Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel.1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- CHO/HPRT Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 1): non mutagenic up to cytotoxic concentrations.
- HL chromosome aberration test (OECD 473, GLP, read-across, K, rel. 2): non clastogenic up to cytotoxic concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 24 to August 8, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected from 2011-06-28 to 2011-06-30/signed on 2011-08-17)
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from male Sprague-Dawley derived rats, dosed i.p. with phenobarbital sodium (30 mg/kg 4 days before killing and 60 mg/kg 1, 2 & 3 days before killing) and 5,6-benzoflavone (80 mg/kg 2 days before killing),
Test concentrations with justification for top dose:
Up to 5000 µg/plate (series of ca half-log10 dilutions)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was assessed at 50 mg/mL in water and in dimethyl sulphoxide (DMSO). It was found to be insufficiently soluble in or miscible with water, but was soluble in DMSO. DMSO (ACS spectrophotometric grade) was, therefore, used as the vehicle for this study.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/1
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
In the absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
In the presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: First test : in agar (plate incorporation); second test : preincubation.

FIRST TEST DURATION
- Exposure duration: ca 72 hours at 37°C

SECOND TEST DURATION
- Preincubation period: 30 minutes at 37°C
- Exposure duration: ca 72 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates per treatment

DETERMINATION OF CYTOTOXICITY
- Method: substantial reduction in mean revertant colony counts or by a sparse or absent background lawn.

STERILITY CHECK
Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer.
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Statistics:
No statistical analysis is performed in case of clear positive results. If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See attached background material
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: not expected (vapor pressure = 1.5 Pa at 25°C)
- Water solubility: not soluble in water, therefore DMSO was used.
- Precipitation: none seen

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.
[Cf Tables in attached background material]

ADDITIONAL INFORMATION:
The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 109/mL in all cases, and therefore met the acceptance criteria.

None

Conclusions:
The test material showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

In this in vitro assessment of the mutagenic potential of the test item, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test material diluted in dimethyl sulphoxide (DMSO). DMSO was also used as a vehicle control. The study was conducted in compliance with OECD 471, EC B.13/14 and OPPTS 870.5100 test guidelines and with GLP.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

Concentrations of the test material up to, and including, 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series ofcahalf-log10dilutions of the highest concentration.

No signs of toxicity towards the tester strains were observed in either mutation test following exposure to the test material.

No evidence of mutagenic activity was seen at any concentration of the test material in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

It was concluded that the test material showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

This study is considered as acceptable and satisfies the requirement for the bacterial gene mutation endpoint.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 August 2012 to 15 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected between 18 and 20 June 2012/signed on 2011-08-17)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cells: CHO-KI cells were obtained from the European Collection of Cell Cultures. The cells are stored at -196°C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO).
- Type and identity of media:
Ham’s Nutrient Mixture F12, supplemented with 2 mM L glutamine and 50 µg/mL gentamicin. The resulting medium is referred to as H0.
H0 medium supplemented with 10% HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.
The selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 µg/mL.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no, assumed to be stable
- Periodically "cleansed" against high spontaneous background: yes, 4 days prior to exposure, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 µg/mL hypoxanthine, 0.3 µg/mL amethopterin and 4 µg/mL thymidine for three days.
All cell cultures are maintained at 37°C in a atmosphere of 5% CO2 in air.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (25% S9-fraction obtained from the liver of male Sprague-Dawley treated with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route 5 days before dosing) (see Table 7.6.1/1)
Test concentrations with justification for top dose:
Preliminary toxicity test: 17.37, 34.73, 69.47, 138.94, 277.88, 555.75, 1111.5 and 2223 µg/mL (up to 10 mM).
Main test 1 (-S9 mix): 10, 30, 35, 40, 45, 50, 55, 60, 65 and 70 µg/mL.
Main test 1 (+S9 mix): 100, 200, 250, 300, 350, 400, 450, 500 and 550 µg/mL.
Additional test 1 (+S9 mix): 100, 120, 140, 160, 180, 200, 240, 280 and 320 µg/mL.
Main test 2 (-S9 mix): 10, 20, 30, 32.5, 35, 37.5, 40 and 45 µg/mL.
Main test 2 (+S9 mix): 100, 120, 130, 140, 150, 160, 170 and 180 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (final concentration 1% v/v)
- Justification for choice of solvent/vehicle: Prior to commencing testing, the solubility of the test substance in a vehicle compatible with this test system was assessed. The test item was found to be soluble at 222.3 mg/mL (1M) in dimethyl sulphoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
250 µg/mL in DMSO
Positive control substance:
ethylmethanesulphonate
Remarks:
in the absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/mL in DMSO
Positive control substance:
3-methylcholanthrene
Remarks:
in the presence of S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
The cells were incubated for approximately 20 hours at 37°C, in an atmosphere of 5% CO2 in air, prior to exposure to the test substance on Day 1.
- Exposure duration: 3 hours.
- Expression time (cells in growth medium): 7 days, at 37°C, in a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: duplicate cultures for each concentration of the test compound and positive controls, four individual cultures for solvent controls.

