2,3-dihydro-2,2,6-trimethylbenzaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro gene mutation study in bacteria (with and without S-9 activation), OECD TG 471, 2019

negative, in vitro cytogenicity / micronucleus study (with and without S-9 activation), OECD TG 487, 2020

negative, in vitro gene mutation study in mammalian cells (with and without S-9 activation), OECD TG 490, 2020

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 28, 2018 to February 22, 2019

Reliability:

1 (reliable without restriction)

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

Target gene:

The tester strains used for the study were Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537 and TA102.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by induced by Aroclor 1254

Test concentrations with justification for top dose:

In the preliminary cytotoxicity assay, no precipitation was observed at the test item highest concentration of 5000 µg/plate. Complete inhibition of background lawn (cytotoxicity) and reduction in the number of revertant colonies were observed at the concentration of 5000, 2500, 1250, 625.0 µg/plate. Similarly, partial inhibition of background lawn was observed at 312.5 µg/plate when compared to vehicle control. Based on the results of preliminary cytotoxicity assay, test item concentrations of 19.53, 39.06, 78.13, 156.3, and 312.5 µg/plate are selected for testing in the main mutagenicity study.

Vehicle / solvent:

Dimethyl sulphoxide (Lot No.SHBJ6335, Sigma-Aldrich) was used as vehicle to prepare different dilutions of the test item. Vehicle control was plated for all strains in the presence and absence of S9 mix.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 were procured from Molecular Toxicology, Inc. PO Box 1189 Boone, NC 28607 USA.
Stock cultures of tester strains in oxoid nutrient broth no. 2 with 10% DMSO are stored in the test facility as frozen permanents at -80±10ºC.

Rationale for test conditions:

The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many test items including a wide range of chemical classes. Additionally, the tester strains of Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 are the recommended by regulatory agencies for conducting Bacterial Reverse Mutation Test.

Evaluation criteria:

The following criteria were used to determine a valid assay before the data evaluation for interpretation of results.
- Regular background growth in the solvent control and negative control (if any).
- To demonstrate that appropriate numbers of bacteria are plated, density of the tester strain cultures must be greater than or equal to 0.5 x 109 bacteria/mL.
- The positive control substances should produce a significant increase in mutant colony frequencies over the vehicle control plates.
- The spontaneous reversion rates in the solvent control and negative control (if any) is in the range of our historical data.
- Minimum five analyzable test item doses should be available to evaluate mutation frequencies.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item, Safranal was assessed for its potential to induce gene mutation by plate incorporation and pre-incubation method using Salmonella typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA102 both in the presence and absence of metabolic activation along with vehicle and positive controls. No significant increase in the revertant colony count was observed in any of the tested concentrations, both in the presence and absence of metabolic activation, when compared to the vehicle control.
The spontaneous revertant colony count of vehicle and positive controls was within the range of laboratory historical control data.

Conclusions:

It is thus concluded that the test item, Safranal is Non-Mutagenic up to the highest tested concentrations of 312.5 µg/plate in the five Salmonella typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) in the presence (10% S9 mix) and absence of metabolic activation, as measured by the Bacterial Reverse Mutation Test, under the conditions of the test employed.

Executive summary:

The test item, Safranal from Organica Aromatics Private Limited, was evaluated in a Bacterial Reverse Mutation Assay as per OECD Guideline No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test item, Safranal was tested for its ability to induce reverse mutations at the histidine locus in tester strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) in the presence and absence of an exogenous mammalian metabolic activation system (S9).

The test item was found to be soluble in DMSO at a concentration of 50 mg/mL and no precipitation was observed at the highest tested concentration i.e. 5000 µg/plate. On the basis of solubility and precipitation tests, the Preliminary Cytotoxicity Test was performed at 39.06, 78.13, 156.3, 312.5, 625.0, 1250, 2500 and 5000µg/plate with Salmonella typhimurium TA100 both in the presence and absence of a metabolic activation system.

