Nonan-4-olide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 July – 17 October 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 474 with minor deviations: relative humidity in the animal rooms ranged between 30 – 114 % instead of 30–70 %; body weight of the animals were not reported

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

relative humidity in the animal rooms ranged between 30 – 114 % instead of 30–70 %; body weight of the animals were not reported

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Nonan-4-olide

EC Number:

203-219-1

EC Name:

Nonan-4-olide

Cas Number:

104-61-0

Molecular formula:

C9H16O2

IUPAC Name:

5-pentyloxolan-2-one

Details on test material:

- Name of test material (as cited in study report): γ-nonalactone (Aldehyde C-18) FFC
- Analytical purity: 99.0 % (GLC)
- Purity test date: 04 March 2009
- Batch No.: 2007302-0001
- Expiration date of the batch: 29 October 2012
- Storage condition of test material: At room temperature under nitrogen

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, Borchen, Germany
- Age at start of acclimatization: 6 - 8 weeks
- Weight at study initiation: Male: 36.5 ± 1.8 g; female: 27.3 ± 2.0 g
- Housing: Animals were housed individually in Makrolon Type I cages, with wire mesh top
- Diet: Pelleted standard diet (Harlan Laboratories GmbH, Borchen, Germany), ad libitum
- Water: Tap water (Gemeindewerke, Rossdorf, Germany), ad libitum
- Acclimation period: Minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-114 %
- Photoperiod: 12 h dark / 12 h artificial light

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Source: Sigma-Aldrich Vertriebs GmbH, Deisenhofen, Germany
- Justification for choice of solvent/vehicle: Nontoxic to animals
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was formulated in corn oil.

Duration of treatment / exposure:

24 h (in all test groups) and 48 h (in 2000 mg/kg bw group)

Frequency of treatment:

Once

Post exposure period:

Not applicable

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

- Pre-experiment 1 and 2: 2/sex/dose
- Main experiment: 6/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Source: Sigma-Aldrich Vertriebs GmbH, Deisenhofen, Germany
- Solvent: Deionised water
- Route of administration: Oral
- Doses: 40 mg/kg bw
- Volume administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:

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

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Dose selection was based on the results of preliminary range-finding tests conducted on 2 mice/sex/dose at 1000 and 2000 mg/kg bw. No significant toxic signs were recorded.

TREATMENT AND SAMPLING TIMES:
- At 24 and 48 h after exposure, animals were sacrificed using CO2 followed by bleeding.
- Femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a syringe.
- Cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was discarded.

DETAILS OF SLIDE PREPARATION:
- A small drop of the re-suspended cell pellet was spread on a slide.
- Smear was air-dried and then stained with May-Grünwald /Giemsa stain.

METHOD OF ANALYSIS:
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives.
- At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei.
- To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.
- Ten animals (5 males, 5 females) per test group were evaluated.

Evaluation criteria:

- A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
- A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical significance at 5 % level (p < 0.05) was evaluated by non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF PRE-EXPERIMENT
- Dose range: 1000 mg/kg bw (first pre-experiment, 2/sex) and 2000 mg/kg bw (second pre-experiment, 2/sex)
- Clinical signs of toxicity in test animals: See table 7.6.2/1

RESULTS OF MAIN EXPERIMENT
- See table 7.6.2/2 for toxic reactions
- See table 7.6.2/3 for frequency of micronuclei in polychromatic erythrocytes and PCE/total erythrocytes ratio

Any other information on results incl. tables

Table 7.6.2/1: Pre-experiment - Toxic reactions

Dose levels (mg/kg bw)	Toxic reactions	hours post-treatment (male/female)
		1 h	2-4 h	6 h	24 h	30 h	48 h
Pre-experiment 1
1000	Reduction of spontaneous activity	0/2	0/0	0/0	0/0	-/-	0/0
	Ruffled fur	2/2	2/2	2/2	2/2	-/-	0/0
Pre-experiment 2
2000	Reduction of spontaneous activity	2/2	2/2	2/2	0/0	0/0	0/0
	Abdominal position	2/2	0/0	0/0	0/0	0/0	0/0
	Ruffled fur	2/2	2/2	2/2	2/2	2/2	2/2
	Apathy	2/2	1/2	0/0	0/0	0/0	0/0

 

 -/- no observation was made

 Table 7.6.2/2: Main experiment - Toxic reactions

Dose levels (mg/kg bw)	No. of animals per sex	hours post-treatment (male/female)	Toxic reactions
			Ruffled fur	Reduction of spontaneous activity	Abdominal position	Tumbling	Apathy
500	6	1 h	2/1	-	-	-	-
		2-4 h	6/2	-	-	-	-
		6 h	6/3	-	-	-	-
		24 h	0/0	-	-	-	-
1000	6	1 h	6/6	4/0	-	-	-
		2-4 h	6/6	4/0	-	-	-
		6 h	6/6	3/1	-	-	-
		24 h	6/6	0/0	-	-	-
2000	12	1 h	12/12	12/12	4/3	7/5	4/6
		2-4 h	12/12	12/12	0/0	0/0	0/0
		6 h	12/12	12/12	0/0	0/0	0/0
		24 h	12/12	0/0	0/0	0/0	0/0
		48 h*	3/4	0/0	0/0	0/0	0/0

 

*: data only from 6 animals per sex.

Table 7.6.2/3: Results of micronucleus test

Test group	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
Vehicle	0	24	0.120	0 - 6	1159
Gamma-nonalactone	500	24	0.140	1 - 5	1195
	1000	24	0.105	0 - 6	1169
	2000	24	0.115	0 - 8	1178
	2000	48	0.160	1 - 6	1088
Positive control	40	24	2.505*	13 - 90	1132

*: p<0.0001

Results of formulation analysis:

- Representative analytical samples of the application formulations were collected and analysed by GC coupled to FID detector.

- The identity of the test material was confirmed by its retention time which was similar to that measured in the working standards and the test item content in all samples was found to be within the accepted range of ±20 % of the nominal content.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the test conditions, γ-nonalactone is not considered as clastogenic in the 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 bone marrow micronucleus assay, performed according to OECD Guideline 474 and in compliance with GLP, groups of NMRI mice (6/sex/dose) were given a single oral dose of γ-nonalactone in corn oil at concentrations of 0, 500, 1000 and 2000 mg/kg bw. Positive control group was treated with cyclophosphamide at 40 mg/kg bw. Bone marrow of 5 mice/sex/dose was extracted after 24 hours (in all test groups) or 48 hours (in 2000 mg/kg bw group) of exposure and the prepared slides were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, PCE:NCE ratio was determined in the same sample and expressed as PCE/2000 erythrocytes. A preliminary range-finding test was also conducted on 2 mice/sex/dose at 1000 and 2000 mg/kg bw in which no toxic sign was recorded.

No statistically significant increases in the frequency of micronucleated PCEs were observed at any dose levels. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. Positive control induced the appropriate response.

Under the test conditions, γ-nonalactone is not considered as clastogenic in the mouse bone marrow micronucleus assay according to the Directive 67/548/EEC and of the Regulation (EC) No. 1272 /2008 (CLP).

This study is considered as acceptable and satisfies the requirement for a micronucleus assay.