Reaction mass of undec-8-enal and undec-9-enal and undec-10-enal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacteria (OECD TG 471, Ames): negative

In vitro micronucleus test: negative (read across from Undecanal tested in OECD TG 487)

Gene mutation in mammalian cells: negative (read across from Undecanal tested in OECD TG 476)

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:

02-12-2016 to 19-01-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan genes

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method)

Without S9: Initial: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate ; Amended: TA100, TA1535 and TA1537 (without S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
With S9: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate for TA100, TA98, TA1537 and E.coli. For TA1535: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Results from the first mutation test showed significant toxicity of the test item to the bacterial tester strains and consequently an insufficient number of non-toxic dose levels.

Experiment 2 (pre-incubation method)

Without S9: 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate (based on results of experiment 1).
With S9: 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate (based on results of experiment 1).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: according to guidelines

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1: Plate incorporation methodology
Experiment 2: Pre-incubation methodology

DURATION
- Preincubation period: with and without S9-mix 20 minutes (prior to exposure in experiment 2 only)
- Exposure duration: 48 hours (experiment 1 and 2)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn measurement and reduction in revertant colonies compared to the controls

Evaluation criteria:

There are several criteria for determining a positive result. Any one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2 uvrA

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 150 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2uvrA

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 5 μg/plate (TA 1535), 15 μg/plate (TA 1537 & TA 100) and 50 μg/plate (TA98 and WP2uvrA).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
-Precipitation: No precipitation was observed.

HISTORICAL CONTROL DATA:
-A history profile of vehicle, untreated and positive control values (reference items) can be found in the attached illustration.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
-In exp 1 (plate incorporation method): the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 μg/plate in the absence of S9-mix and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit was employed as the maximum dose in the second mutation test.
-In exp 2 (pre-incubation method): The test item induced a stronger toxic response in the second mutation test (pre-incubation method), with weakened bacterial background lawns noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA100 and TA1537) and 50 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were noted to all of the tester strains from 150 μg/plate.

ADDITIONAL INFORMATION ON TEST RESULTS
-In exp 1 (plate incorporation method): 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).
-In exp 2 (pre-incubation method): no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). Statistically significant increases in TA1535 revertant colony frequency were observed in the second mutation test at 150 μg/plate in the presence of S9-mix. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted at the same dose level. Therefore these responses would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

Conclusions:

Under the conditions of this study, Intreleven aldehyde was determined to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenic activity of Intreleven aldehyde was evaluated in accordance with OECDG 471 and GLP principles. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of S9-mix. Based on results of experiment 1 (plate incorporation), the dose range used for experiment 2 (pre-incubation method) was between 0.05 and 150 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and background lawn, was observed. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 μg/plate in the absence of S9-mix and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit was employed as the maximum dose in the second mutation test. Weakened bacterial background lawns were noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA100 and TA1537) and 50 μg/plate (TA98 and WP2uvrA) in the second experiment. In the presence of S9-mix, weakened bacterial background lawns were noted to all of the tester strains from 150 μg/plate. These increases were considered to have no biological relevance. No precipitation was observed at any of the concentrations. Adequate negative and positive controls were included. 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) for both experiments. Intreleven aldehyde was therefore considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2010- 8 October 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Intreleven aldehyde.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

other: human

Details on mammalian cell type (if applicable):

- Cell type: lymphocytes
- Type and identity of media: RPMI 1640 medium, containing fetal calf serum, gentamycin and heparin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix, contained 10% v/v S9 fraction from Aroclor induced male Sprague Dawley rat liver fraction, and cofactors

Test concentrations with justification for top dose:

0, 1.25, 2.5, 5.0, 10, 20, 40, 80, 160, 300 and 600 µg/mL (precipitation at 600 µg/mL)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: low water solubility of the test substance

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin and cyclophosphamide

Details on test system and experimental conditions:

CELL CULTURE
Human blood cell cultures were treated for 48 hours with phytohemagglutinin to stimulate lymphocyte division.