Complete inhibition of background lawn and reduction in the number of revertant colonies were observed at the concentration of 5000, 2500, 1250, 625.0 µg/plate. Similarly, partial inhibition of background lawn was observed at 312.5 µg/plate when compared to vehicle control. Therefore, on the basis of preliminary cytotoxicity assay results, the Trial I and Trial II were performed at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate both in the presence and absence of metabolic activation. Vehicle control (dimethyl sulfoxide) and appropriate positive controls (2- nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials “without metabolic activation” and 2- Aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

In Trial I, tester strains TA98, TA100, TA1535, TA1537 and TA102 were treated with test item at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate, along with vehicle and positive controls both in the presence and absence of metabolic activation by plate incorporation method. No significant increase in revertant colony count was observed both in the presence and absence of metabolic activation in any of the tested concentrations up to 312.5 µg/plate, when compared to vehicle control. The partial inhibition of bacterial lawn was observed at 312.5 µg/plate both in the presence and absence of metabolic activation.

In Trial II, all the tester strains, TA98, TA100, TA1535, TA1537 and TA102 were treated with test item at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate by pre-incubation method along with vehicle and positive controls, both in the presence and absence of metabolic activation. In all the tester strains, no significant increase in revertant colony count was observed both in the presence and absence of metabolic activation in any of the tested concentrations up to 312.5 µg/plate, when compared to vehicle control. The partial inhibition of bacterial lawn was observed at 312.5 µg/plate both in the presence and absence of metabolic activation.

The spontaneous revertant frequency of the vehicle control group was within range of historical control data. The positive controls used in the study exhibited significant increase in mean number of revertant frequency respective to their strains, indicating that the sensitivity of the tester strains towards specific mutagens and confirmed that the test conditions were adequate and that the metabolic activation system functioned properly.

It is thus concluded that the test item, Safranal is Non-Mutagenic up to the highest tested concentrations of 312.5 µg/plate in the five Salmonella typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) in the presence (10% S9 mix) and absence of metabolic activation, as measured by the Bacterial Reverse Mutation Test, under the conditions of the test employed.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 6, 2019 to April 20, 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Metabolic activation system:

Name: S-9 mix for chromosomal aberration test
Manufacturer: Idea Trading Corporation
Batches used: RAA201910A (for cell growth inhibition test) and RAA201911B (for micronucleus test)
Animal: Rat, SD; sex: male; age in weeks: 7 weeks old; body weight: 195 - 243g
Inducing substances: Phenobarbital (PB) and 5,6-benzoflavone(5,6-BF)

Test concentrations with justification for top dose:

The dose levels of the test substance were set on the basis of the results of the cell growth inhibition test. The setting doses and the reason for selection are shown below. When RPD or RICC was less than 50%, it was judged that cytotoxicity was defined. In "S9 mix” and “+S9 mix" and 24 hours continuous treatment, the following doses were set to obtain the dose at which RPD or RICC was 40% or more and 50% or less.

Setting doses of test substance for:
1. Short-term treatment, without S9 mix: 2.50, 5.00, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0 and 40.0 μg/mL
2. Short-term treatment, with S9 mix: 20.0, 40.0, 50.0, 60.0, 65.0, 70.0, 75.0, 80.0 and 90.0 μg/mL
3. 24 hours continuous treatment: 0.313, 0.625, 1.25, 2.50, 5.00, 7.50, 10.0, 15.0 and 20.0 μg/mL

Vehicle / solvent:

Name: DMSO
Manufacturer: DOJINDO Laboratories
Lot number: NT029
Purity: 99.9%
Grade: UV spec仕ophotometricgrade

The test substance was insoluble in distilled water but dissolved in DMSO at 150 mg/m.L. The test substance solution of 150 mg/m.L prepared with DMSO was considered to be stable from the facts that there were no change in color, exothermic reaction nor gas generation at room temperature within 2 hours after preparation. Therefore, DMSO was selected as a vehicle.

Negative control substance was stored in a frozen place of cell experimental room No. 1 (permissible range：from 30°C to 10°C).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Positive control used for the short-term treatment without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide monohydrate

Remarks:

Positive control used for the short-term treatment with S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

Positive control used for the 24 hours continuous treatment

Details on test system and experimental conditions:

TEST SYSTEM:
- Cell line: Human lymphoblast cells (TK6 cells), from European Collection of Animal Cell Culture (ECACC)
- Doubling time: about 14 hours
- Mycoplasma: negative
- Storage: Cells were suspended in medium [RPMI 1640 medium (Life Technologies) and 10 vol% heat-inactivated horse serum (HS, PAALaboratories)] including 10 vol% DMSO and were frozen in liquid nitrogen.