NUMBER OF CELLS EVALUATED: 10E06 cells from each culture were seeded to allow expression of the mutant phenotype.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (200 cells/plate)
Evaluation criteria:
- The demonstration of a statistically significant increase in mutant frequency following exposure to the test substance;
- Evidence of a relationship, over at least two dose levels, in any increase in mutant frequency;
- Demonstration of reproducibility in any increase in mutant frequency;
- The mean mutant frequency should fall outside the upper limit of the historical solvent control of 20 mutants per 10E6 survivors with a corresponding survival rate of 20% or greater.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control to solvent control.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See table 7.6.1/2
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 7.6.1/2
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium of more than 1.0 unit compared with the vehicle control were observed at 2223 µg/mL.
- Effects of osmolality: The osmolality of the test substance in medium was tested at a concentration of 2223 µg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Evaporation from medium: not applicable
- Water solubility: not soluble in water
- Precipitation: No precipitate was observed in the absence of S9 either post-dosing or at the end of treatment in both tests. Precipitate was seen in Test 1 (+S9) at 250 μg/mL and above post-dosing and at 200 μg/mL and above the end of treatment. Precipitate was seen in additional test 1 (+S9) at 240 μg/mL and above post-dosing and at 200 μg/mL and above at the end of treatment. No precipitate was seen in Test 2 (+S9) either post-dosing or at the end of treatment.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity test, a four-hour exposure to concentrations from 17.37 to 2223 μg/mL, in the absence or the presence of S9 mix, resulted in Day 1 relative survivals of 95 to 0% and 95 to 0% respectively. Precipitate was seen at 277.88 μg/mL and above post-dosing and at 138.94 μg/mL and above at the end of treatment in both the absence and presence of S9 mix. Concentrations for the main test were based upon these data.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Test 1 (-S9-mix): One of the test item test cultures also had mutation frequencies that exceeded the historical control maximum. However, there was no statistical significance so these data were considered to be acceptable and not to be indicative of a positive response.
All solvent and positive controls (where included) were considered to have met the acceptance criteria and therefore the results were accepted.
- Test 2 (+S9-mix): One of the test item test cultures also had mutation frequencies that exceeded the historical control maximum. However, there was no statistical significance so these data were considered to be acceptable and not to be indicative of a positive response.
All solvent and positive controls (where included) were considered to have met the acceptance criteria and therefore the results were accepted.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Test 1 (-S9 mix): Exposure to the test item resulted in Day 1 relative survivals of between 100 and 0%. The Day 8 cloning efficiencies were 118 to 123% relative to the solvent controls.
- Test 1 (+S9 mix): Exposure to the test item resulted in a decrease in observed cell count on Day 1 that was considered not to achieve the required toxicity range. An additional test was therefore performed.
- Additional Test 1 (+S9 mix): Exposure to the test item resulted in Day 1 relative survivals of between 102 and 27%. The Day 8 cloning efficiencies were 109 to 96 % relative to the solvent control.
- Test 2 (-S9 mix): Exposure to Nirvanol resulted in Day 1 relative survivals of between 97 to 2%. The Day 8 cloning efficiencies were 102 to 96% relative to the solvent control
- Test 2 (+S9-mix): Exposure to Nirvanol resulted in Day 1 relative survivals of between 93 to 14 %. The Day 8 cloning efficiencies were 100 to 94 % relative to the solvent control.

