Indeno[4,5-d]-1,3-dioxin, 4,4a,5,6,7,8,9,9b-octahydro-7,7,8,9,9-pentamethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene Mutation in Bacteria (Bacterial Reverse Mutation Assay/Ames): negative with and without metabolic activation (OECD TG 471) - In vitro chromosomal aberration test: negative with and without metabolic activation (OECD TG 473) - In vivo micronucleus test (in human lymphocytes and hepatocytes): negative (OECD TG 474)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 24 February 2003 and 13 March 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study is performed according to OECD TG 471 under GLP.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test; 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Expt 1: 50, 150, 500, 1500, 5000 µg/plate.
Expt 2: 50, 150, 500, 1500, 5000 µg/plate.

Vehicle / solvent:

Vehicle: Dimethyl sulphoxide

Justification for choice of solvent/vehicle: The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) Experiments 1 and 2

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:

- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2001 and 2002 presented in Appendix 2.

- The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.

- All tester strain cultures should be in the approximate range of 1 to 9.9 X 109 bacteria per mL.

- Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The positive control historical ranges for 2001 and 2002 are presented in Appendix 2.

- There should be a minimum of four non-toxic test material dose levels.

- There should not be an excessive loss of plates due to contamination.

Evaluation Criteria:
The test material may be considered positive in this test system if the following criteria are met:

- The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression (5)) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Standard Deviation
Dunnetts Linear Regression Analysis

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies at 5000 µg/plate both with and without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies at 5000 µg/plate both with and without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECFIC CONFOUNDING FACTORS:
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies at 5000 µg/plate both with and without S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.

STERILITY, VEHICLE AND POSITIVE CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).The S9-mix used in both experiments of the main study was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Study.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 with the results also expressed graphically in Figure 1 to Figure 4.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1.

A history profile of vehicle and positive control values is presented in Appendix 2.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Preliminary Toxicity Test

The test material exhibited toxicity (either as a weakened lawn or a decrease in colony frequency) at 5000 µg/plate to the strains of bacteria used (TA100 and WP2uvrA). The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	92	101	113	108	95	88	101	114	101	74	48*
+	TA100	122	138	128	108	112	110	118	119	98	62	43*
-	WP2uvrA-	27	21	19	22	22	17	20	18	15	12	14
+	WP2uvrA-	27	32	26	38	36	27	27	35	25	16	14

*: Sparse bacterial background lawn

 

Mutation Test

Table 1: Spontaneous Mutation Rates (Concurrent Negative Control)

Range-finding test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA-	TA98	TA1537
84	11	21	17	4
97 (88)	15 (13)	18 (21)	9 (14)	3 (14)
84	13	25	17	5

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA-	TA98	TA1537
143	9	13	11	9
88 (104)	11 (10)	25 (19)	14 (14)	5 (7)
81	11	20	18	7

FOR TABLES OF RESULTS FOR MUTATION TEST:

Please see attached in overall remarks, attachments

 

References:

Ames B N, Durston W E, Yamasaki E and Lee F D (1973b) Proc. Natl. Acad. Sci. (USA), 70,2281-2285.

Ames B N, McCann J and Yamasaki E (1975b), Mutation Research,31,347- 364.

Maron DM and Ames BN (1983) Mutation Research,113,173-215.

Gamer R C, Miller E C, and Miller J A (1972) Cancer Res.,32,2058-2066.

Kirkland D J (Ed) (1989), Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part Ill, Cambridge University Press.

Conclusions:

Interpretation of results (migrated information):
negative

The mutagenicity potential of the test material was assessed according to OECD Guideline 471. The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The mutagenicity in the Ames test was testes according to the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate.The test material caused a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies at 5000 µg/plate both with and without S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames: The mutagenicity in the Ames test was tested according to the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). In a preliminary toxicity assay the doses were derived for the main test (50 to 5000 µg/plate). In this test a visible reduction in the growth of the bacterial background lawn and/or a significant decrease in the frequency of revertant colonies at 5000 µg/plate both with and without S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.The test material was considered to be non-mutagenic under the conditions of this test.

Chromosomal aberrations: The study was performed to assess the potential chromosomal mutagenicity of Nebulone on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to OECD 473. Duplicate cultures of Chinese Hamster Lung (CHL) cells were treated with the test material at several dose levels, together with vehicle and positive controls. Four treatment regimens were used: Experiment 1 included a 6(18)-hours exposure, both with and without the addition of an induced rat liver homogenate metabolising system (5 % S9); Experiment 2 included a 24-hour continuous exposure in the absence of S9 and a repeat of the 6(18)-hours exposure with metabolic activation using a 1 % final concentration of S9.The dose levels used were selected on the basis of molecular weight, solubility and the results of a preliminary toxicity test. The dose range for the 6 (18)-hour short term exposure groups, both with and without S9, was 2.5 to 80 µg/mL and for the 24-hour exposure group the dose range was 2.5 to 30 µg/mL.The vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for the CHL cell line. All of the positive control materials induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant increases in the frequency of cells with aberrations in any of the exposure groups. The dose levels of the test material were shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups.The test material was considered to be non-clastogenic to CHL cells in vitro.

In vivo Micronucleus test: The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice according to OECD TG 474. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) using the maximum tolerated dose (MID) 1000 mg/kg with 250 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later. Thereafter was their bone marrow extracted and smear preparations made as well as stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours. Whilst no statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups, it should be noted that a marked reduction was observed in the 48-hour 1000 mg/kg test material group. With no premature deaths or clinical signs being observed this was taken to indicate that systemic absorption had occurred. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The test material was considered to be non-genotoxic under the conditions of the test.

Justification for selection of genetic toxicity endpoint
The Ames test is one of the three genotoxicity assays available to assess this genotoxicity endpoint. These available genotoxicity tests are adequate for covering this endpoint.

Justification for classification or non-classification

Based on the available information in the dossier, Nebulone does not need to be classified for genotoxicity when considering the criteria outlined in Annex VI of 1272/2008/EC (CLP) and Annex I of 67/548/EEC (DSD).