EXPERIMENTAL CONDITIONS:
Procedure: after a pre-culture, a 50 mL of the cell suspension in the each flask was added to a 50 mL conical tube. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was removed. Fresh medium was added to each 50 mL conical tube to prepare 10 mL of the cell suspension. The cell suspension in all 50 mL conical tube was combined. A 1 mL of the cell suspension was added to each 25 cm2 basal area glass culture flask (KOKURA GARASU) and used for treatment. Because the test substance smelled and the corrosion of plastic was caused by the test substance, the glass culture flask was used for the treatment in this study.
Treatment: One flask was used for each dose. For calculation of Relative Population Doubling (RPD) and Relative Increase in Cell Count (RICC), the number of the cells in one flask were measured at the start of the treatment and the end of the culture using the same procedure as that at the end of the culture. Specimens were not prepared and observation of specimens was not carried out.
(1) For the short-term treatment without S9 mix (-S9 mix), the cells were treated for 3 hours by adding 4 mL of the fresh medium with 50 μL of the test substance solution or the vehicle to the cell suspension.
(2) For the short-term treatment with S9 mix (+S 9 mix), the cells were treated for 3 hours by adding 4.6 mL of fresh medium, 60 μL of the test substance solution or the vehicle, and 0.4 rnL of S9 mix to the cell suspension.
(3) For "- S9 mix” and “+S9 mix", after 3 hours treatment, the cell suspension was centrifuged at 1000rpm for 5 minutes and the supernatant was removed and the cells were washed with 2mL of the fresh medium. After the wash treatment was conducted 3 times, cells were then cultured for another 21 hours in 5 mL of fresh medium.
(4) For the 24 hours continuous treatment, the cells were treated for 24 hours by adding 4 mL of the fresh medium with 50 μL of the test substance solution or the vehicle to the cell suspension.

Evaluation criteria:

1. JUDGEMENT CRITERIA OF TEST RESULTS: the acceptability criteria was considered to be negative, which were either in a) or in b):
a) all results are inside the distribution of the historical data of the negative control group,
b) outside the distribution of the historical data of the negative control group, but none of the doses of the test substance exhibits a statistically significant increase compared with the conc町rentnegative control.
Providing that all acceptability criteria in case of c) to e) or f) were fulfilled, a test substance was considered to be positive:
c) outside the distribution of the historical data of the negative control group,
d) the doses of the test substance exhibits a statistically significant increase compared with the concurrent negative control,
e) the increase of the frequencies of cells with micronuclei is dose-related,
f) both micronucleus test and confirmation test are fulfilled in case of c) and d).

2. ACCEPTABILITY CRITERIA OF THE TEST:
a) The frequencies of cells with micronuclei in the concurrent negative control are "under control" in quality control method used for control chart in the historical data.
b) The frequencies of cells with micronuclei in the concurrent positive control are "under control" in quality control method used for control chart in the historical data.
Concurrent positive controls produce a statistically significant increase compared with the concurrent negative control.
c) The PD is 1.0 or more in the negative control.

Statistics:

Statistical method was performed in order to evaluate the frequencies of cells with micronuclei using the statistical tool “StatLight” (Yukms). Significance level of the test was both sides of 5%.
Fisher's exact test was performed in order to compare in the negative control group with positive control group.
Since the frequencies of cells with micronuclei in each test substance group were inside the distribution of the historical data for the negative control group, Fisher’s exact test was not tested in order to compare in the negative control group with the test substance group and the dose-response relation was not tested by the Cochran-Armitage trend test.

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In each treatment method, the frequencies of cells with micronuclei in the negative and the positive control groups were within the range of the historical data. The frequency of cells with micronuclei in the positive control group showed a statistically significant increase compared with the negative control group. The PD was 1.0 or more in the negative control.
All the acceptability criteria were satisfied, so the present study was judged to be appropriately performed.
As a result of the micronucleus test, the frequencies of cells with micronuclei at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control. Therefore, it was judged be negative.

Conclusions:

It was concluded that Safranal P did not induce the micronuclei under the present test conditions.

Executive summary:

The ability of Safranal P to induce micronuclei was examined by using human lymphoblast cells (TK6 cells).

In the micronucleus test, the observation doses for evaluation were selected to be 10.0, 20.0 and 25.0 μg/mL in the short-term treatment without S9 mix and 20.0, 50.0 and 60.0 μg/mL in the short-term treatment with S9 mix and 2.50, 7.50 and 10.0 µg/mL in the 24 hours continuous treatment.