DURATION
- Exposure duration: 4 hrs (with and without S-9 mix) and 24 hrs in the absence of S-9 mix
- Expression time (cells in growth medium): 24 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): up to 48 hrs

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): acridine orange

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2000 binucleated cells/dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, CBPI (cytokinesis block proliferation index)

OTHER EXAMINATIONS:
- Determination of polyploidy: no

Evaluation criteria:

The vehicle/negative control results should lie within or close to the negative historical control range. The positive controls should produce a substantial increase in the incidence of MBC (at least twice) compared with the concurrent control; values should lay beyond the 99% upper limit of the historical negative/vehicle control range.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 600 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of Results

Treatment		Conc. (mg/mL)		Average CBPI		Cytotoxicity (%)^a		Total No. of BC examined		Total Number of MBC		% MBC
4 hours treatment in the absence of S9 (0S9)
Vehicle		-		1.9		0		2000		12		0.6
n-Undecanal		80.0		1.8		10		2000		14		0.7
		160		1.9		6		2000		10		0.5
		300		1.8		14		2000		8		0.4
		600		1.5		45		1698^b		8		0.5
MMC		0.3		1.6		30		2000		134		6.7*
4 hours treatment in the presence of S9 (+S9)
Vehicle		-		1.9		0		2000		8		0.4
n-Undecanal		160		1.8		11		2000		16		0.8
		300		1.7		24		2000		9		0.5
		600		1.6		30		2000		12		0.6
CP		10		1.5		41		2000		91		4.6*
24 hours treatment in the absence of S9 (0S9)
Vehicle		-		2.0		0		2000		3		0.2
n-Undecanal		40.0		1.9		10		2000		4		0.2
		80.0		1.7		25		2000		10		0.5
		160		1.5		44		2000		15		0.8
		300		1.2		84		NR
MMC		0.2		1.6		42		2000		334		16.7*

CBPI                   Cytokinesis-Block Proliferation Index

BC, MBC            Binucleated cells, micronucleated binucleated cells (%MBC calculated based on rounded values)

NR                        Not reported as considered excessively toxic (Cytotoxicity > 60% relative to vehicle control)

*                            Substantial positive response (at least twice the concurrent vehicle control in terms of %MBC)

a                            Cytotoxicity calculation based on unrounded values

b                            Not enough cells available

 

Conclusions:

Undecanal was not clastogenic in the in vitro micronucleus test using human lymphoctyes, with and without metabolic activation.

Executive summary:

Undecanal was tested in an in vitro micronucleus test using human lymphocytes, with and without metabolic activation, for its potential to induce micronuclei formation. The test was conducted under GLP conditions and according to the OECD TG 487. Cells were treated with the test substance for 4 hours in the presence and absence of metabolic activation and additionally for 24 hours in the absence of metabolic activation. The following concentrations were tested: 40, 80, 160, 300 and 600 µg/mL, which were selected based on preliminary studies. Precipitation was seen at 600 µg/mL. At the end of a 24-hour exposure period, cytochalasin B was added to the cells, which were then harvested and stained and 2000 binucleated cells/dose level were then examined for the occurrence of micronuclei. The results were compared with those of the concurrent and historical vehicle (DMSO) and positive controls (mitomycin and cyclophosphamide). The negative and positive controls performed as expected. Undecanal was tested up into cytotoxic concentrations, but did not induce the formation of micronuclei, with or without metabolic activation. Thus, Undecanal was not genotoxic (clastogenic) in this assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The result derived from read across is sufficiently reliable because all Annex XI criteria are met.

Justification for type of information:

The read across justification is presented in the Endpoint summary and the accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Intreleven aldehyde was found not clastogenic based on read across from Undec-10-enal tested in an in vitro micronucleus test using human lymphoctyes, with and without metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-07-2010 to 21-09-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Intreleven aldehyde.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL) and amphotericin B (1 %).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9 from male Wistar rats

Test concentrations with justification for top dose:

1st experiment:
exposure 4 hours: 0.6-15 µg/mL without S-9 mix // 7.2-230 with S-9 mix

2nd experiment:
exposure 24 hours: 0.6-25 µg/mL without S-9 mix
exposure 4 hours: 7.8-500 µg/mL with S-9 mix

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: low solubility of test article

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: EMS (ethylmethane sulfonate), DMBA (7,12-dimethylbenz(a)anthracene)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hrs in the 1st experiment; 24 hrs without S-9 mix, 4 hrs with S-9 mix, in the 2nd experiment
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 or 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 or 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF EXPERIMENTS: 2

NUMBER OF CELLS EVALUATED: all surviving colonies counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response (i.e. at least 3 times the spontaneous mutation frequency) at one of the test points.

