Cinnamyl alcohol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

In vitro bacterial reverse gene mutation assay for the test chemical was studied using S. typhimurium and Escherichia coli WP2 uvrA strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. Ames test was performed using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 and Escherichia coli WP2 uvrA strains in the presence and absence of S9 metabolic activation system at dose level of 0, 250, 750, 1500 or 3000 µg/plate. For Salmonella strains, the assays without S9 were performed by the plate-incorporation method and the assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. For E. coli strains, the assay was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli. No mutagenicity was noted at the tested dose levels. Based on the observations made, the test chemical was negative in Ames test carried out using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, TA1538 as well in Escherichia coli WP2 uvr A in the presence and absence of S9 metabolic activation. Hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

This information will be submitted later based on ECHA communication/decision number CCH-C-2114632477-44-01/F.

Gene mutation in Mammalian cells:

In vitro gene mutation study in mammalian cells according to OECD TG 476 with the registered substance will be performed if negative results are obtained from OECD 471 and OECD 473 (ongoing) studies and will be submitted later based on ECHA communication/decision number CCH-C-2114632477-44-01/F.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

In vitro gene mutation study in mammalian cells according to OECD TG 476 with the registered substance will be performed if negative results are obtained from OECD 471 and OECD 473 (ongoing) studies and will be submitted later based on ECHA communication/

Data waiving:

other justification

Justification for data waiving:

other:

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

An in vitro cytogenicity study according to OECD TG 473 with the registered substance is ongoing.

Data waiving:

other justification

Justification for data waiving:

other:

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

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:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro bacterial reverse gene mutation assay for test chemical was studied in S. typhimurium and Escherichia coli strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. All tester strains were examined periodically for the markers indicated by Ames et al.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella strains and tryptophan for E. coli strains

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA98, TA1537, TA1538

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 mix (500 µM) consisted of 100µL of S9, 50 µmoles sodium phosphate buffer (pH 7.4), 4 µmoles MgCl2, 16.5 µmoles KCl, 2.5 µmoles G-6-P, 2 µmoles NADH and 2 µmoles NADPH.
- source of S9 : PCB-treated male Sprague-Dawley rats
- method of preparation of S9 mix : No data
- concentration or volume of S9 mix and S9 in the final culture medium : No data
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

0, 250, 750, 1500 or 3000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data available

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

2-nitrofluorene

sodium azide

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
For Salmonella strains:
The assays without S9 were performed by the plate-incorporation method.
The assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977).. Histidine-independent colonies were scored after incubation at 37°C for 48-72 h.

For E. coli strains:
The assay was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli.

DURATION
- Preincubation period: 37°C
- Exposure duration: 48-72 h.

Rationale for test conditions:

No data available

Evaluation criteria:

A bacterial cell line was observed for biologically relevant increase in the number of revertants. Revertants per plate represent average values from 3 to 5 replications.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA100, TA1535, TA98, TA1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

All test compounds showed killing on bacteria at the highest doses used.

Remarks on result:

other: No mutagenic potential

Table: RESULTS OF MUTAGENICITY TEST OF THE TEST CHEMICAL

Chemical	Dose (µg/plate)	Revertants/plate (mean±S.D.)
		Salmonella typhimuriumstrains					Escherichia coli
Without S9
DMSO	50µL	99±6	8±2	24±4	4±2	15±3	55±6
Positive control		769±54	266±34	660±30	822±88	301±33	452±68
Test chemical	250	98±5	10±4	28±1	3±1	9±1	50±9
	750	81±6	5±1	24±1	3±1	10±2	57±6
	1500	81±13	7±1	22±8	5±0	14±0	46±3
	3000	24±13	2±1	20±8	2±2	6±1	33±6
With S9
DMSO	50µL	106±9	10±3	32±4	7±2	21±4	59±5
Positive control		431±92	158±45	180±7	125±37	154±17	753±95
Test chemical	250	87±1	7±2	31±3	9±2	24±8	58±6
	750	130±3	10±1	27±3	7±2	18±4	62±3
	1500	123±12	9±2	24±3	8±3	21±3	42±4
	3000	72±11	12±2	21±2	4±2	11±0	39±8

