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REACH

Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

EC number: 950-718-1 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (OECD TG 471): negative

Chromosome Aberration (OECD TG 473): negative

Link to relevant study records

Reference 1

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 12 December 2018 and 24 December 2018.

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:

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-90-032
Description: Clear colourless liquid
Storage Conditions: Approximately 4 °C, in the dark

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10% liver S9 in standard co-factors

Test concentrations with justification for top dose:

Experiment 1 – Plate Incorporation Method
The maximum concentration was 5000 µg/plate (the OECD TG 471 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.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.
Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in house.

Untreated negative controls:

yes

Remarks:

untreated plates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

In the absence of S9-mix

Untreated negative controls:

yes

Remarks:

untreated plates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Remarks:

In the presence of S9-mix

Details on test system and experimental conditions:

Microsomal Enzyme Fraction
The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized
in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 28 October 2018 was used in this study.

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 7193746 04/2022) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 49292 01/2019 and 49449 01/2019).

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible 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.
The test item was accurately weighed and, on the day of each experiment, approximate
half-log dilutions prepared in pre-dried dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C. No correction for purity was required.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Dose selection
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. 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 °C for between 48 and 72 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 considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C 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 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 °C 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 °C for between 48 and 72 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).

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Typical ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
These values will also be confirmed against current in-house historical control profiles to further validate acceptability. Although the number of spontaneous revertants can be expected to fall within the ranges, they may occasionally fall outside these. Combined historical negative and solvent control ranges for 2016 and 2017 are presented.
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation (S9-mix). The historical ranges of the positive control reference items for 2016 and 2017 are presented.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

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.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

E. coli WP2 uvr A

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:

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) are presented and 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 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 (S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (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 (S9-mix).

Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (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 (S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (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 (S9-mix).

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
121		10		14		21		5
142	(129)	10	(10)	19	(18)	24	(21)	15	(12)
123		9		20		18		17

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
106		14		26		27		10
101	(99)	10	(11)	34	(26)	31	(26)	7	(9)
90		8		18		19		10

Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period

From: 14 December 2018

To: 17 December 2018

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)

106

102

121

(110)

10.0#

(13)

4.0

(23)

6.1

(18)

1.5

(14)

2.1

1.5 µg

119

121

103

(114)

9.9

(15)

2.1

(24)

2.3

(20)

8.0

(16)

6.7

5 µg

107

118

111

(112)

5.6

(14)

0.6

(21)

3.6

(22)

7.5

(16)

0.6

15 µg

125

124

(116)

15.3

(16)

6.9

(27)

5.1

(23)

4.2

(11)

3.2

50 µg

114

121

122

(119)

4.4

(17)

2.5

(27)

4.2

(18)

2.1

(11)

5.5

150 µg

120

105

111

(112)

7.5

(13)

2.1

(22)

6.7

(17)

4.6

(12)

7.1

500 µg

120

115

105

(113)

7.6

(10)

1.2

(21)

3.5

(21)

10.0

(14)

4.6

1500 µg

104

109

(103)

6.0

(15)

1.2

(27)

6.0

(18)

4.6

(8)

4.0

5000 µg

120

107

106

(111)

7.8

(11)

2.9

(22)

1.2

(18)

3.6

(8)

3.5

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

644

640

675

(653)

19.2

778

859

658

(765)

101.1

792

935

1077

(935)

142.5

188

187

198

(191)

6.1

326

505

481

(437)

97.2

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

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

# Standard deviation

Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period

From: 14 December 2018

To: 17 December 2018

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)

123

136

117

(125)

9.7#

(12)

3.5

(32)

3.1

(21)

1.7

(18)

1.7

1.5 µg

116

113

121

(117)

4.0

(14)

2.3

(38)

5.1

(25)

7.0

(15)

9.1

5 µg

136

116

118

(123)

11.0

(9)

2.0

(30)

6.1

(26)

8.5

(16)

3.8

15 µg

118

129

132

(126)

7.4

(13)

1.2

(31)

4.0

(26)

3.5

(20)

6.0

50 µg

127

101

126

(118)

14.7

(12)

0.0

(29)

1.0

(24)

6.0

(13)

3.1

150 µg

122

109

(109)

13.0

(10)

0.0

(30)

3.1

(23)

8.0

(13)

5.5

500 µg

112

119

113

(115)

3.8

(9)

2.3

(29)

7.8

(23)

5.3

(13)

2.3

1500 µg

106

132

118

(119)

13.0

(11)

1.5

(27)

5.0

(21)

4.5

(10)

5.3

5000 µg

102

(88)

12.3

(11)

3.1

(27)

3.2

(25)

5.3

(12)

0.6

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1878

2013

2031

(1974)

83.6

149

100

(114)

30.5

138

176

125

(146)

26.5

116

146

172

(145)

28.0

224

232

266

(241)

22.3

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

# Standard deviation

Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period

From: 21 December 2018

To: 24 December 2018

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)

110

109

105

(108)

2.6#

(19)

7.6

(19)

2.1

(14)

6.0

(15)

3.5

15 µg

(86)

2.5

(12)

4.4

(17)

1.7

(16)

7.0

(10)

1.7

50 µg

(84)

5.5

(15)

4.0

(15)

2.1

(14)

7.1

(11)

5.0

150 µg

102

(82)

18.3

(16)

2.1

(14)

3.8

(14)

5.7

(12)

2.0

500 µg

(70)

14.8

(13)

3.5

(15)

2.5

(13)

5.1

(10)

1.0

1500 µg

(63)

3.5

(22)

3.5

(11)

3.0

(16)

3.1

(8)

1.5

5000 µg

(62)

4.5

(13)

1.0

(12)

4.6

(12)

4.7

(5)

1.2

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

1081

1091

944

(1039)

82.1

1864

1171

1735

(1590)

368.6

1164

960

1114

(1079)

106.3

244

261

238

(248)

11.9

497

280

263

(347)

130.5

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

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

# Standard deviation

Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period

From: 21 December 2018

To: 24 December 2018

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)

100

(94)

5.6#

(9)

2.3

(25)

6.0

(21)

9.8

(7)

1.5

15 µg

102

(91)

9.3

(10)

3.6

(19)

3.5

(27)

2.1

(8)

1.5

50 µg

(86)

5.0

(9)

3.5

(20)

5.5

(20)

2.0

(5)

2.1

150 µg

(92)

6.4

(10)

2.9

(19)

1.5

(24)

5.6

(7)

3.6

500 µg

(84)

8.5

(10)

2.5

(23)

6.5

(25)

10.0

(6)

1.2

1500 µg

(82)

3.6

(8)

1.2

(18)

7.6

(25)

3.8

(5)

3.1

5000 µg

(75)

5.0

(12)

3.2

(19)

6.2

(26)

4.0

(5)

2.9

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

844

869

881

(865)

18.9

169

138

117

(141)

26.2

175

207

187

(190)

16.2

121

105

(107)

13.1

197

245

265

(236)

34.9

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

# Standard deviation

Conclusions:

PG-RAW-90-032 is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay performed according to OECD 471 (1997).

Executive summary:

The mutagenic activity of PG-RAW-90-032 was evaluated in accordance with OECD 471 (1997) and according to GLP principles. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 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 15 to 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxicity of the test item following the change in test methodology.

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 and 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 OECD TG 471 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 (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). 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 (S9-mix).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

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 (S9-mix) 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 (S9-mix) in Experiment 2 (pre‑incubation method).

PG-RAW-90-032was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 10 December 2018 and 15 March 2019.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: PG-RAW-90-032
Description: Clear colourless liquid
Storage Conditions: Approximately 4 °C, in the dark

Species / strain / cell type:

lymphocytes: whole blood cultures

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Induced rat liver homogenate metabolizing system (S9), at a 2% final concentration

Test concentrations with justification for top dose:

The molecular weight of the test item was given as 206.33, therefore, the maximum dose level was 2000 µg/mL, the maximum recommended dose level.

Preliminary Toxicity Test
The dose range of test item used was 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL.

Main Experiment
Three exposure groups were used for the Main Experiment:
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, 4, 8, 16, 32, 40, 48 and 64 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, 16, 32, 48, 64, 72, 80, 88 and 96 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 4, 8, 16, 32, 40, 48 and 64 µg/mL.
The 4(20)-hour exposure in the presence of S9 was initially performed with the dose range 0, 16, 32, 48, 64, 72, 80 and 96 µg/mL but due to the failure of the positive control the exposure was repeated with the revised dose range.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
The test item was insoluble in Minimal Essential Medium at 20 mg/mL but was soluble in dimethyl sulphoxide at 200 mg/mL in solubility checks performed in house.

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Absence of S9-mix

Untreated negative controls:

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Test System and Supporting Information
Cells
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: male, aged 25 years
Main Experiment: male, aged 35 years
Main Experiment Repeat (4(20)-hour with S9): female, aged 35

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).

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/NF S9 31/08/18 was used in this study. A copy of the S9 Certificate of Efficacy is presented in Appendix 2.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (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%.

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The purity of the test item was 99% and therefore no purity correction was required.
Prior to each experiment, the test item was accurately weighed, formulated in DMSO and appropriate 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 pH and osmolality readings are presented in the following table:
Dose level µg/mL 0 7.81 15.63 31.25 62.5 125 250 500 1000 2000
pH 7.34 7.35 7.34 7.37 7.41 7.42 7.41 7.42 7.42 7.44
Osmolality mOsm 488 477 - 484 - 483 485 468 457 446
- = Not performed for this dose concentration
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.

Culture conditions
Duplicate lymphocyte cultures (A and B) for each dose level and quadruplicate for the vehicle were established by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 to 9.08 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.72 - 0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
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 0.1 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 Main Experiment.
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.

4-Hour Exposure Without Metabolic Activation (S9)
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 0.1 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 and 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.

24-Hour Exposure Without Metabolic Activation (S9)
As the exposure was continuous the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 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 all three of the exposure conditions as described for the Main Experiment but using single cultures only for the test item dose levels and duplicate cultures for the vehicle control.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour 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-hour 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, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 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 Experiment.

Main Experiment
Three exposure groups were used for the Main Experiment:
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, 4, 8, 16, 32, 40, 48 and 64 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, 16, 32, 48, 64, 72, 80, 88 and 96 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 4, 8, 16, 32, 40, 48 and 64 µg/mL.
The 4(20)-hour exposure in the presence of S9 was initially performed with the dose range 0, 16, 32, 48, 64, 72, 80 and 96 µg/mL but due to the failure of the positive control the exposure was repeated with the revised dose range.

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.

Evaluation of Response
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 1000 lymphocyte cell nuclei were counted per culture 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, 300 consecutive well-spread metaphases from each concentration were counted (600 from the vehicle control) (150 per replicate), 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. 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:
• 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.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum dose was the maximum recommended 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 125 µg/mL in the 4(20)-hour exposure group in the absence of S9, at and above 250 µg/mL in the 4(20)-hour exposure group in the presence of S9 and at and above 62.5 µg/mL in the 24-hour continuous exposure group.
Hemolysis was observed following exposure to the test item at and above 7.81µg/mL in the 4(20)-hour exposure groups and at and above 62.5 µg/mL in the 24-hour exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. A reduced cell pellet was also noted at the end of exposure at and above 31.25 µg/mL in the 4(20)-hour exposure groups and at and above 62.5 µg/mL in the 24-hour exposure group. This is considered to be indicative of toxicity to the cell population as a whole.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 31.25 µg/mL and 62.5 µg/mL in the 4(20)-hour exposure in the absence and presence of S9, respectively. The maximum dose with metaphases present in the 24 hour continuous exposure was 62.5 µg/mL. The test item induced marked evidence of toxicity in all three exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 64 µg/mL for the exposures in the absence of S9 and 96 µg/mL in the presence of S9.

Chromosome Aberration Test – Main Experiment

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

Exposure Group	Final concentration of PG-RAW-90-032 (µg/mL)
4(20)-hour without S9	0, 4, 8, 16, 32, 40, 48, 64, MMC0.4*
4(20)-hour with S9 (2%)	0, 16, 32, 48, 64, 72, 80, 88,96, CP4*
24-hour without S9	0, 4, 8, 16, 32, 40, 48, 64, MMC 0.2

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity in the absence of S9 was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 48 µg/mL in the 4(20)-hour exposure in the absence of S9 and up to 64µg/mL in the 24-hour exposure. In the presence of metabolic activation (S9), the maximum dose level of the test item with sufficient metaphases suitable for scoring was 88 µg/mL and although there were some metaphases present at 96 µg/mL it was considered there were insufficient for scoring chromosome aberrations.

Precipitate observations were made at the end of exposure and no precipitate was observed in any of the three exposure groups. Hemolysis was observed at and above 32 µg/mL and 16 µg/mL in the 4(20)-hour exposure groups in the absence and presence of S9, respectively. In the 24-hour exposure in the absence of S9, hemolysis was observed at and above 32 µg/mL. Reduced cell pellets were observed at the end of exposure at 64 µg/mL and 48 µg/mL in the 4(20)-hour exposures in the absence and presence of S9, respectively.

The mitotic index data for the Main Experiment are given inTable2andTable3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, optimum toxicity was achieved at 48 µg/mL with 52% mitotic inhibition. In the 24-hour exposure group in the absence of S9, 44% and 68 % mitotic inhibition was achieved at 48µg/mL and 64 µg/mL, respectively. In the presence of S9, 35% mitotic inhibition was achieved at 88 µg/mL and this was the highest dose level with sufficient metaphases for scoring.

The maximum dose level selected for metaphase analysis in each exposure group was based on toxicity and was48 µg/mL for the 4(20)-hour exposure group in the absence of S9, 88 µg/mL for the 4(20)-hour exposure group in the presence of S9 and 64 µg/mL for the 24‑hour exposure group.

The chromosome aberration data are given inTable4,Table5andTable6. 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 in the absence of metabolic activation.

In the presence of metabolic activation there was a small but statistically significant increase in the frequency of cells with aberrations at 80 µg/mL. The response is seen in the middle of the dose range with the dose level below (72 µg/mL) andthe dose level above (88 µg/mL) not showing any increases over the vehicle control. The response at 80 µg/mL is only marginally above the laboratory historical control range for a vehicle and is considered to be spurious and of no toxicological significance.

The 4(20)-hour exposure group in the presence of S9 was initially performed with the test item dose range 0, 16, 32, 48, 64, 72, 80 and 96 µg/mL. The slides were assessed and a mitotic index performed but it was evident from the data that the positive control had failed and therefore the slides were not evaluated for chromosome aberrations. On investigation it was discovered that the failure of the positive control was due to a technical oversight. Since the 80 µg/mL dose level achieved optimum toxicity with 49% mitotic inhibition it was considered acceptable to score this dose level along with the concurrent vehicle control to support the conclusion that the small response seen in the repeat of the 4(20)-hour exposure group in the presence of S9 at 80 µg/mL was of no toxicological significance. The additional score data is presented and demonstrated the absence of any increase in the frequency of cells with aberrations at 80 µg/mL.

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

Mitotic Index - Preliminary Toxicity Test

Dose Level (µg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
Dose Level (µg/mL)	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	6.90	100	7.00	100	6.90	100
0	6.50	100	7.20	100	6.50	100
7.81	6.10 H	91	6.80 H	96	5.20	78
15.63	9.20 H	137	7.50 H	106	6.20	93
31.25	5.80 H R	87	10.40 H R	146	4.20	63
62.5	NM H R	NM	3.90 H R	55	2.10 H R P	31
125	NM H R P	NM	NM H R	NM	NM H R P	NM
250	NM H R P	NM	NM H R P	NM	NM H R P	NM
500	NM H R P	NM	NM H R P	NM	NM H R P	NM
1000	NM H R P	NM	NM H R P	NM	NM H R P	NM
2000	NM H R P	NM	NM H R P	NM	NM H R P	NM

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 observed at the end of exposure

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

4(20)-Hour Without S9					4(20)-Hour With S9
Dose Level (mg/mL)	A	B	Mean	% of Control	Dose Level (mg/mL)	A	B	Mean	% of Control
0	6.00	8.50	6.48	100	0	4.40	6.20	5.18	100
0	5.60	5.80	6.48	100	0	4.80	5.30	5.18	100
4	-	-	-	-	16	-	-	-	-
8	-	-	-	-	32	-	-	-	-
16	-	-	-	-	48	6.10 H R	3.70 H R	4.90	95
32	6.10 H	5.70 H	5.90	91	64	4.40 H R	2.90 H R	3.65	71
40	4.40 H	3.50 H	3.95	61	72	4.30 H R	4.50 H R	4.40	85
48	2.80 H	3.40 H	3.10	48	80	4.30 H R	3.10 H R	3.70	71
64	NM H R	NM H R	NA	NA	88	3.20 H R	3.50 H R	3.35	65
NA	NA	NA	NA	NA	96	4.20 H R	3.50 H R	3.85	74
MMC 0.4	2.40	1.70	2.05	32	CP 6	1.40	1.60	1.50	29

MMC =Mitomycin C

CP =Cyclophosphamide

NA =Not applicable

- =Not assessed for mitotic index

NM =No metaphases suitable for scoring

H =Hemolysisobserved at the end of exposure in blood cultures

R = Reduced cell pellet noted at the end of exposure

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

Dose Level (µg/mL)	24-Hour Without S9
Dose Level (µg/mL)	A	B	Mean	% of Control
0	7.20	7.60	6.70	100
0	6.00	6.00	6.70	100
4	-	-	-	-
8	-	-	-	-
16	-	-	-	-
32	3.80 H	6.00 H	4.90	73
40	5.20 H	3.90 H	4.55	68
48	4.00 H	3.50 H	3.75	56
64	2.10 H	2.20 H	2.15	32
MMC 0.2	2.00	2.00	2.00	30

MMC =Mitomycin C

- =Not assessed for mitotic index

H =Hemolysisobserved at the end of exposure in blood cultures

Mean Frequency of Polyploid Cells (%)

Main Experiment

Dose Level (µg/mL)	Exposure Group
Dose Level (µg/mL)	4(20)-Hour Without S9	4(20)-Hour With S9	24-Hour Without S9
0	0.2	0	0.3
32	0	-	0
40	0	NA	0
48	0.3	-	0.3
64	-	-	0.3
72	NA	0	NA
80	NA	0	NA
88	NA	0.7	NA

MMC 0.4	0	NA	NA
MMC 0.2	NA	NA	0
CP 4	NA	0	NA

MMC Mitomycin C

CP Cyclophosphamide

NA Not applicable

- Not assessed forpolyploidy

Conclusions:

PG-RAW-90-032 did not induce a toxicologically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

An in vitro chromosome aberration test for PG-RAW-90-032 was performed according to OECD guideline 473 and in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. 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:

Exposure Group	Final concentration of test itemPG-RAW-90-032(µg/mL)
4(20)-hour without S9	4, 8, 16, 32, 40, 48, 64
4(20)-hour with S9 (2%)	16, 32, 48, 64, 72, 80, 88, 96
24-hour without S9	4, 8, 16, 32, 40, 48, 64

All vehicle (dimethyl sulphoxide) 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 did not induce any statistically significant increases in the frequency of cells with aberrations, in the absence of S9 using a dose range that included a dose level induced close to optimum toxicity.

In the presence of S9 there was a small but statistically significant increase in the frequency of cells with aberrations in the middle of the dose range at 80 µg/mL. The dose level above and below did not demonstrate any increases in the frequency of cells with aberrations and it was considered that the result was spurious and of no toxicological significance. The reported 4(20)-hour exposure in the presence of S9 was a repeat of the exposure group with a slightly revised dose range due to the failure of the positive control in the initial experiment. However, in the initial experiment optimum toxicity was achieved at 80 µg/mL with 49% mitotic inhibition. The 80 µg/mL dose level from the initial experiment was also assessed for aberrations along with the concurrent vehicle control and it is considered that the absence of any increase in aberrations is further evidence to conclude that the response seen in the repeat experiment in the presence of S9 is of no toxicological significance.

The test item, PG-RAW-90-032 was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed (negative)

Additional information

OECD TG 471 (Ames):

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

PG-RAW-90-032 was considered to be non-mutagenic under the conditions of this test.

OECD 473 (Chromosome aberration study):

Exposure Group	Final concentration of test itemPG-RAW-90-032(µg/mL)
4(20)-hour without S9	4, 8, 16, 32, 40, 48, 64
4(20)-hour with S9 (2%)	16, 32, 48, 64, 72, 80, 88, 96
24-hour without S9	4, 8, 16, 32, 40, 48, 64

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

The test item, PG-RAW-90-032 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 chromosome aberration test, PG-RAW-90-032 does not have to be classified for mutagenicity in accordance with Regulation (EC) No. 1272/2008 and its amendments.

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Diss Factsheets

Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Chromosome Aberration Test – Main Experiment

Endpoint conclusion

Additional information

Justification for classification or non-classification

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