(1S,4R,4aS,9aR)-4,4a,9,9a-tetrahydro-1H-1,4-methanofluorene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 July 1994 - 21 September 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to Japanese Guideline and in compliance with Japanese Good Laboratory Practise. The details provided in this study report were not sufficient to assess the full property of the test material.

Qualifier:

according to guideline

Guideline:

other: Japanese Industrial Safety and Health Law (Notification No.77, 1988)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

S9 Mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, DW

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-finyl)actylamide (AF-2)

Remarks:

without S9 Mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, DW

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 Mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, DW

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 Mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, DW

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 Mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, DW

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 Mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, DW

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 Mix

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

Based on these results, it was concluded that MTF was not mutagenic under the test conditions of this study.

Reference 2

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:

04 July 2002 - 21 February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD, EU, US FDA and Japanese (EPA, MHLW and METI) test guidelines, and in compliance with 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)

Version / remarks:

(Commission Directive 2000/32/EC; L136, 57-64, Annex 4D)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US FDA Administration Center for Food Safey & Applied Nutrition Office of Premarket Approval, Redbook 2000, Toxicological Principles for the Safety of Food Ingredients, IV.C.1.a Bacterial Reverse Mutation Test, July 07, 2000.

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan 287 - EPA", "Eisei 127 - MHLW", and "Heisei 09/10/31 Kikyoku 2 - METI"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

Range finding test:
strains S. typhimurium TA 100 and E. coli WP2 uvrA (with and without methabolic activation): 20.6, 61.8, 185.2, 555.6, 1666.7 and 5000 µg/plate


without metabolic activation:
strains TA 100, TA 102: 1.0, 3.3, 10, 33 and 100 µg/plate
strain TA 1537: 10, 33, 100, 333 and 1000 µg/plate
strains TA 1535, TA 98, WP2 uvrA: 33, 100, 333, 1000 and 2500 µg/plate

with metabolic activation (pre-incubation assay):
strains TA 100, TA 1537, TA 102: 10, 33, 100, 333 and 1000 µg/plate
strains TA 1535, TA 98, WP2 uvrA: 33, 100, 333, 1000 and 2500 µg/plate

Due to a wide range of toxicity in the pre-incubation assay with microsomal activation, additional concentrations were tested with metabolic activation:
strains TA 100, TA 1537: 0.3, 1.0, 3.3, 10 and 33 µg/plate
strains T A 1535, T A 98, T A 102: 3.3, 10, 33, 100 and 333 µg/plate.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-AA

Remarks:

With metabolic activation

Key result

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:

no cytotoxicity, but tested up to precipitating concentrations

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, but tested up to precipitating concentrations

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

During the described mutagenicity test and under the experimental conditions reported, the test item did not induced gene mutations by base-pair changes or frameshifts in the genome of the strains used.


Therefore, MTF is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 September 1996- 18 March 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted according to Japanese Guideline and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals (1987)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: CHL/IU

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Cell growth inhibition test:
Without methabolic activation (24- hour treatment) (µg/ml): 20, 40, 60, 80, 100*, 120*, 140*, 160*;
Without methabolic activation (48- hour treatment) (µg/ml): 20, 40, 60, 80, 100*, 120*, 140*, 160*;
With methabolic activation (µg/ml): 10, 20, 39, 78, 156*, 313*, 625*, 1250*, 2500*, 5000*.
*: Precipitation of the test substance was recognized at the treatment period .

Chromosomal aberration test:
Without methabolic activation (24- hour treatment) (µg/ml): 10.6, 21.3, 42.5, 85
Without methabolic activation (48- hour treatment) (µg/ml): 10.6, 21.3, 42.5, 85
With metabolic activation (µg/ml): 18.8, 37.5, 75, 150(C) (Toxic)
Without metabolic activation (µg/ml): 18.8, 37.5, 75, 150(C)
C: Precipitation of the test substance was recognized at the treatment period.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Key result

Species / strain:

mammalian cell line, other:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

In the chromosomal aberration test, the incidence of cells with structural and numerical aberration was less than 5%.

