1,6,7,8,9,14,15,16,17,17,18,18-dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenic and no toxic effect of Dechlorane Plus was observed in a standard Ames test (Patil 2019) on five different strains of Salmonella typhyrium nor in a micronucleus test on human blood lymphocytes (Tekale 2019).

Also former tests did not show any sign of genetic toxicity.

Link to relevant study records

Reference

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:

short-term in vitro test

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Purity of test substance not given, but study design according to current recommendations

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine dependence/independence reverting from his- to his+

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538

Additional strain / cell type characteristics:

other: Additional mutations rfa, bio, and uvrB, partly plasmid pKM101

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix, arochlor-induced

Test concentrations with justification for top dose:

0, 10, 50, 100, 500, 1000, 5000, 10000 µg/plate

Vehicle / solvent:

DMSO (concentration not reported) in cell culture medium

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

cell culture medium with DMSO

True negative controls:

yes

Remarks:

solvent control

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-anthramine

Remarks:

only 2.anthramine with and without metabolic activation, others without metabolic activation

Details on test system and experimental conditions:

The test was repeated once, for each strain and concentration with and without metabolic activation in both tests, two plates each were evaluated, the plates were incubated for 72 hours

Evaluation criteria:

dose-related increase of mutation frequency

Statistics:

none

Key result

Species / strain:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

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:

No increase in the frequency of mutations in any strain at any concentration with and without metabolic activation, negative and vehicle controls were clearly negative, and positive controls were clearly positive, no cytotoxicity and no precipitations observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Dechlorane Plus (µg/plate)              TA 1535              TA 1537              TA 1538                  TA 98                     TA 100

- S9                     10                     42       42              15       10              21       24              24       30             141       133

- S9                     50                     36       27              17       16              17       17              25       17              146       147

- S9                     100                   39       50              17       19              18       15              32       30              152       146

- S9                     500                   37       35              17       21              13       19              26       27              154       139

- S9                     1,000               46       30              14       17              29       18              35       35              154       138

- S9               5,000                41       34              20       09              22       16              31       21              144       152

+ S9                     10                     22       16              26       33              41       34              45       51              136       128

+ S9                     50                     18       19              29       33              43       35              50       64              137       132

+ S9                     100                   27       20              25       36              48       38              57       44              147       133

+ S9                     500                   18       16              32       39              40       33              44       36              155       152

+ S9                     1,000               22       17              27       27              32       40              41       38              124       140

+ S9                     5,000               32       23              22       19              38       33              41       46              152       128

Repeat study

- S9                      50                     23       18              06       06              19       16              15       35              141       99 

- S9                     100                    26       14              06       11              15       13              32       26              139       117

- S9                     500                    29       20              08       06              10       08              30       45              116       107

- S9                     1,000                 18       19              10       07              12       06              30       31              116       125

- S9                     5,000                 29       21              14       08              16       18              29       25              111       104

- S9                     10,000               37       20              12       09              16       15              42       38              117       110

+ S9                     50                     20       16              23       12              38       14              56       48              134       105

+ S9                     100                   30       11              27       20              45       10              66       30              134       105

+ S9                     500                   31       10              19       17              40       18              66       53              117       92

+ S9                     1,000               30       08              16       18              33       22              73       70              128       114

+ S9                     5,000               14       08              16       13              40       25              55       62              110       93

+ S9                     10,000              16       10              18       18              44       27              56       45              107       106

Conclusions:

negative: no mutagenic effect observed

not mutagenic with and without metabolic activation

Executive summary:

Dechlorane Plus was tested in a standard Ames plate incorporation assay in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 with and without metabolic activation by rat liver S9 -mix at concentrations up to 10,000 µg/plate. All plates were prepared in duplicate. The test was independently repeated once. No mutagenic effect and no cytotoxicity was observed in any strain at any concentration with and without metabolic activation, no precipitation was reported. Dechlorane Plus was essentially nonmutagenic in this test.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

short-term in vitro test

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

purity of test substance not reported, no generally accepted and validated test system, test organism only rarely used

Qualifier:

no guideline available

Principles of method if other than guideline:

Toxicity in vitro to a DNA-repair deficient strain of Salmonella typhimurium and the respective DNA-repair proficient strain - difference in toxicity indicates possible DNA damage resulting in increased toxicity in the repair deficient strain.

GLP compliance:

no

Type of assay:

other: DNA-damage and repair in bacteria, Salmonella typhimurium SL4700

Target gene:

gene for recombination (rec +/-) and gene for excision-repair (uvrB+/-)

Species / strain / cell type:

S. typhimurium, other: SL4700 /SL4525 and TA 1538 / TA 1978

Details on mammalian cell type (if applicable):

The Salmonella typhimurium strain SL4700 is a recombination-deficient (rfa, rec-) derivative of strain SL4525 (rfa, rec+). Strain 1538 is an excision-repair dieficient (uvrB-) strain, and is isogenic to strain TA 1978, the excision repair proficient (uvrB+) strain.

