Instances of data prohibiting definitive judgement about test item activity may be reported as equivocal.

Acceptance Criteria:
The study is considered valid if the following criteria are met:
1. The number of revertants in the vehicle controls is consistent with the historical control data range (information attached to the full study report)
2. The number of revertants in the positive control is higher than the vehicle controls and consistent with the historical control range (information attached to the full study report).

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

See table 1 and 2, without S9 mix activation, slight to moderate cytotoxicity was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

See table 1 and 2, without S9 mix activation, slight to moderate cytotoxicity was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1 – Preliminary Toxicity Test : with and without metabolic activation and results of concurrent positive controls

Strains	Doses µg/plate	Without S9 Mix			With S9 Mix
		T	E	Revertants per plate	T	E	Revertants per plate
TA98	0	0	0	24	0	0	29
	10	0	0	21	0	0	24
	100	0	0	19	0	0	23
	500	0	0	14	0	0	17
	1000	0	0	19	0	0	38
	2500	0	1	23	0	1	20
	5000	0	2	10	0	1	20
TA100	0	0	0	82	0	0	95
	10	0	0	71	0	0	104
	100	0	0	61	0	0	84
	500	0	0	43	0	0	83
	1000	1	0	55	0	0	82
	2500	1	1	65	0	1	90
	5000	1	1	38	0	1	90
WP2 uvrA	0	0	0	29	0	0	40
	10	0	0	18	0	0	40
	100	0	0	22	0	0	30
	500	0	0	31	0	0	30
	1000	0	0	25	0	0	22
	2500	0	1	28	0	1	31
	5000	0	1	21	0	1	34

0 = vehicle control (Ethanol)

Where:

T = toxicity : score = 0, none, 1 = slight, 2 = moderate, 3 = considerable to total

E = Emulsion of test item: score = 0, none, 1 = slight, 2 = moderate, 3 = strong

 

Table 2 : Test Results: Experiment 2 – Main Test (pre-incubation method) : with and without metabolic activation and results of concurrent positive controls

With S9 Mix
Strains	Doses µg/plate	T	E	Revertants per plate			Mean	Standard Deviation
				1	2	3
TA1535	0	0	0	29	30	26	28	2
	312.5	0	0	25	13	30	23	9
	625	0	0	23	24	27	25	2
	1250	0	0	21	13	41	25	14
	2500	0	0	20	31	25	25	6
	5000	0	1	27	29	30	29	2
	2AM(2)	-	-	258	232	226	239	17
TA1537	0	0	0	6	10	7	8	2
	312.5	0	0	8	4	8	7	2
	625	0	0	13	7	7	9	3
	1250	0	0	4	9	4	6	3
	2500	0	0	15	8	7	10	4
	5000	0	1	6	3	5	5	2
	2AM(2)	-	-	81	75	98	85	12
TA98	0	0	0	19	12	21	17	5
	312.5	0	0	14	15	12	14	2
	625	0	0	28	13	16	19	8
	1250	0	0	21	16	17	18	3
	2500	0	0	28	13	14	18	8
	5000	0	1	24	12	18	18	6
	2AM(2)	-	-	1362	1317	1383	1354	34
TA100	0	0	0	139	132	114	128	13
	312.5	0	0	92	81	93	89	7
	625	0	0	93	111	91	98	11
	1250	0	0	76	95	72	81	12
	2500	0	0	80	74	77	77	3
	5000	0	1	65	72	64	67	4
	2AM(2)	-	-	1087	992	921	1000	83
WP2 uvrA	0	0	0	55	44	37	45	9
	312.5	0	0	46	35	32	38	7
	625	0	0	50	40	34	41	8
	1250	0	0	39	26	44	36	9
	2500	0	0	49	42	46	46	4
	5000	0	1	39	32	38	36	4
	2AM(10)	-	-	233	170	179	194	34
Without S9 Mix
TA1535	0	0	0	6	10	12	9	3
	312.5	0	0	5	5	5	5	0
	625	0	0	7	8	6	7	1
	1250	0	0	7	7	5	6	1
	2500	0	0	13	7	7	9	3
	5000	0	1	7	10	8	8	2
	NaN3(1)	-	-	646	616	703	655	44
TA1537	0	0	0	9	14	13	12	3
	312.5	1	0	5	5	5	5	0
	625	1	0	8	10	6	8	2
	1250	2	0	11	7	8	9	2
	2500	2	0	11	7	7	8	2
	5000	2	1	9	6	5	7	2
	9AA(50)	-	-	332	287	435	351	76
TA98	0	0	0	17	16	15	16	1
	312.5	0	0	11	13	10	11	2
	625	0	0	18	10	14	14	4
	1250	1	0	12	17	17	15	3
	2500	1	0	22	14	18	18	4
	5000	2	1	15	20	10	15	5
	2NF(0.5)	-	-	150	164	147	154	9
TA100	0	0	0	107	97	93	99	7
	312.5	0	0	94	80	81	85	8
	625	0	0	124	102	104	110	12
	1250	0	0	83	77	88	83	6
	2500	0	0	119	90	96	102	15
	5000	0	1	106	96	76	93	15
	NaN3(1)	-	-	793	771	773	779	12
WP2 uvrA	0	0	0	30	20	34	28	7
	312.5	0	0	27	24	28	26	2
	625	0	0	31	40	32	34	5
	1250	0	0	35	27	28	30	4
	2500	0	0	40	29	27	32	7
	5000	0	1	27	25	23	25	2
	4NQQ(2)	-	-	1761	1781	1791	1778	15

0 = vehicle control (Ethanol)

Where:

T = toxicity : score = 0, none, 1 = slight, 2 = moderate, 3 = considerable to total

E = Emulsion of test item: score = 0, none, 1 = slight, 2 = moderate, 3 = strong

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, Japanese guidelines for bacterial mutagenicity testing and EU Method B.14, under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix activation system. The test strains were treated in two independent experiments with the test item, utilising the Ames plate incorporation method and then preincubation method, respectively at up to five dose levels, in triplicate, both with and without the addition of a rat liver microsomal metabolic activation system (10% liver S9). The vehicle was ethanol. The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The evaluation of toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The Experiment 1 dose levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated using the same dose range by the pre-incubation method in Experiment 2. All of the positive controls used in the test induced expected increases in the frequency of revertant colonies, both with or without metabolic activation. The vehicle controls were considered acceptable. Therefore the assay was considered valid. The maximum dose level of the test item in the Experiment 1 and Experiment 2 was the maximum recommended dose level of 5000 μg/plate in all tester strains. A slight emulsion was observed in the plates when scoring the revertants mainly at the highest dose-level. Without S9 mix, a slight to moderate toxicity was generally induced, depending on the dose-levels and the tester strains. With S9 mix, except for a slight decrease in the number of revertants, noted in the second experiment (preincubation method) in the TA1537 and TA100 strains at the highest dose-level, no noteworthy toxicity was induced in both experiments. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.