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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
from 2010-04-26 to 2010-07-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to the OECD guideline (No. 471) and is in compliance with GLP.
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: ED50F50R
Purity: 69% (sum of the three main constituents)
Name of test material (as cited in study report): Dipentene multiconstituent
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light

Method

Target gene:
Each strain derived from S. typhimurium LT2 contains one mutation in the histine operon, resulting in a requirement for histidine.
Species / strainopen allclose all
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:
other: additional mutations in rfa and uvrB genes. For the strains TA98 and TA100 presence of an additional plasmid pKM101 in order to enhance their sensitivity of detection of some mutagens. See Table 7.6.1/1
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: mutated in rfa gene and presence of an additional plasmid pKM101 in order to enhance the sensitivity of detection of some mutagens. See Table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
Test concentrations with justification for top dose:
Preliminary test: 10, 100, 500, 1000, 2500, 5000 µg/plate with and without S9 mix.
Mutagenicity experiments: first experiment = 0.8 to 185.2 µg/plate without S9 mix for all strains; 2.3 to 555.6 µg/plate with S9 mix for TA98 and TA100; 6.9 to 1666.7 µg/plate with S9 mix for TA1537 and TA98; 20.6 to 5000µg/plate with S9 mix for TA102. Second experiment = 0.8 to 185.2 µg/plate without S9 mix for TA1535; 0.3 to 61.7 µg/plate without S9 mix for TA1537; 2.3 to 555.6 µg/plate without S9 mix for TA98, TA100; 6.9 to 1666.7 µg/plate without S9 mix for TA102; 2.3 to 555.6 µg/plate with S9 mix for TA1535, TA98, TA100 and TA102; 0.8 to 185.2 µg/plate with S9 mix for TA1537. See details in table 7.6.1/2
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO), batch Nos. K39250750906 and K40013750926 (VWR, Fontenay Sous Bois, France).
- Justification for choice of solvent/vehicle: During the solubility assay, the test item was found freely soluble in the vehicle (DMSO) at 100 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C for without S9 mix efficacy control. 2-Anthramine for with S9 mix efficacy control. See details in Table 7.6.1/3
Remarks:
no remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION:
direct plate incorporation: test item solution (0.05 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
or preincubation method: test item solution (0.05 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 or 72 hours
SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 3 plates/dose/strain. For the mutagenicity experiments, two independent experiments were performed.
NUMBER OF CELLS EVALUATED: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: observation of the decrease in the number of revertant colonies and/or thinning of the bacterial lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: not required
- Determination of endoreplication: not required

OTHER: All the Petri dishes obtained were placed in a sealed jar using one jar for each dose-level tested, one jar for the vehicle control and another jar for the positive controls. The jars were then incubated at 37°C. The precaution of using jars in this study was due to the volatile characteristic of the test item and to limit the oxidation of the test item.
The revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK). Manual counting was used as needed.


Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/4
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/4
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/5
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/5
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/6
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/6
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/7
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/7
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/8
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/8
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: not applicable