Table 7.6.1/2: Summary table

Test Article

Dose Level

µg/mL

Test 1

Test 2

3 hr Treatment ‑S9‑mix

3 hr Treatment +S9‑mix

3 hr Treatment ‑S9‑mix

3 hr Treatment +S9‑mix

Mean Relative Survival (%)

Mean Mutant Frequency (x10-6)

Mean Relative Survival (%)

Mean Mutant Frequency (x10-6)

Mean Relative Survival (%)

Mean Mutant Frequency (x10-6)

Mean Relative Survival (%)

Mean Mutant Frequency (x10-6)

DMSO

0

100

17.63

100

17.09

100

6.99

100

7.46

Nirvanol

10

100

18.22

 

 

90

9.84

 

 

Nirvanol

20

 

 

 

 

97

9.14

 

 

Nirvanol

30

91

19.56

 

 

85

4.36

 

 

Nirvanol

32.5

 

 

 

 

76

13.25

 

 

Nirvanol

35

44

27.67

 

 

72

9.31

 

 

Nirvanol

37.5

 

 

 

 

44

7.78

 

 

Nirvanol

40

11

16.20

 

 

22

11.15

 

 

Nirvanol

100

 

 

102

15.34

 

 

93

7.80

Nirvanol

120

 

 

88

17.47

 

 

70

4.60

Nirvanol

130

 

 

 

 

 

 

54

14.52

Nirvanol

140

 

 

45

23.29

 

 

14

19.48

Nirvanol

160

 

 

27

19.33

 

 

 

 

Ethyl methansulphonate

250

79

291.19***

 

 

72

415.00***

 

 

3-Methylcholanthrene

5

 

 

94

426.06***

 

 

78

548.56***

***       Statistically significant: p < 0.001

Conclusions:
The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation, in both experiments.
Executive summary:

In an in vitro mammalian cell mutation assay performed according to the OECD test guideline No. 476 and in compliance with GLP, Chinese hamster ovary cells (CHO-K1) were exposed to the test material diluted in DMSO, in duplicate in the presence and absence of metabolic activation (S9-mix). The entire test was repeated to confirm the result of the first test. Three-hour exposures were used both with and without activation (S9) in both tests. The highest final concentration used in the preliminary toxicity test was 2223 μg/mL, which was equivalent to the maximum recommended level of 10mM. Cytotoxicity was measured as Day 1 relative survival; values were from 95 to 0%, and 95 to 0%, after exposure to concentrations from 17.37 to 2223 µg/mL in the absence and presence of S9 mix respectively.

In the absence of S9 mix, in Test 1, cells were exposed to concentrations from 10 to 70 μg/mL. Day 1 survival values ranged from 100 to 0% relative to the solvent control. The test material did not cause a statistically significant increase in mutant frequency.

In the presence of S9 mix, in Test 1, cells were exposed to concentrations from 100 to 320 μg/mL. Day 1 survival values ranged from 102 to 27% for relative to the solvent control. The test material did not cause a statistically significant increase in mutant frequency.

In the absence of S9 mix, in Test 2, cells were exposed to concentrations from 10 to 45 μg/mL. Day 1 survival values ranged from 97 to 2% relative to the solvent control. The test material did not cause a statistically significant increase in the mutant frequency.

In the presence of S9 mix, in Test 2, cells were exposed to concentrations from 100 to 180 μg/mL. Day 1 survival values ranged from 93 to 7% relative to the solvent control. The test material did not cause a statistically significant increase in mutant frequency.