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

7.5-20 µg/mL/without S-9 mix); 500 µg/mL with S-9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was seen at 115 µg/mL and above (only with metabolic activation).

Remarks on result:

other: all strains/cell types tested

Tab 1: The cell cultures were evaluated at the following concentrations:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	0.6	1.3	2.5	5.0	7.5	10.0*	15.0*
4 hours	+			28.8	57.5	115.0^P	172.5^P	230.0^P
		Experiment II
24 hours	-			2.5	5.0	10.0	15.0	20.0
4 hours	+			31.3	62.5	125.0^P	187.5^P	500.0^P

P = precipitation

*     mutagenicity evaluation was performed only in culture II

For further details see the Summary of results (attached document)

Conclusions:

Undecanal was not mutagenic in a mammalian cell HPRT assay, with and without metabolic activation.

Executive summary:

Undecanal was tested for its genotoxic potential in mammalian cells in vitro (Chinese hamster, V79 cells) at concentrations from 0.6 to 500 µg/mL, in the presence and absence of metabolic activation (7.2 -500

µg/mL in the presence and 0.6 -25 µg/mL in the absence). The assay was conducted according to the OECD TG 476 and GLP principles. Precipitation of Undecanal was seen at 115 µg/mL and above. In the first experiment, cytotoxicity was seen at 7.5 µg/mL and higher in the absence of metabolic activation (exposure period 4 hours), and no cytotoxicity was seen in the presence of metabolic activation. In the second experiment, cytotoxicity was seen at 20 µg/ml and higher without S-9 mix (exposure period 24 hours), and at 500 µg/mL with S-9 mix (exposure period 4 hours). Undecanal did not increase the mutation frequency to meet the criteria for rating the test substance as positive. Validity criteria were met, because vehicle and positive controls performed as expected and were comparable with historical control data. Therefore, Undecanal was considered to be non-mutagenic in this HPRT assay.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The result derived from read across is sufficiently reliable because all Annex XI criteria are met.

Justification for type of information:

The read across justification is presented in the Endpoint summary and the accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

7.5-20 µg/mL/without S-9 mix); 500 µg/mL with S-9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Intreleven aldehyde was found to be not mutagenic based on the read-across from Undecanal, which was tested in a mammalian cell HPRT assay, with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genotoxic toxicity of Intreleven aldehyde was assessed by an available Ames test and by using read across from Undecanal for the mutagenicity and chromosomal aberrations in mammalian cells. First the available information of Intreleven aldehyde is shown, followed by the description of the studies available for Undecanal. Thereafter, the read across justification is presented, the accompanying files are attached in the present endpoint summary.

Mutagenicity in bacteria with Intreleven aldehyde (Ames test)

The mutagenic activity of Intreleven aldehyde was evaluated in accordance with OECDG 471 and GLP principles. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of S9-mix. Based on results of experiment 1 (plate incorporation), the dose range used for experiment 2 (pre-incubation method) was between 0.05 and 150 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and background lawn, was observed. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 μg/plate in the absence of S9-mix and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit was employed as the maximum dose in the second mutation test. Weakened bacterial background lawns were noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA100 and TA1537) and 50 μg/plate (TA98 and WP2uvrA) in the second experiment. In the presence of S9-mix, weakened bacterial background lawns were noted to all of the tester strains from 150 μg/plate. These increases were considered to have no biological relevance. No precipitation was observed at any of the concentrations. Adequate negative and positive controls were included. 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) for both experiments. Intreleven aldehyde was therefore considered to be non-mutagenic under the conditions of this test.

