Registration Dossier
Registration Dossier
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EC number: 946-670-6 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Genetic toxicity (OECD TG 471): negative (read-across from Cedarwood Virginia oil)
In vitro gene mutation study in mammalian cells (OECD TG 490): negative (read across from Cedarwood Virginia oil)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 29 September 2015 - 23 October 2015
- 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:
- 1997
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor, Batch Number 1002292672
- Expiration date of the lot/batch: 30 November 2016
- Purity test date: 28 May 2015
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in a dark place under nitrogen
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Solutions of the test item, as received, were prepared immediately before use in DMSO. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 1 hour and 29 minutes from the initial formulation. All dose levels in this report are expressed to three significant figures.
OTHER SPECIFICS: UVCB - Target gene:
- Histidine operon
Trypthophan operon - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: All strains: contain the Rfa wall mutation and the uvrB mutation TA98 and TA100 strains: contain the pKM101 plasmid
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- other: uvrA DNA repair deficiency
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 tissue homogenate
- Test concentrations with justification for top dose:
- - Toxicity test:
With and without S9:
5000,1580, 500, 158 and 50.0 μg/plate.
- Main Study I (plate incorporation):
Withhout S9
TA1535: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
TA1537: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
With S9
TA1535: 4000, 2000, 1000, 500, 250 and 125 μg/plate
TA1537: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
- Main Study II (preincubation):
Without S9
TA1535: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
TA1537: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/plate
With S9
TA1535: 2000, 1000, 500, 250, 125, 62,5 and 31.3 μg/plate
TA1537: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
- Main Study II (additional experiment)
Without S9
TA1537: 15.6, 7.8, 3.9, 1.95, 0.975, 0.488 μg/plate
TA100: 15.6, 7.8, 3.9, 1.95, 0.975 μg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was used since it is compatible with the survival of the bacteria and the S9 metabolic activity. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation) if negative or equivocal results are obtained in the first experiment a confirmatory experiment is performed using the pre-incubation method.
- Cell density at seeding (if applicable):
DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: ; other: Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. - Rationale for test conditions:
- The study is designed to comply with the experimental methods indicated in the guidelines
- Evaluation criteria:
- The assay will be considered valid if the following criteria are met:
(i) Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
(ii) The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
(iii) No more than 5%of the plates will be lost through contamination or other unforeseen event The whole assay or only the non-valid treatment series will be repeated in case the acceptance criteria will not be achieved.
Evaluation of data
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. Evaluation of Ames test data based on a ‘doubling rate’ has been shown to be as effective as statistical techniques in allowing the correct interpretation of test results (Chu et al. 1981). Any increase in revertant numbers should lie outside the historical control range to have biological relevance. - Statistics:
- Data are evaluated by performing a regression analysis. This method fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is
expressed as:
y = a + bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
The regression line does not include the untreated control data, but includes the solvent control data. Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Main experiment I, II and III
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Main experiment I, II and III
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the end of the incubation period, precipitation of the test item was observed with WP2 uvrA and TA98, at the highest dose level (5000 μg/plate), in the absence and presence of S9 metabolic activation in main experiment I
RANGE-FINDING/SCREENING STUDIES:
Toxicity, as indicated by thinning of the background lawn, was observed with TA100, TA1535 and TA1537 tester strains, at the two or three highest concentrations tested, both in the absence and presence of S9 metabolism. A
more pronounced toxic effect was observed with TA100 tester strain, showinga marked and dose related reduction in revertant numbers. No toxic effect was noticed with WP2 uvrA and TA98, at any dose level tested, in the absence or presence of S9 metabolism.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached
- Negative (solvent/vehicle) historical control data: see attached - Conclusions:
- Based on the results of this study Cedarwood Virginia Oil does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
Three experiments were performed to test the gene mutation potential of Cedarwood Oil Virginiana according to OECDTG471. In main assay I, using the plate incorporation method, the test item was assayed at dose levels ranging from 16,5-5000µg/plate (concentrations chosen on the basis of the results obtained in a preliminary toxicity test). Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies was observed with TA1535, TA1537 and TA100, starting from 500 μg/plate or 1000 μg/plate, both in the absence and presence of S9 metabolic activation. No toxic effect was noticed with WP2uvrA and TA98. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at dose levels ranging from 7.81-5000µg/plate. No toxicity was noticed with WP2uvrA and TA98, at any dose level. Toxicity, from severe ( TA1537, and TA100) to slight (remaining strains), was observed with the remaining tester strains both in the absence and presence of S9 metabolism. In order to have a sufficient number of analysable concentrations, a further experiment (III), using the pre-incubation method, was performed with TA1537 and TA100 tester strains using dose levels ranging from 0.488-15.6 µg/plate. A slight thinning of the background lawn was observed with both tester strains, at the highest dose level. No relevant increase in the number of revertant colonies was observed, with any tester strain, in the plate incorporation or pre- incubation assay, at any dose level, in the absence or presence of S9 metabolism. The mean plate counts for untreated and positive control plates fell within RTC acceptance criteria based on historical control. It is concluded that the test item Cedarwood Virginia Oil does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Based on the results of this study Cedarwood Virginia Oil does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 January 2017 - 07 March 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro Mammalian Cell Gene Mutation Test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- batch No.of test material: 1002960562, obtained from sponsor
- Expiration date of the lot/batch: 31 August 2017 (expiry date)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. A solubility test was performed. The test item was dissolved in ethanol.
- Final dilution of a dissolved solid, stock liquid or gel: The final concentration of the solvent in the exposure medium was 0.5% (v/v).
OTHER SPECIFICS: Pale yellow to yellow liquid - Target gene:
- thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
(2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).