Based on this result, the test substance was judged to be negative for the ability to induce chromosomal aberration.

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results, the test substance was judged to be negative for the ability to induce chromosomal aberration.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August 2011 - 08 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to official OECD, EU, and US test guidelines, and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A, and ICH (1998) Guideline S2B

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 µg/mL streptomycin and 2.0 mM glutamine.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

First Test (3 hours treatment, with and without S9 mix): 18.37, 30.62, 51.03, 81.05, 141.76, 236.26, 393.77, 656.28, 1093.8, and 1823 µg/mL.
Second test (21 hours treatment in the absence of S9 mix): 15, 20, 25, 30, 35, 40, 42.5, 45, 47.5, 50, and 55 µg/mL.
Second test (3 hours treatment in the presence of S9 mix): 60, 70, 80, 90, 100, 110, 115, 120, 125, 130, and 140 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: MTF was found to be miscible in DMSO.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Used in the absence of S9 mix.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used in the presence of S9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours (first test, and second test in the presence of S9 mix), or 21 hours (second test in the absence of S9 mix).
- Expression time (cells in growth medium): 18 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Cultures were prepared in duplicate.

NUMBER OF CELLS EVALUATED: One hundred metaphase figures examined per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Noted when seen
- Determination of endoreplication: Noted when seen

Evaluation criteria:

Cochran-Armitage test for trend.

Key result

Species / strain:

lymphocytes: Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

First test:

In the absence of S9 mix following 3 hour treatment, MTF caused a reduction in the mitotic index to 53% of the vehicle control value at 60 µg/mL. In the presence of S9 mix (2% v/v) following 3 hour treatment, MTF caused a reduction in the mitotic index to 51% of the vehicle control value at 100 µg/mL. In both the absence and presence of S9 mix, MTF caused no statistically significant increase in the proportion of cells with chromosomal aberrations at any concentration, when compared with the vehicle control.

Second test:

Conclusions:

Interpretation of results (migrated information):
positive

It was concluded that MTF had shown evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix following 21 hour continuous treatment, and in th presence of S9 mix following 3 hours treatment, in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 July 2002-13 January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to the OECD Guideline and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity - In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: "Kanpoan No. 287 - Environmental Protection Agency", "Eisei No. 127 - Ministry of Health & Welfare", "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry"

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

without S9 mix:
Experiment I: 1.3, 2.5, 5.0, 10.0, 15.0, 25.0
Experiment II: 2.5, 5.0, 10.0, 20.0, 30.0, 40.0

with S9 mix:
Experiment I: 3.1, 6.3, 12.5, 25.0, 50.0, 75.0
Experiment II: 3.1, 6.3, 12.5, 25.0, 50.0, 100.00

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

In both experiments, in the absence and presence of S9 mix, no statistically significant and biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.

The aberration rates of the cells after treatment with the test item (0.0- 2.0% aberrant cells, exclusive gaps) were

within the range of the solvent control values (0.0- 3.5% aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item MTF did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 June 2002 - 17 December 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study conducted according to OECD test guideline and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L 1362000, Annex 4E, dated May 19, 2000.

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare, 1988, Guidelines for Toxicity Studies of Drugs, Chapter 4, "Mutagenicity Study".

Qualifier:

according to guideline

Guideline:

other: "U.S. Food and Drug Administration Center , Toxicological Principles for the Safety of Food Ingredients, IV.C.1.c. Mouse Lymphoma Thymidine Kinase Gene Mutation Assay", July 07, 2000.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-test: 3500µg/ml - the highest applied concentration

Main test Experiment 1:
without S9 mix: 3.4, 6.9, 13.8, 27.5, 41.3 and 55.0
with S9 mix: 1.8, 3.4, 6.9, 13.8, 20.7 and 27.5

Experiment II:
without S9 mix: 1.6, 3.1, 6.3, 12.5, 25.0 and 50.0

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, MTF is considered to be non-mutagenic in this mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)