3,7-dimethylocta-1,6-diene

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:

from 2010-04-26 to 2010-08-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to the OECD guideline (No. 471) and is in compliance to the GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethylocta-1,6-diene

EC Number:

219-433-3

EC Name:

3,7-dimethylocta-1,6-diene

Cas Number:

2436-90-0

Molecular formula:

C10H18

IUPAC Name:

3,7-dimethylocta-1,6-diene

Details on test material:

- Name of test material (as cited in study report): Dimethyloctadiene
- Physical state: colorless-slightly yellow liquid
- Lot/batch No.: EC205R
- Expiration date of the lot/batch: 2011/03/18
- Stability under test conditions: to limit the degradation of the products by the oxygen of air and the escape of the Dimethyloctadiene in the ambient air (volatile properties), the test item will be weighed extemporaneously and dissolved or suspended in the vehicle to provide a suitably concentrated stock solution or suspension. The stock solution/suspension and any further dilution will be prepared in tightly sealed tubes within the 2 hours before use and then kept protected from light and at 4°C until use. During the test, all the Petri dishes were placed in a sealed jar using one jar for each dose-level tested, one jar for the vehicle controls and another jar for the positive controls to avoid the escape of test item in the ambient air.
- Storage condition of test material: at 4°C, protected from light and humidity and under nitrogen gas.
- Other: No data

Method

Target gene:

Each strain derived from S. typhimurium LT2 contains one mutation in the histine operon, resulting in a requirement for histidine.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: additional mutations in rfa and uvrB genes. For the strains TA98 and TA100 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 1

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: mutated in rfa gene and presence of an additional plasmid pKM101 in order to enhance the sensitivity of detection of some mutagens. See Table 1

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.

Test concentrations with justification for top dose:

Preliminary test: 10, 100, 500, 1000, 2500, 5000 µg/plate with and without S9 mix.
Mutagenicity experiments: first experiment = 156.3 to 5000 µg/plate without and with S9 mix for all strains. Second experiment = 156.3 to 5000 µg/plate without S9 mix for all strains; 156.3 to 5000 µg/plate with S9 mix for TA1535, TA1537, TA98 and TA102; 78.1 to 2500 µg/plate with S9 mix for TA 100. See details in table 2.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol, batch Nos. V8F771168F and V0F701170F (Carlo Erba, Val de Reuil, France).
- Justification for choice of solvent/vehicle: During the solubility assay, the test item was found freely soluble in the vehicle (Ethanol) at 200 mg/mL. Consequently, with a treatment volume of 25 µL/plate, the highest dose-level to be tested in the preliminary test was 5000 µg/plate, which is the highest dose-level recommended in the international guidelines. Successive dilutions of the stock preparation were also performed in Ethanol and used with a treatment volume of 25 µL/plate. Therefore the dose-levels to be tested in the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C for without S9 mix efficacy control. 2-Anthramine for with S9 mix efficacy control. See details in Table 3

Remarks:

no remarks

Details on test system and experimental conditions:

METHOD OF APPLICATION:
direct plate incorporation: test item solution (0.025 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
or preincubation method: test item solution (0.025 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 or 72 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 3 plates/dose/strain. For the mutagenicity experiments, two independent experiments were performed.
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: not required
- Determination of endoreplication: not required

OTHER: All the Petri dishes obtained were placed in a sealed jar using one jar for each dose-level tested, one jar for the vehicle control and another jar for the positive controls. The jars were then incubated at 37°C. The precaution of using jars in this study was due to the volatile characteristic of the test item and to limit the oxidation of the test item.
The revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK). Manual counting was used as needed.

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

no data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4; 5; 6; 7

Cytotoxicity / choice of top concentrations:

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

Remarks:

See tables 4; 5; 6; 7

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4; 5; 6; 7

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See tables 4; 5; 6; 7

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4; 5; 6; 7

Cytotoxicity / choice of top concentrations:

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

Remarks:

See tables 4; 5; 6; 7

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

See tables 4; 6

Cytotoxicity / choice of top concentrations:

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

Remarks:

See tables 4; 6

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

See tables 5; 7

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only according to the pre-incubation method. See tables 5; 7

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See tables 4; 5; 6; 7

Cytotoxicity / choice of top concentrations:

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

Remarks:

See tables 4; 5; 6; 7

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: not applicable
RANGE-FINDING/SCREENING STUDIES: A first preliminary test was performed testing six dose-levels of the test item (one plate/dose-level) with the TA 98, TA 100 and TA 102 strains, with and without S9 mix. This preliminary test was invalidated. Indeed, the test item used in this test came from a small aliquot taken from one of the flask and stored at +4°C, protected from light and humidity and under nitrogen gas. This conditioning in an inert atmosphere was performed using a layer of parafilm to seal the vessel (as described in CIT’s SOPs). But there were some evidences that this layer of parafilm got deteriorated (most probably by the test item’s emanation) contaminating the aliquot of test item. Because of this contamination, the results of this first preliminary test were invalidated and the study was started all over again using one supplied flask per treatment and avoiding the use of parafilm.
The second preliminary test was performed testing six dose-levels of the test item (two plates/dose-level) with the TA 98, TA 100 and TA 102 strains, with and without S9 mix. No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels. A strong toxicity (observation of a strong thinning of the bacterial lawn) was observed at dose levels 5000 µg/plate with the TA 98 strain without S9 mix and with the TA 100 strain with S9 mix. No noteworthy toxicity was noted in the other tested strains up to the highest dose level of 5000 µg/plate. Since the test item was freely soluble and only toxic in the preliminary test at the highest dose level recommended in the international guidelines, the highest dose-level to be used in the main mutagenicity experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.
COMPARISON WITH HISTORICAL CONTROL DATA: The numbers of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered valid.
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment without S9 mix, a moderate toxicity was noted at dose-levels 1250 µg/plate and above in the TA 100 strain and a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1537 and TA 98 strains (see table 4).
In the second experiment without S9 mix, a moderate toxicity was noted at dose-level 1250 µg/plate and above in the TA 100 strain and at dose-levels 5000 µg/plate in TA98 strain, and a moderate to strong toxicity was noted at dose level 2500 µg/plate and above in the TA1537 strain (see table 6).
In the first experiment with S9 mix (direct plate incorporation), a moderate toxicity was noted at 5000 µg/plate in the TA 98 strain (see table 5).
In the second experiment with S9 mix (preincubation method), a moderate to strong toxicity was noted at dose-level 625 µg/plate and above in the TA 100 strain and from 1250 µg/plate in the TA1537 strain, and a moderate toxicity was noted at dose-level 2500 µg/plate and above in the TA 1535 strain and at 5000 µg/plate in the TA 98 and TA 102 strains (see table 7).

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 4: Number of revertants per plate in the absence of metabolic activation in the first test (direct plate incorporation method)

Dimethyloctadiene Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	23	7	10	3	34	4	142	46	295	9
156.3	16	3	6	1	34	22	108	15	346	41
312.5	19	4	15	8	39	7	123	32	310	29
625	14	5	9	3	51	13	99	45	343	65
1250	23	10	10	2	41	6	127Mt	68	219	20
2500	22	6	5	1	46	24	165Mt	51	243	37
5000	19	2	9St	4	32St	12	128Mt	12	346	42
Positive control**	530	98	377	92	155	3	507	61	1877	210

$: Mean of triplicate

*Solvent control = negative control: ethanol

**Mutagens positive controls:

- NaN₃(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

Mt : Moderate cytotoxicity

St : Strong cytotoxicity

 

 

 

Table 5:Number of revertants per plate in the presence of metabolic activation (S9 mix) in the first test (direct plate incorporation method) 

Dimethyloctadiene Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation	Mean$	Standard deviation
0*	22	3	12	2	41	16	150	58	400	85
156.3	24	5	12	2	28	13	123	20	471	21
312.5	25	4	10	6	49	16	159	48	507	172
625	21	9	10	3	54	5	146	22	468	195
1250	18	7	17	7	40	8	163	33	672	56
2500	19	3	9	2	50	6	132	12	614	96
5000	23	6	8	5	55Mt	13	138	17	596	142
Positive control**	256	69	85	17	577	53	1560	308	958	30

$: Mean of triplicate

*Solvent control = negative control: ethanol

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98

- 2AM (10µg/plate) in TA102 strain

- BAP (5µg/plate) in TA100 strain

Mt : Moderate cytotoxicity

St : Strong cytotoxicity

 

 

Table 6:Number of revertants per plate in the absence of metabolic activation in the second test (direct plate incorporation method)