The positive control treatments, both in the absence and presence of metabolic activation, induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant or dose-related increases in the mutant frequency at the HPRT locus in CHO cells at any dose level, either in the presence or absence of metabolic activation, in both experiments.

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 April 1988 to 20 April 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study performed according to OECD test guideline No. 473 (1983 version) and in compliance with GLP. The exposure time was only 2 hours instead of 3 to 6 hours but the substance was tested up to a toxic limit and the positive control substances worked correctly, therefore this slightly shorter exposure time is not expected to be critical. The 24-hour exposure group is missing, but the test substance does not have any structural alerts, so it should not be positive after a longer exposure period.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(Adopted: 26 May 1983)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not Applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown in Eagle's minimal essential medium (EMEM), supplemented with 15% foetal calf serum (FCS) and antibiotics, at 37'C with 5% C02 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin-M (PHA) at 90 µg/ml final concentration.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (S9-fraction obtained from the livers of male Sprague-Dawley rat injected with Aroclor 1254 at 500 mg/kg bw)
Test concentrations with justification for top dose:
Without S9 : 0*, 78*, 156*, 312*, 625*, 1250, 2500.
With S9 : 0*, 78*, 156*, 312*, 625*, 1250, 2500.
* Dose levels selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none, diluted directly in EMEM medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
EMEM medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in the absence of metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
EMEM medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the presence of metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: mytogenic stimulation of lymphocytes with phytohaemagglutinin-M (PHA)) was performed 48 hours before lymphocyte treatment with the test substance.
- Exposure duration: without and with metabolic activation: 2 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours


SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (Colcemid) 0.1 µg/mL for 2 hours (2 hours before cell harvest)
STAIN (for cytogenetic assays): Gurrs Giemsa R66 (5 % for 5 min)

NUMBER OF CELLS EVALUATED: 200 cells/dose (metaphase analysis)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: YES
- Determination of endoreplication: YES
- Other: any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 45 or more chromosomes,
any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the UKEMS guidelines for mutagenicity testing.
Statistics:
None mentioned in study report
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1250 & 2500 µg/mL
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: not reported
- Evaporation from medium: Vapour pressure of test substance: 1.5 Pa. No relevant impact on the study.
- Water solubility: 13.5 mg/L @ 20°C
- Precipitation: none
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA:
All the negative control cultures gave values of chromosome aberrations within the expected range (See attached document). The frequency of aberrations was consistent between the two negative control groups, the highest frequency (3.5% total aberrations with gaps) being seen in the culture group without S9.
All the positive control cultures gave highly significant increases in the frequency of aberrations (See attached document) indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The maximum two doses used 2500 and 1250 µg/mL, were observed to be haemolytic and also markedly reduced the mitotic index. 625 µg/mL was therefore selected as the highest dose for slide evaluation with 312.5, 156.25 and 78.125 µg/mL as the three lower doses.

In the case of the cultures with S9, there was a non-significant increase in both the number of total aberrations and the % cells with aberrations at the maximum dose level (625 µg/mL) of ST 02 C 88 (See attached document). However, this increase was seen only when gaps were included in the evaluation. There were no increases in the frequency of non-gap aberrations in the presence of S9.

In the absence of S9 there was a small non-significant increase in the number of total aberrations (non-gap) and the %cells with non-gap aberrations at the maximum dose level (625 µg/mL) of ST 02 C 88 when compared to the control incidence (2.5% v 0%, X2 = 3.24 N.S., in both cases). When gap-type aberrations were included in the evaluation the increases became statistically significant (10.5% v 3.5%, X2 = 6.49, p<0.01 and 9.5% v 3.5%, X2 = 4.97, p<0.05, respectively.

ST 02 C 88 did not induce a significant increase in the numbers of hyperdiploid cells at any dose-level in any of the treatment cases. 1% hyperdiploidy was the highest frequency of hyperdiploid cells seen in either the negative controls or the treatment groups and this was within the expected range.