The frequencies of cells with micronuclei at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control.

Therefore, it was judged be negative.

Consequently, it was concluded that Safranal P did not induce the micronuclei under the present test conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 20 November 2019 to November 2020

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:
-Species: Rat
- Strain: Sprague Dawley
- Tissue: Liver
- Inducing Agents: Phenobarbital – 5,6-Benzoflavone
- Producer: MOLTOX,Molecular Toxicology, Inc.
- Batch Number: 3971

The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL):
- S9 tissue fraction: 0.408mL
- NADP (30 mM): 0.204mL
- G-6-P (590 mM): 0.204mL
- KCl (150 mM): 0.204mL
- Complete medium (5%): 8.98mL
- Volume total: 10.0 mL

Test concentrations with justification for top dose:

Solutions of the test item were prepared immediately before use in dimethylsulfoxide (DMSO) on a volume/volume basis. Concentrations were expressed in terms of test item as received. All test item solutions were used within 20 minutes from the initial preparation.
All dose levels in this report are expressed to three significant figures.

Vehicle / solvent:

The solvent used in this study was dimethylsulfoxide (DMSO), batch nos.: H079S and J128BIL obtained from Honeywell.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive controls in the absence of S9 metabolism.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Positive controls in the presence of S9 metabolism.

Details on test system and experimental conditions:

L5178Y TK+/− (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville,Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination. Permanent stocks of the L5178Y TK+/− cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Prior to use, cells were cleansed of pre-existing mutants.

The Study was initially designed in the following way: first the cytotoxicity of the test item was determined, and dose levels were selected for the mutation assay. In the first mutation assay (Main Assay I), the cells were exposed to the test item for a short treatment time (3 hours) both in the absence and presence of S9 metabolism. Due to the equivocal results obtained in the treatment series in the absence of S9 metabolism, an additional mutation assay (Main Assay II) was carried out. Cells were treated for 3 hours in the absence of S9 metabolism and a modified dose-range was used to focus on the highest concentrations that could be tested. Due to the excessive cytotoxicity observed in the second mutation assay, where only two concentrations were analysable, an additional mutation assay was performed. As agreed with the SponsorMain Assay III was carried out in the absence of S9 metabolic activation and cells were exposed to the test item both for a short (3 hours) and a long (24 hours) treatment time.

Evaluation criteria:

The assay was considered valid if the following criteria were met:
1. The cloning efficiencies at Day 2 in the untreated/solvent control cultures fell within the range of 65-120%.
2. The untreated/solvent control suspension growth over 2 days fell within the range: 8-32 (3 hour treatment), 32-180 (24 hour treatment).
3. The mutant frequencies in the untreated/solvent control cultures fell within the range of 50−170×10−6 viable cells.
Every assay was also evaluated as to whether the positive control met at least one of the following two acceptance criteria:
1. The positive control induced a clear increase above the spontaneous background (induced mutent frequency = IMF) of at least 300×10−6. At least 40% of the IMF was reflected in the small colony MF.
2. The positive control induced a clear increase in the small colony IMF of at least 150×10−6.

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126×10−6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis. Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (RobinsonW.D., 1990).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

It is concluded that SAFRANAL P does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item SAFRANAL P was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of rat liver S9 metabolic activation, using a fluctuation method. A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 1500 µg/mL using dimethylsulfoxide (DMSO) as solvent. This concentration corresponds to the upper limit to be tested as indicated in the study protocol, 0.01M. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1500 µg/mL and at a wide range of lower dose levels: 750, 375, 188, 93.8, 46.9, 23.4, 11.7 and 5.86 µg/mL.

In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived treatment from 93.8 µg/mL onwards. At the next lower dose levels from 46.9 to 5.86 µg/mL, a dose related toxicity was observed, reducing relative survival (RS) from 16% to 76% of the concurrent negative control value. Using the 24 hour treatment time, no cells survived treatment from 23.4 µg/mL onwards. At the two next lower dose levels (11.7 and 5.86 µg/mL) the relative survival was reduced to 14 and 41% of the concurrent negative control, respectively. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived treatment from 188 µg/mL onwards. Test item treatment at 93.8 µg/mL yielded severe toxicity reducing RS to 5%, while moderate toxicity was observed at 46.9 µg/mL, RS reduced to 47%. No relevant toxicity was noted over the remaining concentrations tested.