RANGE-FINDING/SCREENING STUDIES: A first preliminary test was performed testing six dose-levels of the test item (one plate/dose-level) with the TA 98, TA 100 and TA 102 strains, with and without S9 mix. This preliminary test was invalidated. Indeed, the test item used in this test came from a small aliquot taken from one of the flask and stored at +4°C, protected from light and humidity and under nitrogen gas. This conditioning in an inert atmosphere was performed using a layer of parafilm to seal the vessel (as described in CIT’s SOPs). But there were some evidences that this layer of parafilm got deteriorated (most probably by the test item’s emanation) contaminating the aliquot of test item. Because of this contamination, the results of this first preliminary test were invalidated and the study was started all over again using one supplied flask per treatment and avoiding the use of parafilm.
The second preliminary test was performed testing six dose-levels of the test item (two plates/dose-level) with the TA 98, TA 100 and TA 102 strains, with and without S9 mix. In this second preliminary test, the results of the vehicle control obtained with the TA 102 strain with S9 mix were found non valid (inconsistent with the historical data range). No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels and in any strains. Without S9 mix, a strong toxicity (observation of the decrease in the number of revertant colonies and of a thinning of the bacterial lawn) was observed at dose-levels ≥ 100 µg/plate with the TA 98, TA 100 and TA 102 strains. With S9 mix, a moderate to strong toxicity was observed at dose-levels ≥ 500 µg/plate with the TA 98 and TA 100 strains. Therefore since the test item was toxic in the preliminary test, the choice of the highest dose-level to be tested in the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants for the vehicle and positive controls was similar to the historical data.
ADDITIONAL INFORMATION ON CYTOTOXICITY: A strong toxicity was observed at dose-levels  185.2 µg/plate in the TA 1537 and TA 98 strains and at dose-levels  555.6 µg/plate in the TA 1535 strain in the second experiment with S9 mix (see table 7.6.1/9). The results of the vehicle control obtained with the TA 100 and TA 102 strains being inconsistent with the historical data range, therefore the data were invalidated. Since too many toxic dose-levels were obtained leading to less than five analysable dose-levels (which is a requirement of the international guidelines), the values were non-retained. The treatment of the second experiment with S9 mix according to the pre-incubation method was repeated using lower ranges of dose-levels.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/4: Results for the strainTA1535

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

0.9

No

0.9

No

-

-

-

-

2.3

1.1

No

1.0

No

1.2

No

1.6

No

6.9

0.9

No

0.9

No

0.7

No

1.2

No

20.6

0.9

No

0.9

No

0.8

No

1.5

No

61.7

0.7

No

0.9

St

0.7

No

1.1

No

185.2

0.4

St

0.2

St

0.6

No

1.2

Mt

555.6

-

-

-

-

0.9

No

0.5

St

Postive controls

 

 

 

 

 

 

 

 

NaN3 (1µg)

28.6

No

17.1

No

-

-

-

 

2AM (2µg)

-

-

-

-

13.6

No

16.5

 

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Table 7.6.1/5: Results for the strainTA1537

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.3

-

-

0.3

No

-

-

-

 

0.8

1.0

No

0.7

No

-

-

1.2

-

2.3

1.1

No

1.1

No

-

-

0.9

No

6.9

0.7

No

0.8

No

1.3

No

1.5

No

20.6

0.7

Mt

0.5

Mt

1.3

No

1.4

No

61.7

0.0

St

1.7

St

1.5

No

1.1

No

185.2

0.0

St

-

-

1.7

No

0.7

Mt

555.6

-

-

-

-

0.9

No

-

-

1666.7

 

 

 

 

1.0

Mt

-

-

Postive controls

 

 

 

 

 

 

 

 

9AA (50µg)

18.1

No

32.9

No

-

-

-

-

2AM (2µg)

-

-

-

-

8.7

No

17.1

No

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Table 7.6.1/6:Results for the strainTA98

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

2.1

No

-

-

-

-

-

-

2.3

1.8

No

0.9

No

-

-

0.9

No

6.9

0.9

No

0.7

No

0.9

No

0.6

No

20.6

0.7

No

0.6

No

1.0

No

0.7

No

61.7

0.9

Mt

0.9

No

1.1

No

0.7

Mt

185.2

0.9

Mt

0.2

Mt

1.7

No

0.0

St

555.6

-

-

0.4

St

0.8

Mt

0.0

St

1666.7

-

-

-

-

0.8

Mt

-

-

Postive controls

 

 

 

 

 

 

 

 

2NF (0.5µg)

7.8

No

4.1

No

-

-

-

 

2AM (2µg)

-

-

-

-

26.4

No

45.6

N o

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Table 7.6.1/7: Results for the strainTA100

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

1.1

No

-

-

-

-

-

-

2.3

1.4

No

0.9

No

1.4

-

1.1

No

6.9

1.0

No

1.1

No

1.5

No

1.1

No

20.6

1.1

No

0.9

No

1.1

No

1.3

No

61.7

0.8

No

0.7

Mt

1.4

No

1.1

No

185.2

0.3

Mt

0.4

St

1.1

No

1.0

Mt

555.6

-

-

0.6

St

1.1

No

0.3

St

Postive controls

 