In vitro micronucleus test with Undecanal tested in OECD TG 487

Undecanal was tested in an in vitro micronucleus test using human lymphocytes, with and without metabolic activation, for its potential to induce micronuclei formation. The test was conducted under GLP conditions and according to the OECD TG 487. Cells were treated with the test substance for 4 hours in the presence and absence of metabolic activation and additionally for 24 hours in the absence of metabolic activation. The following concentrations were tested: 40, 80, 160, 300 and 600 µg/mL, which were selected based on preliminary studies. Precipitation was seen at 600 µg/mL. At the end of a 24-hour exposure period, cytochalasin B was added to the cells, which were then harvested and stained and 2000 binucleated cells/dose level were then examined for the occurrence of micronuclei. The results were compared with those of the concurrent and historical vehicle (DMSO) and positive controls (mitomycin and cyclophosphamide). The negative and positive controls performed as expected. Undecanal was tested up into cytotoxic concentrations, but did not induce the formation of micronuclei, with or without metabolic activation. Thus, Undecanal was not genotoxic (clastogenic) in this assay.

Gene mutation in mammalian cells (Mouse Lymphoma Assay) with Undecanal tested in OECD TG 476

Undecanal was tested for its genotoxic potential in mammalian cells in vitro (Chinese hamster, V79 cells) at concentrations from 0.6 to 500 µg/mL, in the presence and absence of metabolic activation (7.2 -500

µg/mL in the presence and 0.6 -25 µg/mL in the absence). The assay was conducted according to the OECD TG 476 and GLP principles. Precipitation of Undecanal was seen at 115 µg/mL and above. In the first experiment, cytotoxicity was seen at 7.5 µg/mL and higher in the absence of metabolic activation (exposure period 4 hours), and no cytotoxicity was seen in the presence of metabolic activation. In the second experiment, cytotoxicity was seen at 20 µg/ml and higher without S-9 mix (exposure period 24 hours), and at 500 µg/mL with S-9 mix (exposure period 4 hours). Undecanal did not increase the mutation frequency to meet the criteria for rating the test substance as positive. Validity criteria were met, because vehicle and positive controls performed as expected and were comparable with historical control data. Therefore, Undecanal was considered to be non-mutagenic in this HPRT assay.

The in vitro cytogenicity / in vitro gene mutation in mammalian cells of Intreleven aldehyde (CAS 58296-81-4; Target) using read across from Undecanal (CAS 112-44-7; Source)

 

Introduction and hypothesis for the read across

Intreleven aldehyde is a multi-constituent which consists of the following main constituents: Undec-10-enal, (9E) Undec-9-enal, (9Z) Undec-9-enal and (8E) Undec-8-enal. These constituents are aldehydes with a linear carbon backbone and contain one C=C double bond at various positions ranging from C 8 to 10. For this substance an in vitro gene mutation study in bacteria (Ames) is available, which did not indicate any mutagenicity, but no data regarding in vitro cytogenicity or gene mutation in mammalian cells is available.

Therefore, additional information is used in accordance with Article 13 of REACH where it is said that lacking information can be generated whenever possible by means other than experimental testing, i.e. applying SARs, grouping and read-across. For assessing the cytogenicity of Intreleven aldehyde the analogue approach is selected because for one closely related analogue, Undecanal, cytogenicity information is available which can be used for read across.

Hypothesis:

The target substance Intreleven aldehyde is expected to have the same cytogenicity / gene mutation potential in mammalian cells as the analogue Undecanal.

Available information:

For the target, Intreleven aldehyde, negative Ames study is available (OECD TG 471). For the source substance Undecanal, an in vitro mammalian cell mutation (OECD TG 476,K1), and an in vitro micronucleus study (OECD TG 487, K1) are available. In both studies the source Undecanal tested negative for genotoxicity.

Target and Source chemical(s):

Chemical structures of the target chemical and the source chemical are shown in data matrix below, including physico-chemical properties and toxicological information, thought relevant for cytogenicity, of both substances.

Purity / Impurities:

The components and impurities of Intreleven aldehyde do not indicate cytogenicity potential other than indicated by the parent substance. The constituents are known for at least 95% and therefore this substance is well characterized. 