Dimethyloctadiene Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	24	8	10	6	28	2	95	18	452	100
156.3	21	3	7	3	19	2	67	8	463	14
312.5	15	4	9	2	17	12	69	10	474	50
625	16	5	11	5	20	0	71	20	512	19
1250	20	6	8	4	15	5	76Mt	5	512	42
2500	28	17	8Mt	5	19	8	73Mt	15	487	31
5000	23	5	8St	5	18Mt	6	76Mt	35	455	23
Positive control**	745	103	242	55	116	10	815	20	1999	125

$: Mean of triplicate

*Solvent control = negative control: ethanol

- NaN₃(1 µg/plate) in TA1535 and TA100 strains

- 9AA (50 µg/plate) in TA1537 strain

- 2NF (0.5 µg/plate) in TA 98 strain

- MMC ( 0.5 µg/plate) in TA 102 strain

Mt : Moderate cytotoxicity

St : Strong cytotoxicity

 

 

Table 7: Number of revertants per plate in the presence of metabolic activation (S9 mix) in the second test (pre-incubation method)

 

TFE Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		TA 102
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	23	2	9	3	37	9	130	19	476	84
78.1	-	-	-	-	-	-	117	3	-	-
156.3	29	6	9	2	29	4	120	16	437	159
312.5	24	7	11	2	38	3	95	11	350	57
625	26	5	10	7	32	3	88Mt	3	408	56
1250	19	8	9Mt	2	31	5	69St	19	412	96
2500	17Mt	4	8St	3	31	5	82St	23	503	25
5000	21Mt	10	9St	2	35Mt	4	-	-	352Mt	75
Positive control**	139	17	159	36	1497	169	390	34	3418	605

$: Mean of triplicate

*Solvent control = negative control: ethanol

**Mutagens positive controls:

- 2AM (2µg/plate) in TA1535, TA1537, TA98 strains

- 2AM (10µg/plate) in TA102 strain

- BAP (5 µg/plate) in TA100 strain

Mt : Moderate cytotoxicity

St : Strong cytotoxicity

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with or without metabolic activation

Under the test conditions, the test item dimethyloctadiene did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains. Dimethyloctadiene is not considered as mutagenic in this bacterial system according to the criteria of the CLP Regulation (EC) n° 1272/2008 and according to the Annex VI of Directive 67/548/EEC.

Executive summary:

In a reverse gene mutation assay in bacteria performed according to the OECD No.471 and EC No.B13/14 guidelines, dimethyloctadiene (purity of 91.1%) diluted in ethanol was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation or the preincubation method. All the concentrations and dose-levels were expressed as active item, taking into account the purity of the test item (91.1%).

Due to the volatile characteristic of the test item and in order to limit the oxidation of the test item, all the Petri dishes were placed in a sealed jar. One jar was used for each tested dose-level, one jar was used for the vehicle control and another jar for the positive controls. 

Six known mutagens, dissolved in dimethylsulfoxide (except for Mitomycin C which was dissolved in distilled water), were used to check the sensitivity of the test system. The positive controls induced the appropriate responses in the corresponding strains. The number of revertants in the vehicle controls was consistent with the historical data of the testing facility, and the number of revertants in the positive controls was higher than that of the vehicle controls (at least 2-fold increase for the TA 98, TA 100 and TA 102 strains and at least 3-fold increase for the TA 1535 and TA 1537 strains) and was consistent with the historical data of the testing facility. Therefore the study was considered valid.

During the preliminary test, a strong thinning of the bacterial lawn was observed at the highest recommended dose level (5000 µg/plate) in TA98 without metabolic activation and in TA100 with metabolic activation. Since the test item was freely soluble and only toxic in the preliminary test at the highest dose‑level recommended in the international guidelines, the highest dose-level to be used in the main mutagenicity experiments was 5000 µg/plate.

During the main tests, no induced revertant over background was observed in any strains of S. typhimurium whereas the cytotoxic dose-level was reached or the test was conducted up to the highest recommended dose-level (5000 µg/plate).

 

Therefore, under the test conditions, dimethyloctadiene did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhymurium strains. Dimethytloctadiene is not considered as mutagenic according to the criteria of the CLP Regulation (EC) n° 1272/2008 and according to the Annex VI of Directive 67/548/EEC.