Decan-4-olide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

- Ames Tests: non mutagenic (OECD 471, WoE using γ-Undecalactone and γ-Caprolactone data) - MLA/TK Forward Mutation Assay with a Confirmatory Assay (OECD 476, GLP, K, rel. 2): ɣ-Nonalactone non mutagenic up to cytotoxicity limit - In vivo micronucleus test: ɣ-Undecalactone and ɣ-Nonalactone not clastogenic and not aneugenic in mice bone marrow cells up to limit dose (OECD 474, GLP, K, rel. 2) + in vitro CAT, ɣ-Caprolactone not clastogenic up to limit concentration (S, rel.2).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted similarly to OECD Guideline 474 with minor deviations: no data on the housing conditions and individual body weight at the start of the test; number of micronucleated immature erythrocytes for individual animals not reported. 1000 polychromatic erythrocytes (PCE) were analysed per animal, which was a requirement of the old version of the OECD 474 (1983). The new version require the analysis of at least 2000 PCE. γ-Undecalactone, as aliphatic γ-lactone, is considered adequate for read-across purpose (see §"Toxicokinetics").

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

no data on the housing conditions and body weight at the start of the test; number of micronucleated immature erythrocytes for individual animals not reported

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: ddY mice

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka, Japan
- Age at study initiation: 8 weeks
- Diet: Food pellets CE-2 (Japan Clea, Tokyo, Japan), ad libitum
- Water: ad libitum

Route of administration:

intraperitoneal

Vehicle:

Olive oil

Details on exposure:

None

Duration of treatment / exposure:

- 250, 500, 1000 and 2000 mg/kg bw: One day
- 500 mg/kg bw: 4 days

Frequency of treatment:

- 250, 500, 1000 and 2000 mg/kg bw: Single injection
- 500 mg/kg bw: 4 injections with 24 h intervals between the injections

Post exposure period:

24 h

Remarks:

Doses / Concentrations:
250, 500, 1000 and 2000 mg/kg bw
Basis:
other: actual injected

No. of animals per sex per dose:

6 males/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin-C
- Source: Kyowa Hakko Ltd, Tokyo, Japan
- Route of administration: Intraperitoneal
- Doses: 2 mg/kg bw

Tissues and cell types examined:

- Femora bones were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs) and total erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The maximum doses of the test material were set at the supposed maximum tolerated dose referring to LD50.

DETAILS OF SLIDE PREPARATION: Mice were killed by cervical dislocation at 24 h after exposure. Femoral marrow cells were flushed out with fetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 minutes and stained with Giemsa stain.

METHOD OF ANALYSIS:
- 1000 polychromatic erythrocytes (PCEs) per mouse were scored using a light microscope with a high power objective (X 100) and the no. of micronucleated polychromatic erythrocytes (MNPCEs) were recorded.
- Proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was evaluated by observing 1000 erythrocytes on the same slide.

Evaluation criteria:

Result was considered as positive if one or more treatment group(s) showed a statistically significant difference (P < 0.01) from the spontaneous level of MNPCEs and the trend test indicated a positive dose response (P < 0.05).

Statistics:

Two-stage statistical procedure:
- In the first step of the procedure, the frequency of MNPCEs in each treatment group was compared with the binomial distribution specified by historical control data (Hayashi et al. 1985).
- In the second step, the dose-response relationship was tested by the Cochran- Armitage trend test (Armitage, 1955; Cochran, 1954; Margolin and Risko, 1988).

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- See table 7.6.2/1 for MNPCEs (%) and PCEs (%)

Table 7.6.2/1: Results of micronucleus test

Groups	Dose (mg/kg bw)	No. of doses	Time between doses (24 h)	Sampling time (h)	MNPCEs (%)	PCEs (%)	Mortality
Vehicle (Olive oil)	0	1	-	24	0.10 ± 0.06	54.6 ± 6.4	0/6
Undecalactone	250				0.25 ± 0.12	51.6 ± 7.7
	500				0.08 ± 0.08	43.9 ± 7.4
	1000				0.18 ± 0.08	43.7 ± 9.2
	2000				0.25 ± 0.07	52.3 ± 3.0
	500	4	24		0.08 ± 0.10	48.7 ± 8.1
Mitomycin-C	2	1	-		4.78 ± 1.40*	43.5 ± 4.6

 

MNPCEs - Micronucleated polychromatic erythrocytes; PCEs - Polychromatic erythrocytes;

* value of MNPCE differ significantly from the historical control (p < 0.01)

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions, Undecalactone is not considered as clastogenic in an in vivo mouse bone marrow micronucleus assay according to the Directive 67/548/EEC and the Regulation (EC) No. 1272/2008 (CLP).

Executive summary:

In an in vivo mouse bone marrow micronucleus assay performed similarly to OECD Guideline 474, groups of ddY male mice (6/dose) were injected with Undecalactone in olive oil at 250, 500, 1000 and 2000 mg/kg bw (single treatment) and 500 mg/kg bw (4 injections with 24 h intervals between the injections) by intraperitoneal route. The positive control group was injected with mitomycin C at 2 mg/kg bw. Mice were killed by cervical dislocation 24 h after an administration. Femoral marrow cells were flushed out with fetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 minutes and stained with Giemsa stain. 1000 polychromatic erythrocytes (PCEs) per mouse were scored for recording the incidence of micronucleated polychromatic erythrocytes (MNPCEs). The proportion of PCEs among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

Mice treated with Undecalactone did not show any significant increase in the frequency of MNPCEs. Positive control (mitomycin C) induced a statistically significant increase in MNPCEs indicating the validity of the study.