- Methods for maintenance in cell culture if applicable: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 39 – 93%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.2 – 37.7 °C).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
> Horse serum: Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
> Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
> Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
> Exposure medium:
For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
>Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
>Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix: contained per ml: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose- 6-phosphate; 3.4 mg NADP; 4 μmol HEPES. The above solution was filter (0.22 μm)- sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction).
- Test concentrations with justification for top dose:
- First mutagenicity test:
Without S9-mix: 0.63, 1.25, 2.5, 5, 10, 20 and 30 μg/ml exposure medium.
With S9-mix: 6.3, 12.5, 25, 50, 60, 70, 80 and 90 μg/ml exposure medium.
Second mutagenicity test:
Without S9-mix: 1.25, 5, 20, 25, 30, 35, 40 and 45 μg/ml exposure medium.
Justification for top dose:
In the absence of S9-mix (3h exposure), a top dose of 30 µg/mL was chosen since the dose levels of 40 to 70 μg/ml were too toxic for further testing.
In the presence of S9-mix (3h exposure), a top dose of 90 µg/mL was chosen since the dose levels of 100 to 140 μg/ml were too toxic for further testing.
In the absence of S9-mix (24h exposure), a top dose of 45 µg/mL was chosen since the dose levels of 50 and 60 μg/ml were too toxic for further testing. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Per culture 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/ml
for 24 hour treatment) were used.
DURATION
- Exposure duration: 3h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 2 days after the treatment period
- Selection time (if incubation with a selection agent): 11 or 12 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT) selection
STAIN: diphenyltetrazolium bromide (MTT)
NUMBER OF REPLICATIONS: 5
DETERMINATION OF CYTOTOXICITY
- Method: subculture of exposed cells and counting - Rationale for test conditions:
- According to test method described in international guidelines (OECD TG 490)
- Evaluation criteria:
- A mutation assay was considered acceptable if it met the following criteria:
1. The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
2. The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
3. The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
4. The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6). At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control should have an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent olvent/control (a small colony IMF of at least 150 x 10-6).
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126. - Statistics:
- Dose range finding test:
The suspension growth (SG) for the 3 hour treatment= SG = Suspension growth = [Day 1 cell count/1.6 x 105] x [Day 2 cell count/1.25 x 105]
The suspension growth (SG) for the 24 hour treatment= SG = Suspension growth = [Day 0 cell count/1.25 x 105] x [Day 1 cell count/1.25 x 105]
Mutagenicity tests:
The suspension growth (SG) for the 3 hour treatment= [Day 1 cell count/1.6 x 105] x [Day 2 cell count/1.25 x 105]
The suspension growth (SG) for the 24 hour treatment= [Day 0 cell count/1.25 x 105] x [Day 1 cell count/1.25 x 105] x [Day 2 cell count/1.25 x 105]
Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution: P(0) = number of empty wells/total number of wells
The cloning efficiency (CE) was then calculated as follows: CE = -ln P(0)/number of cells plated per well
The relative cloning efficiency (RCE) at the time of mutant selection = CE (test) / CE (controls) x 100
The Relative Total Growth (RTG) was also be calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture: RTG = RSG x RCE/100
The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows: MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 10^6 Small and large colony mutation frequencies were calculated in an identical manner. - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated directly in the exposure medium at concentrations of 78 μg/ml and above. After 3 hour treatment, the test item precipitated in the exposure medium at concentrations of 313 μg/ml and above. The concentration used as the highest test item concentration for the dose range finding test was 200 μg/ml.
RANGE-FINDING/SCREENING STUDIES: The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
FInd below in any other information. - Conclusions:
- Under the conditions of the test, Cedarwood Virginia Oil did not induce mutations in the absence and presence of metabolic activation. It was concluded that the source substance Cedarwood Virginia Oil does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
This study evaluates the effects of Cedarwood Oil Virginia on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test item was dissolved in ethanol. In the first experiment, the test item was tested up to concentrations of 30 and 90μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. In the second experiment, the test item was tested up to concentrations of 45μg/ml in the absence of S9-mix. The incubation time was 24 hours. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency. It is concluded that Cedarwood Virginia oil is not mutagenic in the mouse lymphoma L5178Y test system under the presented experimental conditions, and therefore Cedarwood Virginia oil, does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The full read across justification report is attached under "Attached justification".
18 December 2017 READ-ACROSS STUDY / CW TX OIL TERPENES (1 & 2) / IN VITRO GENE MUTAGENICITY IN BACTERIA I&B9W8768R001F2.0
According to Annex VII, 8.4.1 of the REACh Regulation (EC) No 1907/2006, In vitro gene mutagenicity in bacteria is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).
A read-across approach appears appropriate to predict the endpoint “In vitro gene mutagenicity in bacteria” for the substance Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene) because:
An in vitro gene mutagenicity study in bacteria, according to OECD test guideline 471, is available for the substance Cedarwood Virginia oil, (CW Virginia), which composition is very similar and which slight differences in composition are for constituents that are not notified for mutagenicity and are not corresponding to known structural alerts; therefore no difference in mutagenic potential is expected between the source and target substances.
This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the absence of In vitro gene mutagenicity in bacteria for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Main experiment I, II and III
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Main experiment I, II and III
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the end of the incubation period, precipitation of the test item was observed with WP2 uvrA and TA98, at the highest dose level (5000 μg/plate), in the absence and presence of S9 metabolic activation in main experiment I
RANGE-FINDING/SCREENING STUDIES:
Toxicity, as indicated by thinning of the background lawn, was observed with TA100, TA1535 and TA1537 tester strains, at the two or three highest concentrations tested, both in the absence and presence of S9 metabolism. A
more pronounced toxic effect was observed with TA100 tester strain, showinga marked and dose related reduction in revertant numbers. No toxic effect was noticed with WP2 uvrA and TA98, at any dose level tested, in the absence or presence of S9 metabolism.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached
- Negative (solvent/vehicle) historical control data: see attached - Conclusions:
- Based on the results of the read across study with the source substance Cedarwood Virginia Oil, the target substance Cedarwood Texas Terpenes does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
The skin sensitisition potential of the target substance Cedarwood Texas Terpenes was evaluated by read across with the source study for Cedarwood Virginia oil.
