1,1-diethoxy-3,7-dimethylocta-2,6-diene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation toxicity study was performed to determine the mutagenic nature of citral diethyl acetal. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in ethanol and used at dose levels 0.0, 0.3, 1.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. Citral diethy acetal did not induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to evaluate the mutagenic nature of Citral diethyl acetal

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: Citral diethylacetal
- IUPAC name: 1,1-diethoxy-3,7-dimethylocta-2,6-diene
- Molecular formula: C14H26O2
- Molecular weight: 226.357 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: approx. 79%
- Impurities (identity and concentrations): approx. 21 %

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA100, TA98, TA97 and TA 1535

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers

Test concentrations with justification for top dose:

0.0, 0.3, 1.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/Plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The chemical was soluble in ethanol

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98 and TA1538; -S9) and 2-aminoanthracene (all atrains; +S9)

Details on test system and experimental conditions:

The positive controls in the absence of metabolic activation were sodium azide (TA1535 and TA100), 9-aminoacridine (TA97 and TA 1537), 4-nitro-o-phenylenediamine (TA98 and TA1538), mitomycin C (TA102), and methyl methanesulfonate (TA 104). The positive control for metabolic activation with all strains was 2-aminoanthracene, and either sterigmatocystin or 2-aminoanthracene was used for TA102.

Details on test system and conditions

METHOD OF APPLICATION: pre incubation
DURATION
- Pre incubation period: 20 minutes
- Exposure duration: No data available
- Expression time (cells in growth medium): two days
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available
SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: No data available
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER:METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

Statistics:

Mean ± SEM

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Table: Mutagenic responses (mean ± SEM; three plates) of Salmonella tester strains to test chemicals.

Dose	TA100
	NA(-)		10% HLI        (-)		30% HLI   (-)		10% RLI    (-)		30 % RLI    (-)
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0.000	83	2.4	99	6.2	154	5.5	99	7.9	147	10.7
0.300	95	3.4	102	9.3			98	2.8
1.000	73	13.6	99	18.0			100	13.3
3.300	85	5.6	86	4.1	138	8.9	100	4.9	126	1.9
10.000	79	4.6	79	3.5	132	8.7	89	1.7	142	7.0
33.000	72	5.2	44s	8.2	135	6.3	54s	12.4	138	11.7
100.00					126	1.5			125	8.2
333.000					84s	5.1			102s	8.0
POS	424	10.1	214	5.2	588	25.5	972	29.6	1223	8.1

 

Dose	TA1535
	NA(-)		10% HLI        (-)		30% HLI   (-)		10% RLI    (-)		30 % RLI    (-)
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0.000	20	3.8	21	1.2	23	4.9	22	0.7	30	0.7
0.300	17	0.7	22	2.3			28	0.3
1.000	17	0.9	20	3.2	28	3.9	23	4.6	26	5.9
3.300	17	5.4	18	5.5	23	4.2	26	4.1	27	2.3
10.000	18	0.7	15	2.0	20	2.3	19	1.7	33	3.8
33.000	15	2.5	6s	3.1	23	0.3	16s	2.7	32	2.7
100.00					23	4.1			30	2.6
333.000
POS	310	2.3	103	4.1	114	3.5	333	33.3	128	11.9

 

Dose	TA97
	NA(-)		10% HLI        (-)		30% HLI   (-)		10% RLI    (-)		30 % RLI    (-)
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0.000	92	10.0	119	11.5	145	7.8	126	9.7	203	10.0
0.300	120	6.4	128	10.7			152	10.0
1.000	90	3.8	108	10.4	124	4.4	151	6.2	176	7.3
3.300	101	6.5	113	6.7	158	4.1	145	19.9	156	11.3
10.000	102	4.9	94	5.7	157	8.5	99	13.1	133	5.0
33.000	74	8.2	67s	7.8	141	7.4	60s	12.7	133	5.0
100.00					157	4.2			154	3.8
333.000
POS	391	4.1	637	4.4	947	21.8	2067	124.4	1086	26.7

 

 

 

Dose	TA98
	NA(-)		10% HLI        (-)		30% HLI   (-)		10% RLI    (-)		30 % RLI    (-)
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0.000	26	4.2	42	4.4	39	2.7	37	3.5	44	0.6
0.300	22	3.6	28	12.0			38	5.8
1.000	28	2.6	39	4.2			37	3.7
3.300	23	4.3	41	4.3	41	4.1	35	1.7	39	2.3
10.000	21	2.6	39	4.9	52	4.0	33	5.5	42	3.8
33.000	19	2.9	21s	0.9	46	1.5	16s	1.8	40	3.8
100.00					43	2.6			36	1.2
333.000					28s	4.4			31s	2.9
POS	337	10.1	177	11.9	441	9.8	159	10.2	375	3.1

ET95,95% ethanol (solvent), POS, positive control; NA, not activated;

HLI, Aroclor 1254-induced hamster liver S-9; RLI, Aroclor 1254-induced rat liver S-9; s, slight clearing of background lawn; t, complete clearing of background lawn (colonies not counted); p, precipitate present in plates; x, precipitate present with toxicity; +, mutagenic; +W, weakly mutagenic; ?, questionable response; - , nonmutagenic.

Conclusions:

Citral diethy acetal did not induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of citral diethyl acetal. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in ethanol and used at dose levels 0.0, 0.3, 1.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. Citral diethy acetal did not induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Peer reviewed publications were reviewed to determine the mutagenic nature of Citral diethyl acetal (IUPAC name: 1,1-diethoxy-3,7-dimethylocta- 2,6-diene). The studies are as mentioned below:

Gene mutation toxicity study was performed by Zeiger et al ( Environmental and Molecular Mutagenesis, 1992) to determine the mutagenic nature of citral diethyl acetal. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in ethanol and used at dose levels 0.0, 0.3, 1.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. Citral diethy acetal did not induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence the chemical s not likely to classify as a gene mutant in vitro.

Same study by Zeiger et al (Environmental and Molecular Mutagenesis, 1992) was also performed using DMSO as the solvent and at dose levels of 0, 33, 100, 333, 1000, 1666, 3333, 6666, 10000 µg/plate by the preincubation method. Citral diethy acetal did not induce mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In another study by Zeiger et al (Environmental and Molecular Mutagenesis, 1988) Citral diethyl acetal was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in water and was tested at concentration of 0, 33, 100, 333, 1000, 3333, 4000 or 6666 µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. Citral diethyl acetal did not induce gene mutation in Salmonella typhimurium strains TA98 and TA97 with and without metabolic activation system. It also did not induce gene mutation in strains TA100 and TA1535 upto 1000 µg/plate and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the data available for the target chemical, Citral diethyl acetal does not exhibt gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available for the target chemical, Citral diethyl acetal (CAS no 7492 -66 -2) does not exhibt gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.