Conclusions:
ST 02 C 88 is non-clastogenic to human lymphocytes in-vitro, under the conditions of the test.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 (old version) and in compliance with GLP, human lymphocyte cultures were exposed to the test material at concentrations of 0, 78.125, 156.25, 312.5 and 625 µg/mL both in the presence and absence of metabolic activation (10% rat liver homogenate metabolising system). Cells were exposed 2 hours in the presence and absence of S9-mix and were harvested after a 24-hour expression period.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

In the case of the cultures with S9, there was a non-significant increase in both the number of total aberrations and the % cells with aberrations at the maximum dose level (625 µg/mL) of ST 02 C 88 (See attached document). However, this increase was seen only when gaps were included in the evaluation. There were no increases in the frequency of non-gap aberrations in the presence of S9.

In the absence of S9 there was a small non-significant increase in the number of total aberrations (non-gap) and the %cells with nongap aberrations at the maximum dose level (625 µg/mL) of ST 02 C 88 when compared to the control incidence (2.5% v 0%, X2 = 3.24 N.S., in both cases). When gap-type aberrations were included in the evaluation the increases became statistically significant (10.5% v 3.5%, X2 = 6.49, p<0.01 and 9.5% v 3.5%, X2 = 4.97, p<0.05, respectively.

ST 02 C 88 did not induce a significant increase in the numbers of hyperdiploid cells at any dose-level in any of the treatment cases. 1% hyperdiploidy was the highest frequency of hyperdiploid cells seen in either the negative controls or the treatment groups and this was within the expected range.

Under the test conditions, ST 02 C 88 was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.

Endpoint:
in vitro cytogenicity / chromosome aberration 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:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are trans-3,3-dimethyl-5-(2,2,3-trimethyl-cyclopent-3-en-1-yl)pent-4-en-2-ol. As two asymmetric (chiral) carbon atoms are present in this chemical structure (marked with a * in Table 1), the chemical exists as 2x2=4 enantiomers: RR, RS, SR and SS forms. The target substance is a reaction-mass between two of these stereoisomers (RS and SS) whereas the target substance is a mixture of the two others (SR and RR).

3. ANALOGUE APPROACH JUSTIFICATION
- In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances.
- In vitro gene mutation study in mammalian cells
In the hprt test performed on the target substance, none of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases.
- In vitro cytogenicity study in mammalian cells
In the CAT performed on the source substance, no significant increases in the frequency of cells with aberrations was induced. The CAT test on the target substance provided in this dossier was performed according to OECD TG 473. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the CAT conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.3.

4. DATA MATRIX
Please refer to Iuclid section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1250 & 2500 µg/mL
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: not reported
- Evaporation from medium: Vapour pressure of test substance: 1.5 Pa. No relevant impact on the study.
- Water solubility: 13.5 mg/L @ 20°C
- Precipitation: none
- Other confounding effects: none

COMPARISON WITH HISTORICAL CONTROL DATA:
All the negative control cultures gave values of chromosome aberrations within the expected range (See attached document). The frequency of aberrations was consistent between the two negative control groups, the highest frequency (3.5% total aberrations with gaps) being seen in the culture group without S9.
All the positive control cultures gave highly significant increases in the frequency of aberrations (See attached document) indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The maximum two doses used 2500 and 1250 µg/mL, were observed to be haemolytic and also markedly reduced the mitotic index. 625 µg/mL was therefore selected as the highest dose for slide evaluation with 312.5, 156.25 and 78.125 µg/mL as the three lower doses.

In the case of the cultures with S9, there was a non-significant increase in both the number of total aberrations and the % cells with aberrations at the maximum dose level (625 µg/mL) of ST 02 C 88 (See attached document). However, this increase was seen only when gaps were included in the evaluation. There were no increases in the frequency of non-gap aberrations in the presence of S9.

