4-trans-Methoxy-4-trans-propyl-[1,1-bicyclohexyl]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 the test item was not mutagenic in the absence and presence of a rat liver metabolizing system (S9 mix) (reference 7.6.1-1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 30, 1984 - Jun 26, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRY operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Species / strain / cell type:

other: TA1538

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

trp C 3076, uvrB, rfa

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
S-9 was routinely prepared following the proposals of B.N. Ames (1975)
- method of preparation of S9 mix
Male Emd:Wi-AF/Han (SPF) rats weighing 100 - 160 g (aged 7-8 weeks) are given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) diluted in Miglyol 812 oil. The animals receive drinking water and Altromin standard diet ad libitum. On day 4, about 16 hours before sacrifice, the animals remain without food. On day 5 the animals are sacrificed. Their abdomens are thoroughly disinfected with 70 % ethanol, are opened and the livers removed. The livers are collected in ice-cooled sterilized beakers containing 0.15 M KCl. The beakers, previously tared, are then weighed and the liver transferred to a sterile glass potter homogenizer with Teflon pestle containing 3 mL of 0.15 M KCl per each gram of liver wet-weighed. The homogenate is transferred to sterilized steel centrifuge tubes and spun at 9000 x g for 15 minutes at about 4 °C. Finally, the supernatant fluid is decanted and transferred into sterilized and precooled plastic tubes. The S-9 is then frozen in liquid nitrogen and stored at -196 °C.
- concentration or volume of S9 mix and S9 in the final culture medium
In the series with S9 mix, 10% S9 in the S9 mix were used in the 1st and 2nd series, respectively.
- quality controls of S9
Every solution and S-9 mix used in this experiment was routinely tested by plating 0.1 mL (or 0.5 mL) each on a nutrient broth agar plate. In case of a positive result, the whole experiment would have been considered invalid.

Test concentrations with justification for top dose:

First experiment: 50, 250, 1250, 2500, 5000 and 10000 µg/plate (with and without S9 Mix)
Repeat experiment: 50, 250, 1250, 2500, 5000 and 10000 µg/plate (with and without S9 Mix)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix, TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix, TA1538, TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9-mix, TA1535, TA1538, TA98, TA1537, TA100, TA98, E.coli

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without S9-mix, TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1- Ethyl-2-nitro-3-nitrosoguanidine

Remarks:

without S9 mix, TA1535, E.coli

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix, TA 100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Methyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix, TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Remarks:

without S9 mix, TA1538

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 8 plates (4 with and 4 without S-9) were used for each concentration step of the test article and the control compounds, 16 plates (8 with and 8 without S-9) were used for the negative controls (solvent).

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants, a clearing of the background lawn

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation occured at concentrations or equal 5000 µg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: see attachment

Ames test:
- Signs of toxicity : No toxicity observed.
- Individual plate counts : see attachment
- Mean number of revertant colonies per plate and standard deviation : see attachment

Conclusions:

With and without addition of S-9 as the metabolizing system the substance did not show any mutagenic activity in the concentration range used.

Executive summary:

A study according OECD TG 471 was performed using Salmonella typhimurium TA 100, TA 98, TA 1535, TA 1537, TA 1538, and Escherichia coli WP2 and WP2 uvrA as tester strains. The mutagenic potential was examined in the plate incorporation test with and without addition of a liver postmitochondrial fraction as the in vitro metabolizing system (S-9; male rats, induced with Aroclor 1254).

The substance was tested at the following concentrations:

First experiment: 50, 250, 1250, 2500, 5000 and 10000 µg/plate

Repeat experiment: 50, 250, 1250, 2500, 5000 and 10000 µg /plate

9-Aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, N-methyl-N-nitro-N-nitrosoguanidine, 2-nitrofluorene, and 4-nitro-1,2-phenylene diamine served as positive controls for testing the bacteria. 2-Aminoanthracene was used for testing the bacteria and the induced S-9 preparations. With and without addition of S-9 as the metabolizing system the substance did not show any mutagenic activity in the concentration range used. Inhibition of bacterial growth was not observed. The substances used as positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

A study according OECD TG 471 was performed using Salmonella typhimurium TA 100, TA 98, TA 1535, TA 1537, TA 1538, and Escherichia coli WP2 and WP2 uvrA as tester strains. The mutagenic potential was examined in the plate incorporation test with and without addition of a liver postmitochondrial fraction as the in vitro metabolizing system (S-9; male rats, induced with Aroclor 1254).

The substance was tested at the following concentrations:

First experiment: 50, 250, 1250, 2500, 5000 and 10000 µg/plate

Repeat experiment: 50, 250, 1250, 2500, 5000 and 10000 µg /plate

9-Aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, N-methyl-N-nitro-N-nitrosoguanidine, 2-nitrofluorene, and 4-nitro-1,2-phenylene diamine served as positive controls for testing the bacteria. 2-Aminoanthracene was used for testing the bacteria and the induced S-9 preparations. With and without addition of S-9 as the metabolizing system the substance did not show any mutagenic activity in the concentration range used. Inhibition of bacterial growth was not observed. The substances used as positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.