Three experiments were performed to test the gene mutation potential of Cedarwood Oil Virginiana according to OECDTG471. In main assay I, using the plate incorporation method, the test item was assayed at dose levels ranging from 16,5-5000µg/plate (concentrations chosen on the basis of the results obtained in a preliminary toxicity test). Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies was observed with TA1535, TA1537 and TA100, starting from 500 μg/plate or 1000 μg/plate, both in the absence and presence of S9 metabolic activation. No toxic effect was noticed with WP2uvrA and TA98. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at dose levels ranging from 7.81-5000µg/plate. No toxicity was noticed with WP2uvrA and TA98, at any dose level. Toxicity, from severe ( TA1537, and TA100) to slight (remaining strains), was observed with the remaining tester strains both in the absence and presence of S9 metabolism. In order to have a sufficient number of analysable concentrations, a further experiment (III), using the pre-incubation method, was performed with TA1537 and TA100 tester strains using dose levels ranging from 0.488-15.6 µg/plate. A slight thinning of the background lawn was observed with both tester strains, at the highest dose level. No relevant increase in the number of revertant colonies was observed, with any tester strain, in the plate incorporation or pre- incubation assay, at any dose level, in the absence or presence of S9 metabolism. The mean plate counts for untreated and positive control plates fell within RTC acceptance criteria based on historical control. It is concluded that the test item Cedarwood Virginia Oil does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Based on the results of this study Cedarwood Virginia Oil does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). This result was used for read across to Cedarwood Texas Terpenes
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The full read across justification report is attached under "Attached justification".
10 April 2018 READ-ACROSS STUDY / CW TX OIL TERPENES (1 & 2) / GENETIC TOXICITY IN MAMMALIAN CELLS I&B9W8768R001F1.0
According to Annex VIII, 8.4 of the REACh Regulation (EC) No 1907/2006, Genetic toxicity in mammalian cells is standard information required for the registration of substances manufactured or imported in quantities of ten tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).
A read-across approach appears appropriate to predict the endpoint “Genetic toxicity in mammalian cells” for the substance Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene) because:
Two Genetic Toxicity studies, one according to OECD test guideline 490 and one according OECD test guideline 474, are available for the substance Cedarwood Virginia oil (CW Virginia oil), of which the composition is very similar to Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene).
The compositions of the target and source substance are very similar and any slight differences in composition are for constituents that are not notified for genotoxicity and are not corresponding to known structural alerts; therefore no difference in genotoxic potential is expected between the source and target substances.
This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the absence of Genetic toxicity in mammalian cells for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated directly in the exposure medium at concentrations of 78 μg/ml and above. After 3 hour treatment, the test item precipitated in the exposure medium at concentrations of 313 μg/ml and above. The concentration used as the highest test item concentration for the dose range finding test was 200 μg/ml.
RANGE-FINDING/SCREENING STUDIES: The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
FInd below in any other information. - Conclusions:
- Based on the negative results of an in vitro gene mutation study in mammalian cells performed using the read across source substance Cedarwood Virginia oil, the target substance Cedarwood Texas oil distilled - Terpenes does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
Cedarwood Texas oil distilled - Terpenes is not classified as mutagenic, based on the results from the (OECD TG 490) source study performed with Cedarwood Virginia oil. The justification for this read across is provided in the attached justification for type of information.
This study evaluates the effects of Cedarwood Oil Virginia on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test item was dissolved in ethanol. In the first experiment, the test item was tested up to concentrations of 30 and 90μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. In the second experiment, the test item was tested up to concentrations of 45μg/ml in the absence of S9-mix. The incubation time was 24 hours. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency. It is concluded that Cedarwood Virginia oil is not mutagenic in the mouse lymphoma L5178Y test system under the presented experimental conditions, and therefore Cedarwood Virginia oil does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The full read across justification report is attached under "Attached justification".
18 December 2017 READ-ACROSS STUDY / CW TX OIL TERPENES (1 & 2) / IN VITRO GENE MUTAGENICITY IN BACTERIA I&B9W8768R001F2.0
According to Annex VII, 8.4.1 of the REACh Regulation (EC) No 1907/2006, In vitro gene mutagenicity in bacteria is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).
A read-across approach appears appropriate to predict the endpoint “In vitro gene mutagenicity in bacteria” for the substance Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene) because:
An in vitro gene mutagenicity study in bacteria, according to OECD test guideline 471, is available for the substance Cedarwood Virginia oil, (CW Virginia), which composition is very similar and which slight differences in composition are for constituents that are not notified for mutagenicity and are not corresponding to known structural alerts; therefore no difference in mutagenic potential is expected between the source and target substances.
This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the absence of In vitro gene mutagenicity in bacteria for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Main experiment I, II and III
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Main experiment I and II
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Main experiment I, II and III
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the end of the incubation period, precipitation of the test item was observed with WP2 uvrA and TA98, at the highest dose level (5000 μg/plate), in the absence and presence of S9 metabolic activation in main experiment I
RANGE-FINDING/SCREENING STUDIES:
Toxicity, as indicated by thinning of the background lawn, was observed with TA100, TA1535 and TA1537 tester strains, at the two or three highest concentrations tested, both in the absence and presence of S9 metabolism. A
more pronounced toxic effect was observed with TA100 tester strain, showinga marked and dose related reduction in revertant numbers. No toxic effect was noticed with WP2 uvrA and TA98, at any dose level tested, in the absence or presence of S9 metabolism.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached
- Negative (solvent/vehicle) historical control data: see attached - Conclusions:
- Based on the results of the read across study with the source substance Cedarwood Virginia Oil, the target substance Cedarwood Texas Terpenes does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
The skin sensitisition potential of the target substance Cedarwood Texas Terpenes was evaluated by read across with the source study for Cedarwood Virginia oil.
Three experiments were performed to test the gene mutation potential of Cedarwood Oil Virginiana according to OECDTG471. In main assay I, using the plate incorporation method, the test item was assayed at dose levels ranging from 16,5-5000µg/plate (concentrations chosen on the basis of the results obtained in a preliminary toxicity test). Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies was observed with TA1535, TA1537 and TA100, starting from 500 μg/plate or 1000 μg/plate, both in the absence and presence of S9 metabolic activation. No toxic effect was noticed with WP2uvrA and TA98. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at dose levels ranging from 7.81-5000µg/plate. No toxicity was noticed with WP2uvrA and TA98, at any dose level. Toxicity, from severe ( TA1537, and TA100) to slight (remaining strains), was observed with the remaining tester strains both in the absence and presence of S9 metabolism. In order to have a sufficient number of analysable concentrations, a further experiment (III), using the pre-incubation method, was performed with TA1537 and TA100 tester strains using dose levels ranging from 0.488-15.6 µg/plate. A slight thinning of the background lawn was observed with both tester strains, at the highest dose level. No relevant increase in the number of revertant colonies was observed, with any tester strain, in the plate incorporation or pre- incubation assay, at any dose level, in the absence or presence of S9 metabolism. The mean plate counts for untreated and positive control plates fell within RTC acceptance criteria based on historical control. It is concluded that the test item Cedarwood Virginia Oil does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Based on the results of this study Cedarwood Virginia Oil does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). This result was used for read across to Cedarwood Texas Terpenes
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The full read across justification report is attached under "Attached justification".
10 April 2018 READ-ACROSS STUDY / CW TX OIL TERPENES (1 & 2) / GENETIC TOXICITY IN MAMMALIAN CELLS I&B9W8768R001F1.0
According to Annex VIII, 8.4 of the REACh Regulation (EC) No 1907/2006, Genetic toxicity in mammalian cells is standard information required for the registration of substances manufactured or imported in quantities of ten tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).
A read-across approach appears appropriate to predict the endpoint “Genetic toxicity in mammalian cells” for the substance Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene) because:
Two Genetic Toxicity studies, one according to OECD test guideline 490 and one according OECD test guideline 474, are available for the substance Cedarwood Virginia oil (CW Virginia oil), of which the composition is very similar to Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene).
The compositions of the target and source substance are very similar and any slight differences in composition are for constituents that are not notified for genotoxicity and are not corresponding to known structural alerts; therefore no difference in genotoxic potential is expected between the source and target substances.
This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the absence of Genetic toxicity in mammalian cells for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated directly in the exposure medium at concentrations of 78 μg/ml and above. After 3 hour treatment, the test item precipitated in the exposure medium at concentrations of 313 μg/ml and above. The concentration used as the highest test item concentration for the dose range finding test was 200 μg/ml.
RANGE-FINDING/SCREENING STUDIES: The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
FInd below in any other information. - Conclusions:
- Based on the negative results of an in vitro gene mutation study in mammalian cells performed using the read across source substance Cedarwood Virginia oil, the target substance Cedarwood Texas oil distilled - Terpenes does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
Cedarwood Texas oil distilled - Terpenes is not classified as mutagenic, based on the results from the (OECD TG 490) source study performed with Cedarwood Virginia oil. The justification for this read across is provided in the attached justification for type of information.
This study evaluates the effects of Cedarwood Oil Virginia on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test item was dissolved in ethanol. In the first experiment, the test item was tested up to concentrations of 30 and 90μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. In the second experiment, the test item was tested up to concentrations of 45μg/ml in the absence of S9-mix. The incubation time was 24 hours. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency. It is concluded that Cedarwood Virginia oil is not mutagenic in the mouse lymphoma L5178Y test system under the presented experimental conditions, and therefore Cedarwood Virginia oil does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Data waiving:
- study scientifically not necessary / other information available
- Justification for data waiving:
- an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Referenceopen allclose all
Historical control data of the spontaneous mutation frequencies of the solvent controls for the mouse lymphoma assay
|
Mutation frequency per 10^6 survivors |
||
|
-S9 -mix |
+S9-mix |
|
|
3hour treatment |
24hour treatment |
3hour treatment |
Mean |
86 |
81 |
87 |
SD |
23 |
26 |
28 |
n |
220 |
202 |
273 |
Upper control limit (95%control limits) |
135 |
135 |
145 |
Lower control limit (95%control limits) |
37 |
28 |
28 |
SD=Standard deviation
n=Number of observations
Historicalcontrol data of the spontaneousmutation frequenciesof the positive controls for the mouse lymphomaassay
|
Mutation frequency per 10^6 survivors |
||
|
-S9-mix |
+S9-mix |
|
|
3 hour treatment |
24 hour treatment |
3 hour treatment |
Mean |
857 |
688 |
1710 |
SD |
246 |
187 |
815 |
n |
110 |
102 |
139 |
Upper control limit (95% control limits) |
1425 |
1124 |
4214 |
Lower control limit (95% control limits) |
289 |
253 |
-793 |
SD=Standarddeviation
n=Numberofobservations
Historical control data of the spontaneous mutation frequencies of the solvent controls for the mouse lymphoma assay
|
Mutation frequency per 10^6 survivors |
||
|
-S9 -mix |
+S9-mix |
|
|
3hour treatment |
24hour treatment |
3hour treatment |
Mean |
86 |
81 |
87 |
SD |
23 |
26 |
28 |
n |
220 |
202 |
273 |
Upper control limit (95%control limits) |
135 |
135 |
145 |
Lower control limit (95%control limits) |
37 |
28 |
28 |
SD=Standard deviation
n=Number of observations
Historicalcontrol data of the spontaneousmutation frequenciesof the positive controls for the mouse lymphomaassay
|
Mutation frequency per 10^6 survivors |
||
|
-S9-mix |
+S9-mix |
|
|
3 hour treatment |
24 hour treatment |
3 hour treatment |
Mean |
857 |
688 |
1710 |
SD |
246 |
187 |
815 |
n |
110 |
102 |
139 |
Upper control limit (95% control limits) |
1425 |
1124 |
4214 |
Lower control limit (95% control limits) |
289 |
253 |
-793 |
SD=Standarddeviation
n=Numberofobservations
Historical control data of the spontaneous mutation frequencies of the solvent controls for the mouse lymphoma assay
|
Mutation frequency per 10^6 survivors |
||
|
-S9 -mix |
+S9-mix |
|
|
3hour treatment |
24hour treatment |
3hour treatment |
Mean |
86 |
81 |
87 |
SD |
23 |
26 |
28 |
n |
220 |
202 |
273 |
Upper control limit (95%control limits) |
135 |
135 |
145 |
Lower control limit (95%control limits) |
37 |
28 |
28 |
SD=Standard deviation
n=Number of observations
Historicalcontrol data of the spontaneousmutation frequenciesof the positive controls for the mouse lymphomaassay
|
Mutation frequency per 10^6 survivors |
||
|
-S9-mix |
+S9-mix |
|
|
3 hour treatment |
24 hour treatment |
3 hour treatment |
Mean |
857 |
688 |
1710 |
SD |
246 |
187 |
815 |
n |
110 |
102 |
139 |
Upper control limit (95% control limits) |
1425 |
1124 |
4214 |
Lower control limit (95% control limits) |
289 |
253 |
-793 |
SD=Standarddeviation
n=Numberofobservations
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In vivo mammalian cytogenicity study (OECD TG 474): negative (read across from Cedarwood Virginia oil)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The full read across justification report is attached under "Attached justification".
10 April 2018 READ-ACROSS STUDY / CW TX OIL TERPENES (1 & 2) / GENETIC TOXICITY IN MAMMALIAN CELLS I&B9W8768R001F1.0
According to Annex VIII, 8.4 of the REACh Regulation (EC) No 1907/2006, Genetic toxicity in mammalian cells is standard information required for the registration of substances manufactured or imported in quantities of ten tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).
A read-across approach appears appropriate to predict the endpoint “Genetic toxicity in mammalian cells” for the substance Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene) because:
Two Genetic Toxicity studies, one according to OECD test guideline 490 and one according OECD test guideline 474, are available for the substance Cedarwood Virginia oil (CW Virginia oil), of which the composition is very similar to Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene).
The compositions of the target and source substance are very similar and any slight differences in composition are for constituents that are not notified for genotoxicity and are not corresponding to known structural alerts; therefore no difference in genotoxic potential is expected between the source and target substances.
This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the absence of Genetic toxicity in mammalian cells for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Key result
- Sex:
- female
- Genotoxicity:
- ambiguous
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Remarks on result:
- other: see Remarks
- Remarks:
- In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group.
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
A summary of the results are given in the tables below. The test material did not induce the formation of micronuclei, as measured in the blood mature erythrocytes. Comparison was performed with the vehicle controls. No data on historical controls are presented.
No significant increases in micronucleated erythrocytes (normochromatic erythrocytes; NCEs) were observed in blood samples from male B6C3F1/N mice following 3 months of dermal exposure to cedarwood oil (6.25% to 50%) (Table E2). In female B6C3F1/N mice treated with cedarwood oil for 3 months, small increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses (25% and 50%), but the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Because the trend test was significant (P=0.011), the results of the micronucleus assay in female mice were judged to be equivocal. No significant alterations in the percentage of micronucleated reticulocytes (polychromatic erythrocytes; PCEs) were seen in male or female mice, suggesting that Cedarwood Virginia oil applied dermally did not induce bone marrow toxicity. - Conclusions:
- The cytogenicity of Cedarwood Texas oil distilled - Terpenes is read across from the in vivo mammalian erythrocyte micronucleus source study performed with Cedarwood Virginia. The source substance did not induce any significant micronuclei formation in the mature blood erythrocytes of male mice, and though a trend was visible it did not induce the formation of micronucleated erythrocytes female mice compared to the control group. Therefore the overall cytogenicity protential for Cedarwood Virginia oil, and the read across target substance Cedarwood Texas oil distilled - Terpenes are considered to be negative.
- Executive summary:
The cytogenicity of Cedarwood Texas oil distilled - Terpenes is based on the results from the (similar to OECD TG 474) source study performed with Cedarwood Virginia oil. The justification for this read across is provided in the attached justification for type of information.
In a peripheral blood micronucleus assay using B6C3F1 mice, Cedarwood Virginia oil was administered dermally to groups of male and female animals at doses of 0, 120, 240, 480, 960, 1290 mg/kg bw (5 animals/sex). This examination was performed as part of 90 day repeated dose toxicity study by NTP. Negative control groups were treated with vehicle only (ethanol). The animals were sacrificed at the end of the treatment period and blood samples were collected for the micronucleus assay investigation. Slides of mature erythrocytes (NCEs) were prepared and stained with acridine orange. No deaths were observed in the test substance-dosed groups, or vehicle control group.
No significant alterations in the percentage of micronucleated reticulocytes were seen in male or female mice, suggesting that the exposure did not induce bone marrow toxicity. No significant increases in micronucleated erythrocytes were observed in blood samples from male mice following 3 months of dermal exposure. In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Therefore the overall cytogenicity protential for Cedarwood Virginia oil, and the read across target substance Cedarwood Texas oil distilled - Terpenes are considered to be negative.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- The full read across justification report is attached under "Attached justification".
10 April 2018 READ-ACROSS STUDY / CW TX OIL TERPENES (1 & 2) / GENETIC TOXICITY IN MAMMALIAN CELLS I&B9W8768R001F1.0
According to Annex VIII, 8.4 of the REACh Regulation (EC) No 1907/2006, Genetic toxicity in mammalian cells is standard information required for the registration of substances manufactured or imported in quantities of ten tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).
A read-across approach appears appropriate to predict the endpoint “Genetic toxicity in mammalian cells” for the substance Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene) because:
Two Genetic Toxicity studies, one according to OECD test guideline 490 and one according OECD test guideline 474, are available for the substance Cedarwood Virginia oil (CW Virginia oil), of which the composition is very similar to Cedarwood Texas oil distilled - Terpenes (Cedrene and Thujopsene).
The compositions of the target and source substance are very similar and any slight differences in composition are for constituents that are not notified for genotoxicity and are not corresponding to known structural alerts; therefore no difference in genotoxic potential is expected between the source and target substances.
This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the absence of Genetic toxicity in mammalian cells for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Key result
- Sex:
- female
- Genotoxicity:
- ambiguous
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Remarks on result:
- other: see Remarks
- Remarks:
- In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group.
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
A summary of the results are given in the tables below. The test material did not induce the formation of micronuclei, as measured in the blood mature erythrocytes. Comparison was performed with the vehicle controls. No data on historical controls are presented.
No significant increases in micronucleated erythrocytes (normochromatic erythrocytes; NCEs) were observed in blood samples from male B6C3F1/N mice following 3 months of dermal exposure to cedarwood oil (6.25% to 50%) (Table E2). In female B6C3F1/N mice treated with cedarwood oil for 3 months, small increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses (25% and 50%), but the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Because the trend test was significant (P=0.011), the results of the micronucleus assay in female mice were judged to be equivocal. No significant alterations in the percentage of micronucleated reticulocytes (polychromatic erythrocytes; PCEs) were seen in male or female mice, suggesting that Cedarwood Virginia oil applied dermally did not induce bone marrow toxicity. - Conclusions:
- The cytogenicity of Cedarwood Texas oil distilled - Terpenes is read across from the in vivo mammalian erythrocyte micronucleus source study performed with Cedarwood Virginia. The source substance did not induce any significant micronuclei formation in the mature blood erythrocytes of male mice, and though a trend was visible it did not induce the formation of micronucleated erythrocytes female mice compared to the control group. Therefore the overall cytogenicity protential for Cedarwood Virginia oil, and the read across target substance Cedarwood Texas oil distilled - Terpenes are considered to be negative.
- Executive summary:
The cytogenicity of Cedarwood Texas oil distilled - Terpenes is based on the results from the (similar to OECD TG 474) source study performed with Cedarwood Virginia oil. The justification for this read across is provided in the attached justification for type of information.
In a peripheral blood micronucleus assay using B6C3F1 mice, Cedarwood Virginia oil was administered dermally to groups of male and female animals at doses of 0, 120, 240, 480, 960, 1290 mg/kg bw (5 animals/sex). This examination was performed as part of 90 day repeated dose toxicity study by NTP. Negative control groups were treated with vehicle only (ethanol). The animals were sacrificed at the end of the treatment period and blood samples were collected for the micronucleus assay investigation. Slides of mature erythrocytes (NCEs) were prepared and stained with acridine orange. No deaths were observed in the test substance-dosed groups, or vehicle control group.
No significant alterations in the percentage of micronucleated reticulocytes were seen in male or female mice, suggesting that the exposure did not induce bone marrow toxicity. No significant increases in micronucleated erythrocytes were observed in blood samples from male mice following 3 months of dermal exposure. In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Therefore the overall cytogenicity protential for Cedarwood Virginia oil, and the read across target substance Cedarwood Texas oil distilled - Terpenes are considered to be negative.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Study performed by the NTP, widely recognised a high quality laboratory with extensive experience in toxicity studies. The study is considered acceptable. The test was performed according to the methods described by MacGregor et al. (1990).
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- The historical database is not presented in the data given for this study, i.e. abscence of control data (historical negative and positive controls). Administration of test item for 90 days, no details on occlusion. Blood samples taken only once.
- Principles of method if other than guideline:
- Peripheral blood miconucleus test on mice treated in 13 week toxicity study of the NTP as part of the bioassay programme
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Texarome, Inc., lot T122303DP
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 5° C in the original sealed amber glass shipping bottles
- Stability under test conditions: Stability was confirmed for at least 2 weeks for samples stored at temperatures up to 25° C in sealed amber glass vials. Freeze/thaw analyses indicated no decomposition due to repeated freezing and thawing.
- Solubility and stability of the test substance in the solvent/vehicle: Homogeneity was confirmed, and cedarwood oil formulations were stable for up to 3 hours under simulated animal room conditions.
OTHER SPECIFICS: Further information regarding the test material is available from the National Institute of Environmental Health Sciences - Species:
- mouse
- Strain:
- B6C3F1
- Remarks:
- /N
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Assigned to test groups randomly: yes
- Housing: mice were housed individually
- Diet: ad libitum
- Water: ad libitum - Route of administration:
- dermal
- Vehicle:
- - Vehicle(s)/solvent(s) used: ethanol
- Concentration of test material in vehicle: 6.25%, 12.5%, 25%, and 50%
- Lot/batch no. (if required): TP0179
- Purity: 95% - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared four times by mixing cedarwood oil and 95% ethanol to give the required concentrations. The dose formulations were stored at approximately 25° C in amber glass bottles sealed with Teflon®-lined lids for up to 34 days.
- Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- 5 days per week
- Post exposure period:
- none
- Dose / conc.:
- 0 mg/kg bw/day
- Dose / conc.:
- 120 mg/kg bw/day
- Dose / conc.:
- 240 mg/kg bw/day
- Dose / conc.:
- 480 mg/kg bw/day
- Dose / conc.:
- 960 mg/kg bw/day
- Dose / conc.:
- 1 290 mg/kg bw/day
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- none
- Tissues and cell types examined:
- erythrocytes from blood samples
- Details of tissue and slide preparation:
- TREATMENT AND SAMPLING TIMES: the animals were treated dermally (5 days/week) with the test item or vehicle control for 90 consecutive days. At termination (90 days) blood samples were collected from each animal.
DETAILS OF SLIDE PREPARATION:
The slides were air-dried, fixed, and stained with acridine orange that easily illuminates any micronuclei that may be present. 2000 mature erythrocytes (NCEs) were scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. - Evaluation criteria:
- In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 and the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups. Trials with either a significant trend or a significant dose are judged to be equivocal. The absence of a trend and a significant dose results in a negative call.
- Statistics:
- The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the vehicle control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Key result
- Sex:
- female
- Genotoxicity:
- ambiguous
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Remarks on result:
- other: see Remarks
- Remarks:
- In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group.
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
A summary of the results are given in the tables below. The test material did not induce the formation of micronuclei, as measured in the blood mature erythrocytes. Comparison was performed with the vehicle controls. No data on historical controls are presented.
No significant increases in micronucleated erythrocytes (normochromatic erythrocytes; NCEs) were observed in blood samples from male B6C3F1/N mice following 3 months of dermal exposure to cedarwood oil (6.25% to 50%) (Table E2). In female B6C3F1/N mice treated with cedarwood oil for 3 months, small increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses (25% and 50%), but the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Because the trend test was significant (P=0.011), the results of the micronucleus assay in female mice were judged to be equivocal. No significant alterations in the percentage of micronucleated reticulocytes (polychromatic erythrocytes; PCEs) were seen in male or female mice, suggesting that Cedarwood Virginia oil applied dermally did not induce bone marrow toxicity. - Conclusions:
- Under the conditions of this study, the test material Cedarwood Virginia did not induce any significant micronuclei formation in the mature blood erythrocytes of male mice, and though a trend was visible it did not induce the formation of micronucleated erythrocytes female mice compared to the control group.
- Executive summary:
In a peripheral blood micronucleus assay using B6C3F1 mice, Cedarwood Virginia oil was administered dermally to groups of male and female animals at doses of 0, 120, 240, 480, 960, 1290 mg/kg bw (5 animals/sex). This examination was performed as part of 90 day repeated dose toxicity study by NTP. Negative control groups were treated with vehicle only (ethanol). The animals were sacrificed at the end of the treatment period and blood samples were collected for the micronucleus assay investigation. Slides of mature erythrocytes (NCEs) were prepared and stained with acridine orange. No deaths were observed in the test substance-dosed groups, or vehicle control group. No significant alterations in the percentage of micronucleated reticulocytes were seen in male or female mice, suggesting that the exposure did not induce bone marrow toxicity. No significant increases in micronucleated erythrocytes were observed in blood samples from male mice following 3 months of dermal exposure. In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Therefore the overall cytogenic potential for Cedarwood Virginia oil is considered to be negative.
Referenceopen allclose all
Table 1. Summary of given scores for male animals
Male mice |
Dose (mg/kg bw/day) |
Mean MN-NCE/1000 NCE ±SEM |
Pairwise P |
Vehicle control |
0 |
2.000 ± 0.320 |
|
0 |
2.000 ± 0.320 |
|
|
Test substance |
120 |
2.000 ± 0.520 |
0.5 |
240 |
2.700 ± 0.200 |
0.1533 |
|
480 |
2.200 ± 0.300 |
0.3787 |
|
960 |
2.200 ± 0.410 |
0.3787 |
|
1290 |
0 |
|
Trend P=0.209
Table 2. Summary of given scores for female animals
Female mice |
Dose (mg/kg bw/day) |
Mean MN-NCE/1000 NCE ±SEM |
Pairwise P |
Vehicle control |
0 |
1.600 ± 0.370 |
|
0 |
1.600 ± 0.370 |
|
|
Test substance |
120 |
1.500 ± 0.270 |
0.5713 |
240 |
1.300 ± 0.410 |
0.7114 |
|
480 |
2.600 ± 0.370 |
0.0612 |
|
960 |
2.400 ± 0.290 |
0.1027 |
|
1290 |
0 |
|
Trend P=0.011
Table 1. Summary of given scores for male animals
Male mice |
Dose (mg/kg bw/day) |
Mean MN-NCE/1000 NCE ±SEM |
Pairwise P |
Vehicle control |
0 |
2.000 ± 0.320 |
|
0 |
2.000 ± 0.320 |
|
|
Test substance |
120 |
2.000 ± 0.520 |
0.5 |
240 |
2.700 ± 0.200 |
0.1533 |
|
480 |
2.200 ± 0.300 |
0.3787 |
|
960 |
2.200 ± 0.410 |
0.3787 |
|
1290 |
0 |
|
Trend P=0.209
Table 2. Summary of given scores for female animals
Female mice |
Dose (mg/kg bw/day) |
Mean MN-NCE/1000 NCE ±SEM |
Pairwise P |
Vehicle control |
0 |
1.600 ± 0.370 |
|
0 |
1.600 ± 0.370 |
|
|
Test substance |
120 |
1.500 ± 0.270 |
0.5713 |
240 |
1.300 ± 0.410 |
0.7114 |
|
480 |
2.600 ± 0.370 |
0.0612 |
|
960 |
2.400 ± 0.290 |
0.1027 |
|
1290 |
0 |
|
Trend P=0.011
Table 1. Summary of given scores for male animals
Male mice |
Dose (mg/kg bw/day) |
Mean MN-NCE/1000 NCE ±SEM |
Pairwise P |
Vehicle control |
0 |
2.000 ± 0.320 |
|
0 |
2.000 ± 0.320 |
|
|
Test substance |