Under the test conditions, Undecalactone is not considered as clastogenic in an in vivo mouse bone marrow micronucleus assay according to the criteria of the Annex VI of the Directive 67/548/EEC and the Annex I of the Regulation (EC) No. 1272/2008 (CLP).

γ-Undecalactone, as aliphatic γ-lactone, is considered adequate for read-across purpose (see §"Toxicokinetics").

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Table 7.6/1: Summary of genotoxicity tests

 

Test n°	Test / Guideline Reliability	Focus	Strains / Cells tested	Metabolic activation	Test concentration	Statement
1   Ishidate, 1984	Ames Test (eq. OECD 471) WoE, rel. 4	γ-Undecalactone   Gene mutation	TA 1535, TA 1537, TA92, TA94, TA 98, TA 100	-S9 +S9	Up to 5000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
2   Masayuki Kato, 2002	Ames Test (eq. OECD 471) WoE, rel. 2	γ-Caprolactone   Gene mutation	TA 1535, TA 1537, TA 98, TA 100, WP2uvrA	-S9 +S9	Up to 5000 µg/plate	-S9 : non mutagenic +S9 : non mutagenic
3 Woods, 2012	ML/TK test (OECD 476) K, rel. 1	γ-Nonalactone   Gene mutation	mouse lymphoma L5178Y cells	-S9 +S9	Up to 800 µg/mL –S9 Up to 1562 µg/mL (10mM) +S9	-S9 : non mutagenic +S9 : non mutagenic
4   Honarvar, 2009	In vivo micronucleus (OECD 474, WoE, rel.2)	γ-Nonalactone   Chromosomal aberration	Bone marrow cells (femur)	NA	Up to 2000 mg/kg bw	Not clastogenic
5   Hayashi, 1988	In vivo micronucleus (eq. OECD 474) WoE, rel. 2	γ-Undecalactone   Chromosomal aberration	Bone marrow cells (femur)	NA	Up to 2000 mg/kg bw	Not clastogenic
6   Kawamura, 2002	CHL/CAT S, rel.2	γ-Caprolactone   Chromosomal aberration	Chinese Hamster lung fibroblasts	-S9 +S9	Up to 5000 µg/mL (cytotoxic)	-S9 : non clastogenic +S9 : non clastogenic

  

Gene mutation Assays (Tests n° 1-3):

Two Bacterial Reverse mutation Assays(Ames test)were performed similarly to the OECD 471 test guideline with the read-across substance γ-Undecalactone and γ-Caprolactone (See §”Toxicokinetics” for read-across justification). Both tests were used in a weight-of evidence approach as only four strains of bacteria were used in the Test n°1 whereas all required strains were used in Test n°2. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both tests, with any dose of the test materials up to limit concentration, either in the presence or absence of metabolic activation. The tests indicate that γ-Undecalactone and γ-Caprolactone did not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.Based on this weight-of evidence,γ-Decalactone is therefore considered as non-mutagenic according to the Ames test.

Inability to produce gene mutation was confirmed in mammals using anin vitroforward mutation assay in mouse lymphoma TK L5178Y cells(ML/TK test)(Test n°3). None of the dose levels up to the cytotoxicity limit with the read-across substance, ɣ-Nonalactone, either with or without metabolic activation, induced significant mutant frequency increases in the initial, or repeat tests. ɣ-Nonalactone does not induce forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. ɣ-Nonalactoneis therefore considered as negative for inducing forward mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay.This result confirms the results of both Ames tests and extends the non-mutagenic effect of ɣ-lactones to mammalian cells.

 

Chromosomal aberration (Tests n°4-5)

The clastogenic and aneugenic potential of the read-across substances, γ-Nonalactone and γ-Undecalactone were determined using the in vivo mammalian erythrocytes micronucleus assay (Test n°4 & 5), which measured the potential of a substance to increase the incidence of the structural and numerical chromosome aberrations in mice bone marrow erythrocytes. In both tests, none of the dose level, up to the limit dose of 2000 mg/kg bw, induced increase in the frequency of micronucleated polychromatic erythrocytes (fMPCE) in bone marrow, whereas positive control chemical induced significant increase in the fMPCE. γ-Nonalactone and γ-Undecalactone were therefore considered as negative for inducing chromosomal aberrations in mice bone marrow erythrocytes under the conditions used in the studies.

This result is supported by anin vitrochromosome aberration tests in Chinese hamster lung fibroblast, conducted on the read-across substance, γ-Caprolactone (Test n°6). In this test, none of the dose levels up to the cytotoxicity limit, either in the presence of absence of metabolic activation, induced significant increases in the frequency of cells with aberrations.

Based on this weight of evidence, γ-Decalactone is therefore considered as non-clastogenic and non-aneugenic.

Justification for selection of genetic toxicity endpoint
See Table 7.6/1 below.

Justification for classification or non-classification

Harmonized classification:

γ-Decalactone has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 including the ATP2.

Self classification:

Based on the available data, no additional classification is proposed according to the Regulation (EC) No. 1272/2008 (CLP) and the Directive 67/548/EEC.