Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 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:
April - May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
January 2015
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and Tryptophane
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats
Test concentrations with justification for top dose:
- TA1535, TA1537, TA98, TA100 and E. Coli WP2: 50, 150, 500, 1500, 3500 and 5000 μg/plate, with and without S9-mix
At 5000 µg/plate important bacteriostatic activity was observed, thus a dose at 3500 µg/plate was also used.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Antramine (2µg/plate with S9 metablica activation), 9-Aminoacridine (50µg/plate without metabolic activation), cis-Platinum (II) Diammine dichloride (1µg/plate without metabolica activation)
Details on test system and experimental conditions:
SOURCE OF THE TEST SYSTEM: Strains were obtained from MOLTOX TM.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

NUMBER OF REPLICATES: Controls and treatment were performed in triplicate.

DURATION
- Preincubation period: 30 minutes at 37 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 °C in the dark for 48-72 hour in both direct plate and preincubation methods.

Evaluation criteria:
The following criteria were checked to validate the study:
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
- Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).
Statistics:
No data
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate thinning of the background bacterial lawn at 3500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate thinning of the background bacterial lawn at 3500 and 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate thinning of the background bacterial lawn at 3500 and 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate thinning of the background bacterial lawn at 3500 and 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate thinning of the background bacterial lawn at 3500 and 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary cytotoxicity testing (Strain TA100):
Bacteriostatic test has been performed, in case of bacteriostatic activity the concentration, the highest concentration that will be retained for the study is the concentration that induices a bacteriostatic activity of 75% or less.
Bacteriostatic activity has been observed at 500µg/plate
Conclusions:
Under the test conditions, the test item is not considered as mutagenic in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA-) (pKM 101) strains without, or with metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium(TA1535, TA1537, TA98, TA100 and E.coli WP2) were exposed to test item, cistus oil (N0300) at the following concentrations: 

- TA1535, TA1537, TA98, TA100 and E.coli WP2: 50, 150, 500, 1500, 3500 and 5000 μg/plate, with and without S9-mix

 

Metabolic activation system used in this test 10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats . Vehicle, negative and positive control groups were also included in mutagenicity tests.

 

In Experiments , following the treatment,evidence of toxicity was observed at 5000 μg/plate and/or 3500 μg/plate in all strains in the absence and presence of S-9.

 

The mean numbers of revertant colonies are fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Under the test conditions, test item is not considered as mutagenic in this bacterial system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity test

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

Savineau, 2016

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100,

WP2 uvrA-

-S9

+S9

Up to cytotoxic or highest recommended concentration

-S9 : non mutagenic

+S9 : non mutagenic

 

 

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA1535, TA1537, TA98, TA100 and E.coli WP2) were exposed to test item, cistus oil (N0300) at the following concentrations: 

- TA1535, TA1537, TA98, TA100 and E.coli WP2: 50, 150, 500, 1500, 3500 and 5000 μg/plate, with and without S9-mix

 

Metabolic activation system used in this test 10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats . Vehicle, negative and positive control groups were also included in mutagenicity tests.

 

In Experiments, following the treatment, evidence of toxicity was observed at 5000 μg/plate and/or 3500 μg/plate in all strains in the absence and presence of S-9.

 

The mean numbers of revertant colonies are fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Under the test conditions, test item is not considered as mutagenic in this bacterial system.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, the registered substance does not require classification for mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP).