Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of the test substance, IFF 215 (Floriane) was assessed and considered to be non-mutagenic according to OECD 471 using the Bacterial Reverse Mutation method. The genotoxic potential of the test substance, IFF 215 (Floriane) was also assessed according to OECD Test Guideline 473 using an In Vitro Mammalian Chromosome Aberration method. The test item, IFF 215 (Floriane), did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 17 June 2014 and 13 November 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 473 using an In Vitro Mammalian Chromosome Aberration method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.


Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

4 (20)-hour without S9: 0, 7.5, 15, 30, 60, 80, 100, 120 µg/mL.
4 (20)-hour with S9: 0, 7.5, 15, 30, 60, 80, 100, 120 µg/mL.
24-hour without S9: 0, 3.75, 7.5, 15, 30, 60, 80, 100, 120 µg/mL.
4 (20)-hour with S9 (1 %): 0, 3.75, 7.5, 15, 30, 45, 60, 80 µg/mL.

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Without S9: Mitomycin C at 0.4 and 0.2 µg/mL in Experiment 1 and 2 respectively dissolved in Minimal Essential Medium. With S9: cyclophosphamide at 5 µg/mL dissolved in dimethyl sulphoxide.

Details on test system and experimental conditions:

Test Item
The molecular weight of the test item was given as 196.2, therefore, the maximum dose level was 1962 µg/mL, which was calculated to be equivalent to the 10mM dose level. The purity of the test item was 87.4 % and was accounted for in the test item formulations.

The test item was insoluble in aqueous media at 19.62 mg/mL and in dimethyl sulphoxide at 196.2 and 98.1 mg/mL, however, the test item was miscible in acetone at 196.2 and 392.4 mg/mL in solubility checks performed in house. Acetone is toxic to human lymphocytes at 100 µL; therefore all of the formulations were prepared at concentrations two times greater than required in the culture flasks. To compensate, each formulation was dosed using 50 µL (0.05 mL) aliquots. The highest achievable concentration was 392.4 mg/mL which when dosed into culture flasks, gave a concentration of 1962 µg/mL. Prior to each experiment, the test item was accurately weighed and formulated in acetone and serial dilutions prepared.

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Microsomal Enzyme Fraction
Lot No’s. PB/βNF S9 01/06/14 and PB/βNF S9 27/07/14 were used in this study. The S9 Microsomal fraction was prepared in house from male rats induced with Phenobarbitone/β Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (10 to 20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2% in the Preliminary Toxicity Test and Experiment 1 and 1% in Experiment 2.

This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.

Test Procedure
Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood


With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.

In Experiment 2, 1 mL of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.

The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

The dose range of test item used was 0, 7.66, 15.33, 30.66, 61.31, 122.63, 245.25, 490.5, 981, 1962 µg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1
Two exposure groups were used for Experiment 1:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 30, 60, 80, 100 and 120 µg/mL.

ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 30, 60, 80, 100 and 120 µg/mL.


Experiment 2
Two exposure groups were used for Experiment 2:
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 3.75, 7.5, 15, 30, 60, 80 and 120 µg/mL.

ii) 4-hour exposure to the test item with S9-mix (1%) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 3.75, 7.5, 15, 30, 45, 60 and 80 µg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) and cells with endoreduplicated chromosomes was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

The following criteria were used to determine a valid assay:

Negative Control
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.

Positive Control
All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 7.66 to 1962 µg/mL. The maximum dose was the 10 mM concentration.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 245.25 µg/mL in the 4(20)-hour exposure group in the absence of S9-mix, 490.5 µg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at and above 122.63 µg/mL in the continuous exposure group.

Haemolysis was also observed following exposure to the test item at and above 30.66 µg/mL in the 4(20)-hour exposure group in the absence of S9-mix, at and above 15.33 µg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at and above 61.31 µg/mL in the 24-hour continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 61.31 µg/mL in all three exposure groups. The mitotic index data confirms that the test item induced excessive toxicity with a sudden onset in all of the exposure groups. Based on these observations it was considered that it would be difficult to achieve optimum toxicity.

The selection of the maximum dose level was based on toxicity for both Experiments 1 and 2.


Chromosome Aberration Test - Experiment 1
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 30 µg/mL in both exposure groups.
No precipitate observations were made at the end of exposure in either exposure group. Haemolysis was observed following exposure to the test item at and above 7.5 µg/mL in both exposure groups.

The mitotic index data confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed prior to the excessive toxicity observed at and above 60 µg/mL. Therefore, the maximum dose level selected for metaphase analysis was 30 µg/mL in both the absence and presence of S9-mix.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The qualitative assessment of the slides determined that the toxicity was not as severe as the previous experiment and there were metaphases suitable for scoring present up to 120 µg/mL and 80 µg/mL in the absence and presence of S9-mix, respectively.

No precipitate of the test item was observed at the end of exposure, however, haemolysis was observed at and above 30 µg/mL and 15 µg/mL in the absence and presence of S9-mix, respectively.

The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in both exposure groups. In the absence of S9-mix 38% and 64% mitotic inhibition was achieved at 60 and 80 µg/mL, respectively. In the presence of S9-mix, 40% mitotic inhibition was achieved at 60 µg/mL only.

The maximum dose level selected for metaphase analysis was, therefore, based on the concentrations approaching the optimum toxicity limit of 50% and was 60 µg/mL in both exposure groups.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test item are given in the table below:

 

Group	Final concentration ofIFF215 (Floriane)(µg/mL)
4(20)-hour without S9	0*, 7.5*, 15*, 30*, 60, 80, 100, 120, MMC 0.4*
4(20)-hour with S9 (2%)	0*, 7.5*, 15*, 30*, 60, 80, 100, 120, CP 5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test item are given in the table below:

 

Group	Final concentration ofIFF215 (Floriane)(µg/mL)
24-hour without S9	0*, 3.75, 7.5, 15*, 30*, 60*, 80, 120, MMC0.2*
4(20)-hour with S9	0*, 3.75, 7.5, 15, 30*, 45*, 60*, 80, CP5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Mitotic Index - Preliminary Toxicity Test

4-Hour Treatment, 20-Hour Recovery -S9

4-Hour Treatment, 20-Hour Recovery +S9

24-Hour Treatment -S9

 

se Level (µg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	2.10	100	3.20	100	5.20	100
7.66	-	-	-	-	-	-
15.33	2.30	110	2.15 H	67	3.60	69
30.66	2.90 H	138	2.40 H	75	2.85	55
61.31	1.75 H	83	3.80 H	119	2.60 H	50
122.63	- NM H	-	- NM H	-	- NM H P	-
245.25	- NM H P	-	- NM H	-	- NM H P	-
490.5	- NM H P	-	- NM H P	-	- NM H P	-
981	- NM H P	-	- NM H P	-	- NM H P	-
1962	- NM H P	-	- NM H P	-	- NM H P	-

Mitotic Index - Experiment 1

Dose Level (mg/mL)	4(20)-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	2.15	3.35	2.75	100	5.10	5.15	5.13	100
7.5	2.30 H	2.25 H	2.28	83	6.20 H	7.05 H	6.63	129
15	1.20 H	3.20 H	2.20	80	9.10 H	8.15 H	8.63	168
30	1.45 H	3.50 H	2.48	90	3.80 H	5.35 H	4.58	89
60	- NM H	- NM H	-	-	- NM H	- NM H	-	-
80	- NM H	- NM H	-	-	- NM H	- NM H	-	-
100	- NM H	- NM H	-	-	- NM H	- NM H	-	-
120	- NM H	- NM H	-	-	- NM H	- NM H	-	-
MMC 0.4	1.55	2.25	1.90	69	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	0.65	0.70	0.68	13

Mitotic Index - Experiment 2

Dose Level (µg/mL)	24-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	9.15	9.55	9.35	100	5.70	6.55	6.13	100
3.75	-	-	-	-	-	-	-	-
7.5	-	-	-	-	-	-	-	-
15	9.95	12.45	11.20	120	- H	- H	-	-
30	9.55 H	8.50 H	9.03	97	4.70 H	6.05 H	5.38	88
45	NA	NA	NA	NA	5.85 H	5.60 H	5.73	93
60	5.75 H	5.80 H	5.78	62	4.55 H	2.85 H	3.70	60
80	3.45 H	3.20 H	3.33	36	- NM H	- NM H	-	-
120	- NM H	- NM H	-	-	NA	NA	NA	NA
MMC 0.2	2.90	3.70	3.30	35	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	1.50	1.85	1.68	27

Mean Frequency of Polyploid Cells (%)

Experiment 1 

Dose Level (µg/mL)	Exposure Group
	4(20)-Hour Without S9	4(20)-Hour With S9
0	0	0
7.5	0	0.5
15	0	0
30	0	0
MMC 0.4	0	NA
CP 5	NA	0