120 |
2.000 ± 0.520 |
0.5 |
240 |
2.700 ± 0.200 |
0.1533 |
|
480 |
2.200 ± 0.300 |
0.3787 |
|
960 |
2.200 ± 0.410 |
0.3787 |
|
1290 |
0 |
|
Trend P=0.209
Table 2. Summary of given scores for female animals
Female mice |
Dose (mg/kg bw/day) |
Mean MN-NCE/1000 NCE ±SEM |
Pairwise P |
Vehicle control |
0 |
1.600 ± 0.370 |
|
0 |
1.600 ± 0.370 |
|
|
Test substance |
120 |
1.500 ± 0.270 |
0.5713 |
240 |
1.300 ± 0.410 |
0.7114 |
|
480 |
2.600 ± 0.370 |
0.0612 |
|
960 |
2.400 ± 0.290 |
0.1027 |
|
1290 |
0 |
|
Trend P=0.011
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
The available information on genetic toxicity for Cedarwood Texas Terpenes is based on read-across from Cedarwood Virginia oil. The executive summary of the experimental study is presented, and the read across justification is attached to the target study record.
Ames test(s) (OECD TG 471)
Three experiments were performed to test the gene mutation potential of Cedarwood oil Virginiana according to OECDTG471. In main assay I, using the plate incorporation method, the test item was assayed at dose levels ranging from 16,5-5000 µg/plate (concentrations chosen on the basis of the results obtained in a preliminary toxicity test). Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies was observed with TA1535, TA1537 and TA100, starting from 500 μg/plate or 1000 μg/plate, both in the absence and presence of S9 metabolic activation. No toxic effect was noticed with WP2uvrA and TA98. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of main assay II. The test item was assayed at dose levels ranging from 7.81-5000µg/plate. No toxicity was noticed with WP2uvrA and TA98, at any dose level. Toxicity, from severe ( TA1537, and TA100) to slight (remaining strains), was observed with the remaining tester strains both in the absence and presence of S9 metabolism. In order to have a sufficient number of analysable concentrations, a further experiment (III), using the pre-incubation method, was performed with TA1537 and TA100 tester strains using dose levels ranging from 0.488-15.6 µg/plate. A slight thinning of the background lawn was observed with both tester strains, at the highest dose level. No relevant increase in the number of revertant colonies was observed, with any tester strain, in the plate incorporation or pre- incubation assay, at any dose level, in the absence or presence of S9 metabolism. The mean plate counts for untreated and positive control plates fell within RTC acceptance criteria based on historical control. It is concluded that the test item Cedarwood Virginia Oil does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Based on the results of this study Cedarwood Virginia oil does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). This result was used for read-across to Cedarwood Texas oil distilled - Terpenes.
In vitro gene mutation study inmammalian cells (OECD TG 490) (read across from Cedarwood Virginia oil)
Cedarwood Texas oil distilled - Terpenes is not classified as mutagenic, based on the results from the (OECD TG 490) source study performed with Cedarwood Virginia oil. The justification for this read across is provided in the attached justification for type of information.
This study evaluates the effects of Cedarwood Oil Virginia on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test item was dissolved in ethanol. In the first experiment, the test item was tested up to concentrations of 30 and 90μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. In the second experiment, the test item was tested up to concentrations of 45μg/ml in the absence of S9-mix. The incubation time was 24 hours. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency. It is concluded that Cedarwood Virginia oil is not mutagenic in the mouse lymphoma L5178Y test system under the presented experimental conditions, and therefore Cedarwood Virginia oil, and its read across target Cedarwood Texas oil distilled - Terpenes:, do not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
In vivo mammalian cytogenicity study (OECD TG 474) (read across from Cedarwood Virginia oil)
The cytogenicity of Cedarwood Texas oil distilled - Terpenes is based on the results from the (similar to OECD TG 474) source study performed with Cedarwood Virginia oil. The justification for this read across is provided in the attached justification for type of information.
In a peripheral blood micronucleus assay using B6C3F1 mice, Cedarwood Virginia oil was administered dermally to groups of male and female animals at doses of 0, 120, 240, 480, 960, 1290 mg/kg bw (5 animals/sex). This examination was performed as part of 90 day repeated dose toxicity study by NTP. Negative control groups were treated with vehicle only (ethanol). The animals were sacrificed at the end of the treatment period and blood samples were collected for the micronucleus assay investigation. Slides of mature erythrocytes (NCEs) were prepared and stained with acridine orange. No deaths were observed in the test substance-dosed groups, or vehicle control group.
No significant alterations in the percentage of micronucleated reticulocytes were seen in male or female mice, suggesting that the exposure did not induce bone marrow toxicity.
No significant increases in micronucleated erythrocytes were observed in blood samples from male mice following 3 months of dermal exposure. In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Therefore the overall cytogenicity protential for Cedarwood Virginia oil, and the read across target substance Cedarwood Texas oil distilled - Terpenes are considered to be negative.
Justification for classification or non-classification
Based on the available information and read across assessment, Cedarwood Texas oil distilled - Terpenes does not need to be classified for genetic toxicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).
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