Based on cytotoxicity results, three independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

Assay No.:	S9	Treatment time (hours)	Dose level (µg/mL)
1	-	3	48.0, 40.0, 20.0, 10.0, 5.00, 2.50 and 1.25
1	+	3	80.0, 57.1, 40.0, 20.0, 10.0 and 5.00
2	-	3	58.0, 52.7, 47.9, 43.6, 39.6 and 19.8
3	-	3	58.0, 54.0, 47.0, 40.8, 35.5, 20.4, 10.2 and 5.10
3	-	24	14.0, 11.7, 9.72, 7.78, 4.86, 2.43, 1.22 and 0.608

Negative and positive control treatments were included in each mutation assay in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. The study was accepted as valid.

Adequate levels of cytotoxicity, covering a range from the maximum toxicity, RTG value between 10-20 %, to slight or no toxicity, were observed in Main Assay I for the 3 hour treatment time in the presence of S9 metabolism. In Main Assay I for the 3 hour treatment time in the absence of S9 metabolism, no adequate level of cytotoxicity were obtained at the highest concentration tested, where the RTG value was 24% of the concurrent negative control (acceptability range 10-20%). For this reason, an additional mutation assay (Main Assay II) was carried out. Due to the excessive cytotoxicity observed in the second mutation assay, where only two concentrations were analysable, an additional mutation assay was performed. As agreed with the Sponsor, Main Assay III was carried out in the absence of S9 metabolic activation and cells were exposed to the test item both for a short (3 hours) and a long (24 hours) treatment time. Adequate levels of cytotoxicity, covering a range from the maximum toxicity, RTG value between 10-20 %, to slight or no toxicity, were observed in Main Assay III, both treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. It is concluded that SAFRANAL P does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study : OECD TG 471, 2019: The test item, Safranal from Organica Aromatics Private Limited, was evaluated in a Bacterial Reverse Mutation Assay as per OECD Guideline No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997.

The test item, Safranal was tested for its ability to induce reverse mutations at the histidine locus in tester strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) in the presence and absence of an exogenous mammalian metabolic activation system (S9).

The test item was found to be soluble in DMSO at a concentration of 50 mg/mL and no precipitation was observed at the highest tested concentration i.e. 5000 µg/plate. On the basis of solubility and precipitation tests, the Preliminary Cytotoxicity Test was performed at 39.06, 78.13, 156.3, 312.5, 625.0, 1250, 2500 and 5000µg/plate with Salmonella typhimurium TA100 both in the presence and absence of a metabolic activation system.

Complete inhibition of background lawn and reduction in the number of revertant colonies were observed at the concentration of 5000, 2500, 1250, 625.0 µg/plate. Similarly, partial inhibition of background lawn was observed at 312.5 µg/plate when compared to vehicle control. Therefore, on the basis of preliminary cytotoxicity assay results, the Trial I and Trial II were performed at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate both in the presence and absence of metabolic activation. Vehicle control (dimethyl sulfoxide) and appropriate positive controls (2- nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials “without metabolic activation” and 2- Aminoanthracene for trials “with metabolic activation”) were tested simultaneously.

In Trial I, tester strains TA98, TA100, TA1535, TA1537 and TA102 were treated with test item at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate, along with vehicle and positive controls both in the presence and absence of metabolic activation by plate incorporation method. No significant increase in revertant colony count was observed both in the presence and absence of metabolic activation in any of the tested concentrations up to 312.5 µg/plate, when compared to vehicle control. The partial inhibition of bacterial lawn was observed at 312.5 µg/plate both in the presence and absence of metabolic activation.

In Trial II, all the tester strains, TA98, TA100, TA1535, TA1537 and TA102 were treated with test item at 19.53, 39.06, 78.13, 156.3 and 312.5 µg/plate by pre-incubation method along with vehicle and positive controls, both in the presence and absence of metabolic activation. In all the tester strains, no significant increase in revertant colony count was observed both in the presence and absence of metabolic activation in any of the tested concentrations up to 312.5 µg/plate, when compared to vehicle control. The partial inhibition of bacterial lawn was observed at 312.5 µg/plate both in the presence and absence of metabolic activation.

The spontaneous revertant frequency of the vehicle control group was within range of historical control data. The positive controls used in the study exhibited significant increase in mean number of revertant frequency respective to their strains, indicating that the sensitivity of the tester strains towards specific mutagens and confirmed that the test conditions were adequate and that the metabolic activation system functioned properly.

It is thus concluded that the test item, Safranal is Non-Mutagenic up to the highest tested concentrations of 312.5 µg/plate in the five Salmonella typhimurium tester strains (TA98, TA100, TA1535, TA1537 and TA102) in the presence (10% S9 mix) and absence of metabolic activation, as measured by the Bacterial Reverse Mutation Test, under the conditions of the test employed.

Key study : OECD TG 487, 2020: The ability of Safranal P to induce micronuclei was examined by using human lymphoblast cells (TK6 cells).

In the micronucleus test, the observation doses for evaluation were selected to be 10.0, 20.0 and 25.0 μg/mL in the short-term treatment without S9 mix and 20.0, 50.0 and 60.0 μg/mL in the short-term treatment with S9 mix and 2.50, 7.50 and 10.0 µg/mL in the 24 hours continuous treatment.

The frequencies of cells with micronuclei at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control.

Therefore, it was judged be negative. Consequently, it was concluded that Safranal P did not induce the micronuclei under the present test conditions.

Key study : OECD TG 490, 2020: The test item SAFRANAL P was examined for mutagenic activity by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of rat liver S9 metabolic activation, using a fluctuation method. A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 1500 µg/mL using dimethylsulfoxide (DMSO) as solvent. This concentration corresponds to the upper limit to be tested as indicated in the study protocol, 0.01M. On the basis of this result, a cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1500 µg/mL and at a wide range of lower dose levels: 750, 375, 188, 93.8, 46.9, 23.4, 11.7 and 5.86 µg/mL.

In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived treatment from 93.8 µg/mL onwards. At the next lower dose levels from 46.9 to 5.86 µg/mL, a dose related toxicity was observed, reducing relative survival (RS) from 16% to 76% of the concurrent negative control value. Using the 24 hour treatment time, no cells survived treatment from 23.4 µg/mL onwards. At the two next lower dose levels (11.7 and 5.86 µg/mL) the relative survival was reduced to 14 and 41% of the concurrent negative control, respectively. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived treatment from 188 µg/mL onwards. Test item treatment at 93.8 µg/mL yielded severe toxicity reducing RS to 5%, while moderate toxicity was observed at 46.9 µg/mL, RS reduced to 47%. No relevant toxicity was noted over the remaining concentrations tested.

Based on cytotoxicity results, three independent assays for mutation at the TK locus were performed using the dose levels described in the following table:

Assay No.:	S9	Treatment time (hours)	Dose level (µg/mL)
1	-	3	48.0, 40.0, 20.0, 10.0, 5.00, 2.50 and 1.25
1	+	3	80.0, 57.1, 40.0, 20.0, 10.0 and 5.00
2	-	3	58.0, 52.7, 47.9, 43.6, 39.6 and 19.8
3	-	3	58.0, 54.0, 47.0, 40.8, 35.5, 20.4, 10.2 and 5.10
3	-	24	14.0, 11.7, 9.72, 7.78, 4.86, 2.43, 1.22 and 0.608

Negative and positive control treatments were included in each mutation assay in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. The study was accepted as valid.

Adequate levels of cytotoxicity, covering a range from the maximum toxicity, RTG value between 10-20 %, to slight or no toxicity, were observed in Main Assay I for the 3 hour treatment time in the presence of S9 metabolism. In Main Assay I for the 3 hour treatment time in the absence of S9 metabolism, no adequate level of cytotoxicity were obtained at the highest concentration tested, where the RTG value was 24% of the concurrent negative control (acceptability range 10-20%). For this reason, an additional mutation assay (Main Assay II) was carried out. Due to the excessive cytotoxicity observed in the second mutation assay, where only two concentrations were analysable, an additional mutation assay was performed. As agreed with the Sponsor, Main Assay III was carried out in the absence of S9 metabolic activation and cells were exposed to the test item both for a short (3 hours) and a long (24 hours) treatment time. Adequate levels of cytotoxicity, covering a range from the maximum toxicity, RTG value between 10-20 %, to slight or no toxicity, were observed in Main Assay III, both treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. It is concluded that SAFRANAL P does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity