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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

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 26 January 2012 and 14 February 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Kanpoan No. 287 -- Environment Protection Agency
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Eisei No. 127 -- Ministry of Health & Welfare
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Heisei 09/10/31 Kikyoku No. 2 -- Ministry of Economy, Trade & Industry
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction mass of 2,4-dimethyl-6-phenyl-3,6-dihydro-2H-pyran and 2-methyl-4-methylene-6-phenyltetrahydro-2H-pyran and 4,6-dimethyl-2-phenyl-3,6-dihydro-2H-pyran
Molecular formula:
C13H160
IUPAC Name:
Reaction mass of 2,4-dimethyl-6-phenyl-3,6-dihydro-2H-pyran and 2-methyl-4-methylene-6-phenyltetrahydro-2H-pyran and 4,6-dimethyl-2-phenyl-3,6-dihydro-2H-pyran
Test material form:
other: Liquid
Details on test material:
- Name of test material: Pelargene
- Physical state: liquid
- Lot no. / Batch no. NX0002642
- Purity: 99.03 %
- Expiration date of the lot/batch: 2012-08-14
- Storage condition of test material: At room temperature, light protected

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E. coli
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Base on the toxic effects observed eight concentrations were tested and 2500 µg/plate were chosen as maximal concentration for the Salmonella typhimurium strains and 5000 µg/plate were chosen as maximal concentration for the E. coli strain in experiment II.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
S. typhimurium strains: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
E. coli strain: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation

Migrated to IUCLID6: sodium azide, NaN3 used with Strains TA 1535, TA 100 @ 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine, 4-NOPD used with Strains: TA 1537, TA 98 @ 10 µg/plate in TA 98, 50 µg/plate in TA 1537
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation

Migrated to IUCLID6: methyl methane sulfonate, MMS used with Strains: WP2 uvrA @ 3 µL/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 2-AA used with Strains:TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA @ 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100),10 µg/plate (WP2 uvrA)
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major draw¬back an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 27.7 mg/mL (lot no. R 041111) in both experiments.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.



Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
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:
TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate in experiment I with and without S9 mix and at 2500 and 5000 µg/plate in experiment II with S9 mix . No precipitation of the test item in the overlay agar was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Reduced background growth was observed at the following concentrations (µg/plate):

Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 333 - 5000 1000 - 500 100 - 5000 100 - 5000
TA 1537 333 - 5000 333 - 5000 100 - 5000 100 - 5000
TA 98 333 - 5000 1000 - 5000 100 - 5000 100 - 5000
TA 100 333 - 5000 333 - 5000 100 - 5000 100 - 5000
WP2 uvrA / 2500, 5000 / /
/ = no reduced background growth observed


Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 333 - 5000 1000 - 5000 333 - 2500 333 - 2500
TA 1537 333 - 5000 1000 - 5000 100, 2500 1000, 5000
TA 98 333 - 5000 2500, 5000 333 - 2500 1000, 5000
TA 100 333 - 5000 2500, 5000 2500 1000, 5000
WP2 uvrA / 5000 / /
/ = no toxic effects observed
Remarks on result:
other: other: reverse mutation assay
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 Summary of Results Pre-Experiment and Experiment I

Study Name: 1455002

Study Code: Harlan CCR 1455002

Experiment: 1455002 VV Plate

Date Plated: 26/01/2012

Assay Conditions:

Date Counted: 01/02/2012

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

13 ± 1

17 ± 3

32 ± 2

89 ± 4

53 ± 2

Untreated

 

 

12 ± 5

15 ± 5

25 ± 3

91 ± 6

45 ± 7

Pelargene

3 µg

 

15 ± 2

16 ± 3

25 ± 4

83 ± 3

47 ± 3

 

10 µg

 

15 ± 1

15 ± 5

21 ± 6

77 ± 7

54 ± 2

 

33 µg

 

14 ± 2

12 ± 4

28 ± 2

73 ± 9

55 ± 13

 

100 µg

 

16 ± 0

13 ± 6

23 ± 4

84 ± 11

45 ± 12

 

333 µg

 

5 ± 3M R

5 ± 1M R

12 ± 2M R

38 ± 6M R

37 ± 9

 

1000 µg

 

0 ± 0M R

0 ± 0M R

10 ± 1M R

15 ± 3M R

37 ± 5

 

2500 µg

 

0 ± 0M R

0 ± 0M R

6 ± 2M R

0 ± 0M R

36 ± 1

 

5000 µg

 

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

29 ± 4

NaN3

10 µg

 

1817 ± 63

 

 

2160 ± 39

 

4-NOPD

10 µg

 

 

 

296 ± 7

 

 

4-NOPD

50 µg

 

 

64 ± 2

 

 

 

MMS

3.0 µL

 

 

 

 

 

1267 ± 109

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

22 ± 7

19 ± 2

44 ± 3

113 ± 3

71 ± 8

Untreated

 

 

27 ± 1

24 ± 4

48 ± 10

111 ± 6

87 ± 6

Pelargene

3 µg

 

26 ± 3

20 ± 4

42 ± 7

107 ± 9

72 ± 15

 

10 µg

 

23 ± 2

29 ± 1

37 ± 4

111 ± 12

67 ± 4

 

33 µg

 

24 ± 2

23 ± 5

45 ± 7

112 ± 29

69 ± 7

 

100 µg

 

28 ± 2

23 ± 7

40 ± 8

124 ± 4

66 ± 4

 

333 µg

 

16 ± 1

19 ± 6R

36 ± 9

86 ± 3R

62 ± 2

 

1000 µg

 

5 ± 2M R

5 ± 2M R

21 ± 2M R

68 ± 8M R

41 ± 5

 

2500 µg

 

0 ± 0M R

0 ± 0M R

15 ± 1M R

0 ± 0M R

38 ± 5R

 

5000 µg

 

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

31 ± 3M R

2-AA

2.5 µg

 

300 ± 9

270 ± 12

1720 ± 259

2127 ± 92

 

2-AA

10.0 µg

 

 

 

 

 

264 ± 12

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

M

R

Manual count

Reduced background growth

 

1.1     Summary of Results Experiment II

Study Name: 1455002

Study Code: Harlan CCR 1455002

Experiment: 1455002 HV2 Pre

Date Plated: 08/02/2012

Assay Conditions:

Date Counted: 14/02/2012

 

Metabolic

Activation

Test

Group

Dose Level

(per plate)

 

Revertant Colony Counts (Mean ±SD)

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

TA 1537

TA 98

TA 100

WP2 uvrA

 

 

 

 

 

 

 

 

 

Without Activation

DMSO

 

 

23 ± 3

13 ± 2

26 ± 8

88 ± 2

41 ± 14

Untreated

 

 

24 ± 2

13 ± 3

24 ± 4

97 ± 5

48 ± 8

Pelargene

1 µg

 

24 ± 7

13 ± 2

26 ± 1

88 ± 6

 

 

3 µg

 

23 ± 6

12 ± 6

25 ± 3

102 ± 4

47 ± 7

 

10 µg

 

27 ± 1

15 ± 4

28 ± 6

100 ± 10

41 ± 3

 

33 µg

 

23 ± 6

10 ± 2

26 ± 8

96 ± 7

39 ± 2

 

100 µg

 

11 ± 5R

4 ± 3R

17 ± 8R

67 ± 6R

31 ± 7

 

333 µg

 

4 ± 1M R

4 ± 2M R

9 ± 4R M

71 ± 10R

25 ± 3

 

1000 µg

 

0 ± 0M R

0 ± 0M R

0 ± 0M R

56 ± 9M R

27 ± 5

 

2500 µg

 

0 ± 0M R

0 ± 0M R

0 ± 0M R

7 ± 3M R

24 ± 2

 

5000 µg

 

 

 

 

 

29 ± 6

NaN3

10 µg

 

1794 ± 60

 

 

2241 ± 151

 

4-NOPD

10 µg

 

 

 

348 ± 10

 

 

4-NOPD

50 µg

 

 

80 ± 7

 

 

 

MMS

3.0 µL

 

 

 

 

 

275 ± 22

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With Activation

DMSO

 

 

32 ± 5

21 ± 4

38 ± 10

123 ± 9

53 ± 7

Untreated

 

 

30 ± 1

23 ± 5

45 ± 4

119 ± 8

60 ± 12

Pelargene

1 µg

 

30 ± 6

20 ± 9

40 ± 3

102 ± 9

 

 

3 µg

 

38 ± 12

19 ± 7

35 ± 5

134 ± 15

56 ± 5

 

10 µg

 

35 ± 5

16 ± 4

36 ± 5

128 ± 6

55 ± 4

 

33 µg

 

38 ± 7

20 ± 4

48 ± 4

108 ± 9

56 ± 6

 

100 µg

 

20 ± 1R

17 ± 7R

40 ± 1R

109 ± 17R

47 ± 7

 

333 µg

 

12 ± 6M R

9 ± 3M R

23 ± 4M R

83 ± 4R

48 ± 7

 

1000 µg

 

0 ± 0M R

1 ± 1M R

2 ± 3M R

14 ± 2M R

36 ± 5

 

2500 µg

 

0 ± 0M R

0 ± 0M R

0 ± 0M R

0 ± 0M R

40 ± 3

 

5000 µg

 

 

 

 

 

29 ± 4

2-AA

2.5 µg

 

263 ± 24

217 ± 18

1691 ± 63

1901 ± 33

 

2-AA

10.0 µg

 

 

 

 

 

357 ± 42

 

 

 

 

 

 

 

 

 

 

Key to Positive Controls

Key to Plate Postfix Codes

 

 

NaN3

2-AA

4-NOPD

MMS

sodium azide

2-aminoanthracene

4-nitro-o-phenylene-diamine

methyl methane sulfonate

R

M

Reduced background growth

Manual count

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Pelargene is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Pelargene to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II,  S. typhimurium strains: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
                         E. coli strain:
3; 10; 33; 100; 333; 1000; 2500;and 5000 µg/plate

The plates incubated with the test item showed reduced background growth at higher concentrations with and without metabolic activation in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at higher concentrations with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pelargene at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct in­crease of induced revertant colonies.