 

Experiment 2 

Dose Level (µg/mL)	Exposure Group
	24-Hour Without S9	4(20)-Hour With S9
0	0	0
15	0	NA
30	0	0
45	NA	0
60	0	0
MMC 0.2	0	NA
CP 5	NA	0

Conclusions:

Interpretation of results (migrated information):
negative

The test item, IFF 215 (Floriane), did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The genotoxic potential of the test substance, IFF 215 (Floriane) was assessed according to OECD Test Guideline 473 using an In Vitro Mammalian Chromosome Aberration method. The test item, IFF 215 (Floriane), did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 21 May 2014 and 17 May 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliability 1 as it has been conducted according to OECD 471 using the Bacterial Reverse Mutation method and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine
E. coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.


Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.


With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.


Incubation and Scoring
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).


Test for Mutagenicity (Experiment 2) – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.


Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 50 to 5000 µg/plate.


Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.


With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 °C± 3 °C for 20 minutes (with shaking) and addition of molten amino-acid supplemented media. All testing for this experiment was performed in triplicate.


Incubation and Scoring
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to revertant colonies spreading slightly, thus distorting the actual plate count.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

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

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test (pre-incubation method). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre incubation method).

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
127		16		20		33		15
114	(115)	20	(17)	19	(23)	27	(27)	15	(15)
104		15		29		21		15

 

 

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
103		21		16		21		15
76	(94)	19	(19)	27	(21)	15	(23)	16	(17)
103		17		19		32		19

 

 

Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 05 June 2014					To: 08 June 2014
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	95 114 119	(109) 12.7#	17 20 16	(18) 2.1	29 24 23		(25) 3.2	29 29 35	(31) 3.5	15 20 15	(17) 2.9
	1.5 µg	108 115 124	(116) 8.0	11 17 15	(14) 3.1	21 21 17		(20) 2.3	20 16 27	(21) 5.6	21 15 15	(17) 3.5
	5 µg	94 87 92	(91) 3.6	16 25 16	(19) 5.2	29 29 29		(29) 0.0	31 29 20	(27) 5.9	16 13 15	(15) 1.5
	15 µg	88 75 107	(90) 16.1	11 13 11	(12) 1.2	20 29 15		(21) 7.1	27 28 27	(27) 0.6	13 20 20	(18) 4.0
	50 µg	75 118 96	(96) 21.5	16 12 16	(15) 2.3	17 24 28		(23) 5.6	20 27 15	(21) 6.0	20 15 19	(18) 2.6
	150 µg	83 114 84	(94) 17.6	13 17 13	(14) 2.3	12 19 23		(18) 5.6	23 19 35	(26) 8.3	16 19 13	(16) 3.0
	500 µg	92 96 99	(96) 3.5	19 13 13	(15) 3.5	19 21 17		(19) 2.0	19 23 17	(20) 3.1	11 11 9	(10) 1.2
	1500 µg	104 98 110	(104) 6.0	17 17 16	(17) 0.6	23 20 28		(24) 4.0	20 19 21	(20) 1.0	19 13 16	(16) 3.0
	5000 µg	94 136 106	(112) 21.6	15 15 19	(16) 2.3	32 27 23		(27) 4.5	25 20 27	(24) 3.6	12 12 9	(11) 1.7
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		1016 1058 1172	(1082) 80.7	234 202 188	(208) 23.6	651 569 617		(612) 41.2	212 182 335	(243) 81.1	1354 1417 1284	(1352) 66.5

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

# Standard deviation

 

Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 05 June 2014					To: 08 June 2014
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	126 130 116	(124) 7.2#	11 11 13	(12) 1.2	29 23 32		(28) 4.6	36 21 29	(29) 7.5	11 11 12	(11) 0.6
	1.5 µg	119 118 128	(122) 5.5	7 12 11	(10) 2.6	28 31 24		(28) 3.5	27 20 29	(25) 4.7	12 15 13	(13) 1.5
	5 µg	128 124 107	(120) 11.2	12 13 13	(13) 0.6	28 23 24		(25) 2.6	20 35 28	(28) 7.5	13 13 13	(13) 0.0
	15 µg	100 131 130	(120) 17.6	9 7 17	(11) 5.3	27 12 40		(26) 14.0	32 29 35	(32) 3.0	8 11 12	(10) 2.1
	50 µg	131 116 119	(122) 7.9	7 9 8	(8) 1.0	33 29 36		(33) 3.5	32 23 32	(29) 5.2	9 11 12	(11) 1.5
	150 µg	104 123 124	(117) 11.3	7 11 8	(9) 2.1	23 19 39		(27) 10.6	29 24 25	(26) 2.6	12 11 11	(11) 0.6
	500 µg	111 128 119	(119) 8.5	11 13 12	(12) 1.0	25 32 31		(29) 3.8	23 32 24	(26) 4.9	12 9 9	(10) 1.7
	1500 µg	114 114 123	(117) 5.2	15 11 8	(11) 3.5	27 25 27		(26) 1.2	28 32 25	(28) 3.5	9 7 11	(9) 2.0
	5000 µg	128 123 124	(125) 2.6	8 8 15	(10) 4.0	27 32 21		(27) 5.5	28 28 33	(30) 2.9	12 8 9	(10) 2.1
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		830 825 835	(830) 5.0	274 251 246	(257) 14.9	303 309 303		(305) 3.5	269 246 196	(237) 37.3	361 342 338	(347) 12.3

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

# Standard deviation

Conclusions:

Interpretation of results (migrated information):
negative

IFF 215 (Floriane) was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The genetic toxicity of the test substance, IFF 215 (Floriane) was assessed and considered to be non-mutagenic according to OECD 471 using the Bacterial Reverse Mutation method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A mutation is a permanent change in the amount or structure of the genetic material in a cell. The term “mutation” applies to both heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known, including specific base pair changes and chromosomal translocations. The term “mutagenic” and “mutagen” are used for agents giving rise to an increased occurrence of mutations in populations of cells or organisms.

The more generic terms “genotoxic” and “genotoxicity” apply to agents or processes which alter the structure, information content or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non-physiological manner temporarily alter its replication. Genotoxicity test results are usually taken as indicators for mutagenic effects.

The in vitro gene mutation study (Ames) was designed to assess the mutagenic potential of the test material using a bacterial test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al. In this study mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. This method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test".

 

The Salmonella strains used in the test are incapable of synthesising histidine and are therefore dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent, these bacteria may undergo a reverse mutation to histidine independent form which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional strains have been introduced. These include a deletion though the excision repair gene (uvrB- Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. A mutant strain of E. coli (WP2uvrA-) which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence was used to complement the salmonella strains. This strain also has a deletion in the excision repair gene (uvrA-). Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a compound known to induce an elevated level of these enzymes.

From the results of the Ames test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested, with any dose of the test item, either with or without metabolic activation. The test item, IFF 213 (Floriane) was therefore considered to be non-mutagenic in vitro.

 

The purpose of the chromosome aberration study was to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocytes. The detection of structural chromosomal aberrations in cultured mammalian cells supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). Human peripheral blood lymphocytes are recognized in the OECD 473 guideline as being a suitable cell line for the Mammalian Chromosome Aberration Test. Numerical and structural chromosome aberrations are implicated in the pathology of neoplasia (Radmanet al., 1982; Cairns, 1981) and also occur in a high proportion of spontaneous abortions and abnormal live births (Chandley, 1981). Furthermore, most carcinogens are capable of inducing such changes in chromosome fidelity. Metaphase analysis in vitro involves recording such structural and numerical aberrations in the chromosomes of exposed cells. Many of these changes are lethal to the cells in which they occur and are therefore not of heritable significance. However, it is assumed that agents capable of inducing gross chromosomal changes also induce more subtle changes (translocations, inversions and small deletions) which are not cell lethal, and therefore represent a hazard. The ability to induce chromosome aberrations also correlates well with the induction of gene mutations (Hollsteinet al., 1979). This study was designed to be compatible with OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test".

 

It was established in the chromosome aberration test that the test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations. The test item, IFF 213 (Floriane) was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Justification for selection of genetic toxicity endpoint
The study was conducted in vitro, in an appropriate test species and according to internationally recognised guidelines.

Justification for classification or non-classification

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.

The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.

To this end, the test substance has been assessed according to internationally recognized guidelines in two in vitro tests. Firstly, an in vitro gene mutation study in bacteria (Ames test) and secondly an in vitro mammalian chromosome aberration study.

In the in vitro gene mutation study in bacteria (Ames) no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation.

In the in vitro mammalian chromosome aberration study, the test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, either with or without metabolic activation and was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Based on two negative results in vitro, the test item is considered non-mutagenic.