In the absence of S9 there was a small non-significant increase in the number of total aberrations (non-gap) and the %cells with non-gap aberrations at the maximum dose level (625 µg/mL) of ST 02 C 88 when compared to the control incidence (2.5% v 0%, X2 = 3.24 N.S., in both cases). When gap-type aberrations were included in the evaluation the increases became statistically significant (10.5% v 3.5%, X2 = 6.49, p<0.01 and 9.5% v 3.5%, X2 = 4.97, p<0.05, respectively.

ST 02 C 88 did not induce a significant increase in the numbers of hyperdiploid cells at any dose-level in any of the treatment cases. 1% hyperdiploidy was the highest frequency of hyperdiploid cells seen in either the negative controls or the treatment groups and this was within the expected range.

Conclusions:
Based on the available data on the source substance, the target substance is considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No 473 (old version) and in compliance with GLP, human lymphocyte cultures were exposed to the source substance at concentrations of 0, 78.125, 156.25, 312.5 and 625 µg/mL both in the presence and absence of metabolic activation (10% rat liver homogenate metabolising system). Cells were exposed 2 hours in the presence and absence of S9-mix and were harvested after a 24-hour expression period.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

In the case of the cultures with S9, there was a non-significant increase in both the number of total aberrations and the % cells with aberrations at the maximum dose level (625 µg/mL) of ST 02 C 88 (See attached document). However, this increase was seen only when gaps were included in the evaluation. There were no increases in the frequency of non-gap aberrations in the presence of S9.

In the absence of S9 there was a small non-significant increase in the number of total aberrations (non-gap) and the %cells with nongap aberrations at the maximum dose level (625 µg/mL) of ST 02 C 88 when compared to the control incidence (2.5% v 0%, X2 = 3.24 N.S., in both cases). When gap-type aberrations were included in the evaluation the increases became statistically significant (10.5% v 3.5%, X2 = 6.49, p<0.01 and 9.5% v 3.5%, X2 = 4.97, p<0.05, respectively.

ST 02 C 88 did not induce a significant increase in the numbers of hyperdiploid cells at any dose-level in any of the treatment cases. 1% hyperdiploidy was the highest frequency of hyperdiploid cells seen in either the negative controls or the treatment groups and this was within the expected range.

Based on the available data on the source substance, the target substance was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

HLS, 2012

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100,

WP2 uvrA

-S9

+S9

Up to limit concentrations

-S9 : non mutagenic

+S9 : non mutagenic

2

 

HLS, 2012

CHO/HPRT test (OECD 476)

K, rel. 1

Gene mutation

Chinese hamster ovary cells (CHO-K1)

-S9

+S9

Up to cytotoxic concentrations

-S9 : non mutagenic

+S9 : non mutagenic

3

(Read-across)

 

Safepharm, 1988

HL CAT

(OECD 473)

K, rel. 2

Chromosomal aberration

Human lymphocytes

 

-S9

+S9

Up to cytotoxic concentrations

-S9 : non clastogenic

+S9 : non clastogenic

 

Gene mutation Assays (Tests n° 1 -2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with the substance (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria under the test conditions whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammalian cells using an in vitro gene mutation assay in Chinese hamster ovary cells (CHO/HPRT test) (Test n°2). None of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments. The substance does not induce forward mutations at the HPRT locus in CHO cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore the substance is considered as negative for inducing gene mutations at the HPRT locus in CHO cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

 

Chromosomal aberration (Test n°3)

No study was available on the substance itself to assess clastogenicity potential, therefore a read-across approach was used. The source substance is considered adequate for read-across purposes (see Iuclid section 13 for additional justification).

The clastogenic potential of the test material was determined with the supporting substance using an in vitro chromosome aberration test in Human lymphocytes, which measures the potential of a substance to increase the incidence of structural chromosome aberrations in Human lymphocytes. None of the dose levels up to the cytotoxicity limit with the supporting substance, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The supporting substance does not induce structural aberrations in the chromosomes of Human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.

Therefore both the supporting substance and the registered substance are considered as negative for inducing chromosomal mutations in Human lymphocytes under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

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

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).