Conclusions:

The test chemical was negative in Ames test carried out using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, TA1538 as well in Escherichia coli WP2 uvr A in the presence and absence of S9 metabolic activation. Hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In vitro bacterial reverse gene mutation assay for test chemical was studied using S. typhimurium and Escherichia coli WP2 uvrA strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. Ames test was performed using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 and Escherichia coli WP2 uvrA strains in the presence and absence of S9 metabolic activation system at dose level of 0, 250, 750, 1500 or 3000 µg/plate. For Salmonella strains, the assays without S9 were performed by the plate-incorporation method and the assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. For E. coli strains, the assay was performed in the same manner as with the Salmonella assay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored with E. coli. No mutagenicity was noted at the tested dose levels. Based on the observations made, the test chemical was negative in Ames test carried out using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537, TA1538 as well in Escherichia coli WP2 uvr A in the presence and absence of S9 metabolic activation. Hence it 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:

Data available from various sources for the target chemical and the closely related test chemicals was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

Ames assay:

Study 1

In vitro bacterial reverse gene mutation assay for test chemical was studied using S. typhimurium and Escherichia coli WP2 uvrA strains. The mutagenicity assay was conducted as described by Ames et al. with slight modifications. Ames test was performed using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 and Escherichia coli WP2 uvrA strains in the presence and absence of S9 metabolic activation system at dose level of 0, 250, 750, 1500 or 3000 µg/plate. For Salmonella strains, the assays without S9 were performed by the plate-incorporation method and the assays with S9 were conducted by the pre-incubation method described by Yahagi et al. (1975). The pre-incubation method is useful in detecting weak mutagenicity in samples (Yahagi et al., 1977). Histidine-independent colonies were scored after incubation at 37°C for 48-72 h. ForE. colistrains, the assay was performed in the same manner as with theSalmonellaassay except that the supplement of 0.1 µmole histidine plus 0.1 mole biotin in the soft agar was replaced with a supplement of 0.1 µmole of tryptophan. Tryptophan-independent revertant colonies were scored withE. coli.No mutagenicity was noted at the tested dose levels. Based on the observations made, the test chemical was negative in Ames test carried out usingSalmonella typhimuriumstrains TA100, TA1535, TA98, TA1537, TA1538 as well inEscherichia coliWP2 uvr A in the presence and absence of S9 metabolic activation. Hence it is not likely to classify as a gene mutant in vitro.

Study 2:

Bacterial gene mutation assay was performed to evaluate the mutagenic potential of the test material. The test chemical was non mutagenic at concentrations up to 500–4000 µg/plate in E. coli strain WP2 uvrA (trp-) and hence, according to CLP criteria, it can be concluded that the test chemical was non mutagenic in nature.

Study 3:

Ames assay as per OECD 471 was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.0(VC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. B  

Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0(VC),  0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.  

The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data.  

The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method.  

Conclusion

In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Study 4:

Bacillus subtilis recombination assay was performed to evaluate the mutagenic potential of the test material. The study was performed using B. subtilis M45 (rec-) & H17 (rec+) strains in the absence of S9 metabolic activation system. In Bacillus subtilis recombination assay, the test chemical at dose of 21 µg/disk was not mutagenic in B. subtilis M45 (rec-) & H17 (rec+) without metabolic activation and hence it 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 and its closely related test chemicals, the target chemical Cinnamyl alcohol (CAS 104-54-1) is likely to be “Non-mutagenic” based on 471 (Bacterial gene mutation studies). Also, the structurally and functionally closely related test chemicals support the non-mutagenic classification in vitro. Hence the test chemical is “Not classified” for gene mutation end point as per the criteria mentioned in CLP regulation.