 

 

 

 

 

 

 

NaN3 (1µg)

4.7

No

7.7

No

-

-

-

 

BAP (5µg)

-

-

-

-

8.6

No

5.6

N o

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Table 7.6.1/8:Results for the strainTA102

Dipentene concentration plate (µg/plate)

Without metabolic activation

With metabolic activation

First experiment (direct plate incorporation)

Second experiment (direct plate incorporation)

First experiment (direct plate incorporation)

Second experiment (pre-incubation)

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

Ratio mean revertant treated/revertant solvent

Cytotoxicity*

0

-

No

-

No

-

No

-

No

0.8

1.4

No

-

-

-

-

-

-

2.3

1.8

No

-

-

-

-

0.8

No

6.9

1.9

No

1.0

No

-

-

1.1

No

20.6

1.3

No

1.0

No

1.0

No

1.1

No

61.7

1.2

No

0.9

No

1.5

No

1.0

No

185.2

1.6

No

0.7

Mt

1.3

No

1.0

No

555.6

-

-

0.6

Mt

1.6

No

0.6

Mt

1666.7

-

-

0.4

Mt

1.0

No

-

-

5000

-

-

-

-

1.4

No

-

-

Postive controls

 

 

 

 

 

 

 

 

MMC (0.5µg)

7.6

No

5.2

No

-

-

-

 

2AM (10µg)

-

-

-

-

3.8

No

5.5

N o

*: based on the thining of the bacterial lawn, Mt: Moderate toxicity; St: Strong toxicity

Applicant's summary and conclusion

Conclusions:
Test item dipentene multiconstitent did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium. Therefore, dipentene multiconstituent is not considered as mutagenic in this bacterial system according to CLP Regulation (EC) No 1272/2008 and Directive 67/548/EEC.
Executive summary:

In a reverse gene mutation assay in bacteria (No. 36799 MMO), performed according to the OECD No. 471 and EC No. B13/14 guidelines, dipentene multiconstiuent (purity of 48.4%) diluted in Dimethylsulfoxide (DMSO) was tested in S. typhimurium TA1535, TA1537, TA100, TA98 and TA102 in the presence and the absence of mammalian metabolic activation (S9) using the direct plate incorporation or the preincubation method.The dipentene content (48.4%) was not taken into account. As a consequence, no correction factor was applied to express the concentrations and dose-levels of test item.

Due to the volatile characteristic of the test item and in order to limit the oxidation of the test item, all the Petri dishes were placed in a sealed jar. One jar was used for each tested dose-level, one jar was used for the vehicle control and another jar for the positive controls. 

Six known mutagens, dissolved in dimethylsulfoxide (except for Mitomycin C which was dissolved in distilled water), were used to check the sensitivity of the test system. The positive controls induced the appropriate responses in the corresponding strains. The number of revertants in the vehicle controls was consistent with the historical data of the testing facility, and the number of revertants in the positive controls was higher than that of the vehicle controls (at least 2-fold increase for the TA 98, TA 100 and TA 102 strains and at least 3-fold increase for the TA 1535 and TA 1537 strains) and was consistent with the historical data of the testing facility. Therefore the study was considered valid.

During the preliminary test, the decrease in the number of revertant colonies and/or thinning of the bacterial lawn showed that test item dipentene multiconstituent was cytotoxic for bacteria. Thus, the choice of the highest dose-level to be tested in the main test was based on the level of toxicity, according to the criteria specified in the international guidelines. During the main test, no induced revertant over background was observed in any strains of S. typhimurium whereas the cytotoxic dose-level was reached.

Therefore, dipentene multiconstituent did not show any mutagenic activity in the bacterial reverse mutation test using Salmonella typhimurium. Dipentene multiconstituent is not considered as mutagenic in this bacterial system according to CLP Regulation (EC) No 1272/2008 and Directive 67/548/EEC.

 

This study is considered as acceptable as it satisfied the criteria of Guideline OECD No. 471.