Analogue justification

According REACH Annex XI an analogue approach and structural alert information can be used to replace testing when information from different sources provides sufficient evidence to conclude that this substance has or does not have a particular dangerous property. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

Analogue selection: Undecanal was selected as analogue because of the close structural similarity and because adequate experimental information is available for this substance.

Structural similarities and differences: The target chemical, Intreleven aldehyde, and source chemical, Undecanal, both have the same linear carbon backbone (C11) and functional aldehyde group. The difference is that Undecanal does not have a C=C double bond which is present in Intreleven aldehyde. This difference is not anticipated to influence the cytogenicity and gene mutation potential of the source substance.

Toxicokinetics: Absorption of Intreleven aldehyde (target) and Undecanal (source) will be alike based on the similarity in being liquids, having similar molecular weights (168.28 and 170.30 g/mol, respectively) and similar water solubility (26.1 and 120 mg/l, respectively). Intreleven aldehyde has a slightly higher log Kow in comparison with Undecanal (4.47 vs 3.84), which can be regarded as an experimental error because the calculated values are almost the same, reflecting the unsaturated vs. saturated chain. Both substances are equally absorbed orally. The substances would pass through the biological cell membrane, and are likely to distribute into cells.

Metabolism: The target substance Intreleven aldehyde and its constituents are likely to be metabolized via a pathway analogous to that of unsaturated fatty acids, meaning beta oxidation in the liver with the formation of H₂O and CO₂. The metabolism of the source Undecanal substance will take place through beta-oxidation only, as it is saturated. These metabolic pathways are very similar and are unlikely to lead to differences in toxicity.

Toxicodynamics - genotoxicity

The target chemical Intreleven aldehyde and source chemical Undecanal are expected to have the same reactivity, based on the fact that both have the same functional aldehyde group. The similarity of these substances is supported further by negative results in their respective Ames tests.

Remaining uncertainties: There are no remaining uncertainties as presented above.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data matrix.

Conclusions for in vitro cytogenicity / in vitro gene mutation in mammalian cells

For the target substance Intreleven aldehyde, the potential for in vitro cytogenicity / in vitro gene mutation in mammalian cells was derived from Undecanal using read across. The results of this read-across assessment based on in vitro mammalian cell mutation study (OECD TG 476), as well as an in vitro micronucleus (OECD TG 487) indicated no genotoxic potential. This information can also be used for Intreleven aldehyde based on the similarity in structure, toxicokinetics and reactivity.

Final conclusion on hazard and application in the risk assessment:

Intreleven aldehyde is negative for cytogenicity and gene mutation in mammalian cells.

 

Data matrix for the read across from Undecanal to Intreleven aldehyde

Name of substance	Intreleven aldehyde, (Undec-8-enal)	Undecanal
Chemical structure
Cas no.	58296-81-4	112-44-7
REACH	To be registered (Annex VIII)	Registered (Annex VIII)
Molecular formula	C11H20O	C11H22O
Mol weight	168.28	170.30
Physico-chemical properties
Appearance	Liquid	Liquid
Melting point (^oC)	<-20 (IFF, 2016)	-10 (ECHA dissemination)
Boiling point (^oC)	239.1 (IFF, 2016)	225 (ECHA dissemination)
Vapour pressure (Pa at 25^oC)	6.04 (IFF, 2016)	8 (C) 38 (at 20^oC) (ECHA dissemination)
Water solubility (mg/l)	26.1 (IFF, 2016)	120 (ECHA dissemination)
Log Kow	4.04 (C) 4.47 (IFF, 2017)	4.25 (C) 3.84 (ECHA dissemination)
Human health
Genotoxicity – Ames test	Negative (OECD TG 471)	Negative (OECD TG 471)
Genotoxicity – in vitro mammalian cell gene mutation test	Read Across from Undecanal	Negative (OECD TG 476)
Genotoxicity – in vitro cytogenicity	Read Across from Undecanal	Negative (OECD TG 487)

For testing data, see the IUCLID under the relevant endpoint.

 (C) =Calculated with EpiSuite version 4.11

Justification for classification or non-classification

Based on the available information, Intreleven aldehyde does not need to be classified for mutagenicity in accordance with the criteria outlined in the EU CLP Regulation (EC No. 1272/2008 and its updates).