Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a chromosome aberration test performed according to OECD 473 PG-RAW-0004 was considered to be non-clastogenic to human lymphocytes in vitro.

In a bacterial reverse mutation test performed according to OECD 471 PG-RAW-0004 was considered to be non-mutagenic under the conditions of this test

Link to relevant study records

Referenceopen allclose all

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
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
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.
Target gene:
not applicable
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.
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.
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.

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

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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 18 October 2017 Experimental completion date: 07 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
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 colourless liquid
Storage Conditions: Approximately 4 °C in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content of the test item.
Target gene:
- S. typhimurium: Histidine gene
- E.coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Test for Mutagenicity: Experiment 1 - Plate Incorporation 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 used

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was initially 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
However, results from the second experiment showed that the toxicity of the test item yielded results that differed slightly from the Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA100, TA1537 and TA1535 (absence of S9-mix only). Therefore, these strains were repeated employing an amended test item dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 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 in solubility checks performed in house. Dimethyl sulphoxide was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
80 µg/plate for TA1537
Positive control substance:
9-aminoacridine
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/plate for TA98
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Presence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate for TA98
Positive control substance:
benzo(a)pyrene
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or 0.1 mL of 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 ± 3 ºC 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 the result of Experiment 1 was deemed negative, Experiment 2 (and the subsequent repeat assay) was performed using the pre-incubation method in the presence and absence of metabolic activation.

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 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace 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 (see 3.3.3.2) 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 ± 3 ºC for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC 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). Two manual counts were performed due to colonies spreading and artefacts on the plate, 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.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns at 5000 µg/plate to TA100, TA1535 and TA1537 in the absence of S9 and to TA100 in the presence of S9. No toxicity was noted to any of the remaining bacterial strains. Consequently, the same maximum dose level (5000 µg/plate) was initially employed as the maximum concentration in the second mutation test. However, after incorporating the pre incubation modification in the second mutation test, the test item induced significant toxicity as weakened bacterial background lawns and substantial reductions in the revertant colony frequency of a number of tester strains to the extent where a number of strains required repeat analysis employing an amended test item dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 µg/plate) or the toxic limit of the test item was employed as the maximum concentration in the second mutation test. Results from the second mutation test showed that the test item induced a stronger toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 µg/plate (TA100), 15 µg/plate (TA1535 and TA1537) and 500 µg/plate (TA98 and WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 500 µg/plate (TA100 and TA1537), 1500 µg/plate (TA98 and TA1535) and at 5000 µg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.
In the first mutation test (plate incorporation method) a light test item precipitate (globular in appearance) was noted by eye at 5000 µg/plate to all of the strains dosed in the absence and presence of S9-mix. However, after employing the pre-incubation modification in the second mutation test no test item precipitate was noted at any test item dose level.
There were no toxicologically meaningful 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 (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no 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 (S9-mix) in Experiment 2 (pre incubation method). Minor statistical values were noted in Experiment 1 (TA100 at 15 µg/plate in the presence of S9-mix), however these responses were within the in-house historical vehicle/untreated control values and were, therefore considered of no biological relevance.
Conclusions:
The substance is not mutagenic in the Salmonella typhimurium and E.coli reverse mutation assay performed according to OECD 471 (1997).
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was initially 5 to 5000 µg/plate. However, results from the second mutation test (pre-incubation method) showed that the toxicity of the test item yielded results that differed significantly from Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA100, TA1535 and TA1537 in the absence of S9‑mix. Therefore, these strains were repeated employing an amended test item dose range of 0.15 to 150 µg/plate. Up to eighttest item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.Adequate negative and positive controls were included.

There were no toxicologically meaningful 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 (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no 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 (S9-mix) in Experiment 2 (pre-incubation method). Therefore, PG-RAW-0004was considered to be non-mutagenic under the conditions of this test.

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

Additional information

Ames test:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was initially 5 to 5000 µg/plate. However, results from the second mutation test (pre-incubation method) showed that the toxicity of the test item yielded results that differed significantly from Experiment 1 and consequently an insufficient number of non-toxic dose levels were attained for TA100, TA1535 and TA1537 in the absence of S9‑mix. Therefore, these strains were repeated employing an amended test item dose range of 0.15 to 150 µg/plate. Up to eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.Adequate negative and positive controls were included.

There were no toxicologically meaningful 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 (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no 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 (S9-mix) in Experiment 2 (pre-incubation method). Therefore, PG-RAW-0004was considered to be non-mutagenic under the conditions of this test.

Chromosome aberration test:

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.


Justification for classification or non-classification

Based on the results of the Ames test and in vitro chromasome aberration test, the substance does not have to be classified for mutagenicity in accordance with Regulation (EC) No. 1272/2008 and its amendments.