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Genetic toxicity: in vitro

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Administrative data

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:
Experimental start date 26 October 2017 Experimental completion date 14 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD guideline 473 without any deviation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene
EC Number:
950-170-3
Molecular formula:
C10H18O
IUPAC Name:
Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene
Specific details on test material used for the study:
Identification: PG-RAW-0004
Chemical name: Reaction mass of 2-methoxy-6-methylocta-1,5-diene and 2-methoxy-6-methylocta-2,5-diene
Physical state/Appearance: Clear colorless liquid
Batch: RDRW004-3
Purity: 95.5%
Expiry Date: 01 September 2019
Storage Conditions: Approximately 4 °C in the dark
Intended use/Application: Fragrance ingredient
Formulated concentrations were adjusted to allow for the stated water/impurity content (4.5%) of the test item.

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fractions
Test concentrations with justification for top dose:
Preliminary Toxicity Test
The molecular weight of the test item was given as 154, therefore, the maximum dose level was 1540 μg/mL, the maximum recommended dose level. The purity of the test item was 95.5 % and was accounted for in the test item formulations.
The dose levels of test item used were 0, 6.01, 12.03, 24.06, 48.13, 96.25, 192.5, 385, 770 and 1540 μg/mL.

Main Experiment
The selection of the maximum dose level for the Main Experiment was based on toxicity for all three exposure groups and was 64 μg/mL and 96 μg/mL for the exposure groups in the absence and presence of S9, respectively.
4-hour exposure to the test item without S9-mix: 12, 16, 24, 32, 48 and 64 μg/mL.
4-hour exposure to the test item with S9-mix (2%): 0, 12, 16, 24, 32, 48, 64 and 96 μg/mL.
24-hour continuous exposure to the test item without S9-mix: 0, 8, 12, 16, 24, 32, 48 and 64 μg/mL.
Vehicle / solvent:
The test item was immiscible in MEM at 15.4 mg/mL but was miscible in DMSO at 154 mg/mL in solubility checks performed in-house.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 μg/mL for 4-hour exposure 0.2 μg/mL for 24-hour exposure
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
4 μg/mL for 4-hour exposure
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
PREPARATION OF CULTURES
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) 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. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: female, aged 22 years Main Experiment: female, aged 26 years
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).

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air
- Exposure duration: 4 or 24 hrs 37 ºC with 5% CO2 in humidified air
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hrs

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 μg/mL) 2.5 hours before the required harvest time
STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS: Duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 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. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: 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.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. 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 (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 1.
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed

Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.
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).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
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:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 6.01 to 1540 μg/mL. The maximum dose was the recommended maximum dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 192.5 μg/mL and 385 μg/mL in the absence and presence of metabolic activation (S9), respectively. Hemolysis was observed following exposure to the test item at and above 24.06 μg/mL in the 4(20)-hour exposure groups and at and above 96.25 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. Additionally, a reduced cell pellet was observed at 96.25 μg/mL in all three exposure groups indicating that maximum exposure was occurring at the onset of toxicity.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 48.13 μg/mL in all three exposure groups. The test item induced marked evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity for all three exposure groups and was 64 μg/mL and 96 μg/mL for the exposure groups in the absence and presence of S9, respectively.

Chromosome Aberration Test – Main Experiment
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 64 μg/mL in all three exposure groups.
No precipitate observations were made at the end of exposure in blood cultures. Hemolysis was observed following exposure to the test item at and above 32 μg/mL in the 4(20)-hour exposure group in the absence of S9, at and above 48 μg/mL in the presence of S9 and at and above 24 μg/mL in the 24-hour continuous exposure group. Additionally, a reduced cell pellet was observed at 32 μg/mL in the 4(20)-hour exposure group in the absence of S9, at and above 64 μg/mL in the 4(20)-hour in the presence of S9 and 48 μg/mL in the 24-hour exposure group indicating that maximum exposure was occurring.
They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, particularly in the 4(20)-hour exposure groups.
In the 4(20)-hour exposure group in the absence of S9, 33% and 43% mitotic inhibition was achieved at 48 and 64 μg/mL, respectively. Therefore, the maximum dose level selected for metaphase analysis was the maximum dose level, 64 μg/mL, because it approached the range for optimum toxicity as defined in the OECD 473 guideline (55±5%).
In the presence of S9, an inhibition of mitotic index of 42%, 36% and 49% was noted at 32, 48 and 64 μg/mL, respectively. Above this dose level, there were no scorable metaphases available for analysis. Therefore, the maximum dose level selected for metaphase analysis was 64 μg/mL, because it approached the range for optimum toxicity as defined in the OECD 473 guideline (55±5%).
In the 24-hour continuous exposure group, a very modest mitotic inhibition was observed at 64 μg/mL (16%). Therefore, the maximum dose level selected for metaphase analysis was the maximum dose level, 64 μg/mL.
The assay was considered valid as it met all of the following criteria:
• The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range
• All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed
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 polyploid cell frequency data are given in Table 7. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in all of the exposure groups.

Any other information on results incl. tables

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

Group

Final concentration of test itemPG-RAW-0004 (µg/mL)

4(20)-hour without S9

0, 8, 12, 16, 24, 32, 48, 64

4(20)-hour with S9 (2%)

0, 12, 16, 24, 32, 48, 64, 96

24-hour without S9

0, 8, 12, 16, 24, 32, 48, 64

Mitotic Index - Preliminary Toxicity Test

Dose 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

4.65

100

1.75

100

6.55

100

6.01

-

-

-

-

-

-

12.03

3.80

82

4.60

263

7.00

107

24.06

2.40 H

52

3.10 H

177

2.80

43

48.13

2.20 H

47

4.45 H

254

3.15

48

96.25

NM H R

-

NM H R

-

NM H R

-

192.5

NM H R P

-

NM H R

-

NM H R P

-

385

NM H R P

-

NM H R P

-

NM H R P

-

770

NM H R P

-

NM H R P

-

NM H R P

-

1540

NM H R P

-

NM H R P

-

NM H R P

-

- = Not assessed for mitotic index

NM = No metaphases or insufficient metaphases suitable for scoring

P = Precipitate observed at end of exposure period in blood-free cultures

H = Hemolysis observed at the end of exposure in blood cultures

R = Reduced cell pellet

Mitotic Index – Main Experiment (4(20)-hour Exposure Groups)

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

4.50

7.35

5.93

100

10.95

13.40

12.18

100

8

-

-

-

-

NA

NA

NA

NA

12

-

-

-

-

-

-

-

-

16

5.15

5.30

5.23

88

-

-

-

-

24

8.55

9.45

9.00

152

-

-

-

-

32

6.25 H R

6.20 H R

6.23

105

10.15

4.05

7.10

58

48

3.55 H R

4.55 H R

3.95

67

10.50 H

5.10 H

7.80

64

64

3.30 H R

3.50 H R

3.40

57

6.90 H R

5.55 H R

6.23

51

96

NA

NA

NA

NA

NM H R

NM H R

-

-

MMC 0.2

4.50

2.95

3.73

63

NA

NA

NA

NA

CP 4

NA

NA

NA

NA

5.35

2.30

3.83

31

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

H = Hemolysis

R = Reduced cell pellet

Mitotic Index – Main Experiment (24-hour Exposure Group)

Dose Level

(µg/mL)

24-Hour Without S9

A

B

Mean

% of Control

0

6.20

7.50

6.85

100

8

-

-

-

-

12

-

-

-

-

16

-

-

-

-

24

- H

 H

-

-

32

7.35 H

8.30 H

7.83

114

48

6.95 H R

8.25 H R

7.60

111

64

5.65 H R

5.80 H R

5.73

84

MMC 0.2

3.35

3.55

3.45

50

MMC = Mitomycin C

- = Not assessed for mitotic index

H = Hemolysis

R = Reduced cell pellet

Mean Frequency of Polyploid Cells (%)

Dose Level

(µg/mL)

24-Hour Without S9

A

B

Mean

% of Control

0

6.20

7.50

6.85

100

8

-

-

-

-

12

-

-

-

-

16

-

-

-

-

24

- H

 H

-

-

32

7.35 H

8.30 H

7.83

114

48

6.95 H R

8.25 H R

7.60

111

64

5.65 H R

5.80 H R

5.73

84

MMC 0.2

3.35

3.55

3.45

50

MMC Mitomycin C

CP Cyclophosphamide

NA Not applicable

Applicant's summary and conclusion

Conclusions:
Under the test conditions, PG-RAW-0004 is not considered as clastogenic in human lymphocytes according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to PG-RAW-0004 in DMSO. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Experiment were as follows:

4(20)-hour without S9: 0, 8, 12, 16, 24, 32, 48, 64 μg/mL

4(20)-hour with S9 (2%): 0, 12, 16, 24, 32, 48, 64, 96 μg/mL

24-hour without S9: 0, 8, 12, 16, 24, 32, 48, 64 μg/mL

All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced 55±5% mitotic inhibition or was at the limit of exposure.

The test item, PG-RAW-0004 was considered to be non-clastogenic to human lymphocytes in vitro.