Additional strain / cell type characteristics:

other: rfa

Metabolic activation:

without

Test concentrations with justification for top dose:

0.5 or 1.0 mg per disc

Vehicle / solvent:

none

Untreated negative controls:

yes

Remarks:

kanamycin

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

The test substance as liquid was given on a filter disc which was placed on a bacterial culture in semisolid agar. The growth inhibition zone around the filter was measured as sign of cytotoxicity and genotoxicity. Incubation was 17-17 hours.

Evaluation criteria:

Measurable difference between growth inhibition zones in repair deficient and repair proficient strains.

Statistics:

none

Key result

Species / strain:

S. typhimurium, other: SL4700, SL4525, TA 1538, TA 1978

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

no growth inhibition in any strain

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In the test substance-exposed cultures, no growth inhibition was seen in repair-proficient and repair-deficient strains, no conclusion can be derived. Results with positive and negative controls are not reported.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation no conclusion possible

No conclusion possible

Executive summary:

When Dechlorane Plus was incubated with cultures of Salmonella typhimurium recombination proficient (SL4700) or recombination deficient (SL4525, or with Salmonella typhimurium excision-repair proficient (TA1978) or excision-repair deficient (TA 1538), no growth inhibition at all was observed. Therefore no difference in growth inhibition could be delineated, and no conclusion with respect to genotoxicity could be derived.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Purity and physical chemical properties of the test substance not reported, origin of the test system (cell line) not reported.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

no further details reported

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

0.5, 1, 5, 10, 20, 40, 80, 100, 130, 150 µg/ml

Vehicle / solvent:

DMSO in cell culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO in cell culture medium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Remarks:

negative controls were clearly negative, positive controls were clearly positive

Details on test system and experimental conditions:

The expression period was 2 days, 3 x 1,000,000 cells were seeded for cloning, concentrations higher than 20 µg/ml without metabolic activation and 10 µg/ml with metabolic activation precipitated severely thus making the evaluation of the cultures impossible.

Evaluation criteria:

At least two fold increase of mutant frequency above negative controls, dose-dependent increase

Statistics:

No statistics was performed

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Cultures with concentrations above 20 µg/ml without metabolic activation and 10 µg/ml with metabolic activation could not be evaluated due to excessive precipitation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Dechlorane Plus       Total viable       Total mutant       Cloning efficiency       Mutant frequency       Induced mutations       S9 -mix

(µg/ml)                     clones                  clones               relative %                     (x 1 000 000)             (x 1 000 000)    

0                             504                         108                     100%                            43                            0                            No

EMS 500 µg/ml       319 / 322              937 / 931         63.3 / 63.9                   587 / 578               544 / 535                   No

0.5                          619 / 514              139 / 84           122.8 / 102.0               45 / 33                      2 / 0                      No

1.0                          505 / 440              111 / 141        100.2 / 87.3                  44 / 64                      1 / 21                      No

5.0                          557 / 504              142 / 118        110.5 / 100.0                51 / 47                      8 / 4                         No 

10.0                        478 / 556              148 / 126          94.8 / 110.3               62 / 45                      19 / 2                      No

20.0                      529 / 553              140 / 116          105.0 / 109.7               53 / 42                      10 / 0                       No

40.0                            no evaluation possible due to severe precipitation                                                                           No

80.0                            no evaluation possible due to severe precipitation                                                                           No

100.0                          no evaluation possible due to severe precipitation                                                                           No

130.0                          no evaluation possible due to severe precipitation                                                                           No

150                             no evaluation possible due to severe precipitation                                                                           No

0                     722 / 595              199 / 131              109.6 / 90.4              55 / 44              0 / 0                                   Yes

3 -MCA 4.0 µg/ml 363 / 355       484 / 533              55.1 / 53.9               267 / 300           217 / 251                           Yes

0.5                   618 / 487              175 / 131              93.8 / 74.0              57 / 54               7 / 4                                Yes

1.0                   554 / 515              158 / 127              84.1 / 78.2              57 / 49                    7 / 0                               Yes

5.0                   585 / 567              127 / 113              88.8 / 86.1              43 / 40                     0 / 0                              Yes

10.0                 574 / 561              115 / 121              87.2 / 85.2              40 / 43                     0 / 0                              Yes

20.0                            no evaluation possible due to severe precipitation                                                                         Yes

40.0                            no evaluation possible due to severe precipitation                                                                         Yes

80.0                            no evaluation possible due to severe precipitation                                                                         Yes

100.0                          no evaluation possible due to severe precipitation                                                                       Yes

130.0                          no evaluatio possible due to severe precipitation                                                                           Yes

150.0                          no evaluation possible due to severe precipitation                                                                       Yes    

     

Conclusions:

Interpretation of results (migrated information):
negative

Not mutagenic in mammalian cells with and without metabolic activation

Executive summary:

Dechlorane Plus was tested in the standard mouse lymphoma assay using L5178Y mouse lymphoma cells with and without metabolic activation by rat liver S9 -mix. Concentrations up to 20 µg/ml without metabolic activation and up to 10 µg/ml with metabolic activation were not mutagenic and not cytotoxic. Higher concentrations could not be evaluated due to severe precipitation. Solvent controls were clearly negative and positive controls were clearly positive. Dechorane Plus was essentially nonmutagenic in this test.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 05 to July 20, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Jiangsu Anpon Electrochemical Co., Ltd, batch no. 20190519024
- Expiration date of the lot/batch: May 18, 2020
- Purity: 99.2 % w/w

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and Glucose-6-phosphate to form the “S9 mix”. This mix is added to the top agar in this activated assay. The S9 fraction procured from Dr. G. P Meshram, Nagpur, and (Lot N ° MWR/ARI/S9F/ 01/18 APPENDIX 7) was used in the study.
A volume of 0.1 mL of S9 mix (5% v/v S9 mix for initial toxicity-mutation test and 10% v/v S9 mix for confirmatory mutation test) prepared for treatment was added aseptically to 2 mL of top agar, mixed thoroughly and this mixture was poured onto MGA plate.

Test concentrations with justification for top dose:

Test Concentrations (µg/plate) in the absence and presence of the metabolic activation system (10% v/v S9 mix):
A (Stock ID): 5000 µg/plate
B (Stock ID): 2500 µg/plate
C (Stock ID): 1250 µg/plate
D (Stock ID): 625 µg/plate
E (Stock ID): 312.5 µg/plate
F (Stock ID): 156.25 µg/plate

Vehicle / solvent:

Vehicle / solvent: A volume of 0.1 mL of dimethyl sulfoxide, used as vehicle was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto MGA plate.
The test item was insoluble in distilled water (stock A, 50000 µg/mL), while it was found to be soluble in dimethyl sulfoxide (stock B, 50000 µg/mL), hence dimethyl sulfoxide was selected as the vehicle for treatment.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Positive Controls used in Absence of Metabolic Activation System

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Positive Control used in the Presence of Metabolic Activation System

Positive control substance:

other: 2-Aminoanthracene

Details on test system and experimental conditions:

The test evaluates the test item for its ability to induce reverse mutations in the tester strains and restore the functional capability of the bacteria to synthesize an essential amino acid. In this case, the revertant bacteria are detected by their ability to grow in the absence of histidine, which is required by the Salmonella typhimurium tester strains.
The tester strains used for the bacterial reverse mutation test were Salmonella typhimurium TA1537, TA1535, TA98, TA100 and TA102.
Solubility and Precipitation Test
The test item was insoluble in distilled water (stock A, 50000 µg/mL), while it was found to be soluble in dimethyl sulfoxide (stock B, 50000 µg/mL), hence dimethyl sulfoxide was selected as the vehicle for treatment. A volume of 100 µL of the test item from stock B (50000 µg/mL) was added to 2 mL of top agar, to assess the precipitation. Precipitation was observed at the tested concentration of 5000 µg/plate, But which did not interfere with the colony count. Hence, 5000 µg/plate was selected as the highest concentration to be tested for initial toxicity-mutation test both in the absence and presence (5% v/v S9 mix) of the metabolic activation.
Top Agar
Top agar prepared and used for the study (2 mL) was poured aseptically onto MGA plate.
S9 Mix
A volume of 0.1 mL of S9 mix (5% v/v S9 mix for initial toxicity-mutation test and 10% v/v S9 mix for confirmatory mutation test) prepared for treatment was added aseptically to 2 mL of top agar, mixed thoroughly and this mixture was poured onto MGA plate.
Solvent
A volume of 0.1 mL of dimethyl sulfoxide, used as vehicle was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto MGA plate.
Test Item
A volume of 0.1 mL of the test item stock solution of the highest concentration was added aseptically to 2 mL of top agar, mixed well and this mixture was poured onto MGA plate.
ONB Solution
A volume of 0.1 mL of ONB solution was added aseptically to 2 mL of top agar, mixed well and the mixture was poured on to MGA plate.
0.2 M Sodium Phosphate Buffer
A volume of 0.1 mL of 0.2 M of sodium phosphate buffer was added aseptically to 2 mL of top agar, mixed well and the mixture was poured on to MGA plate.
MGA Plate
Blank MGA plate was maintained for the sterility check.
Confirmatory Mutation Test
Based on the result of initial toxicity mutation test, the confirmatory mutation test was performed both in the absence and presence of the metabolic activation system (10% v/v S9 mix). The treatments were performed by plate incorporation technique as described in the initial toxicity-mutation test.
The tester strains TA1537, TA1535, TA98, TA100, and TA102 were exposed to the test concentration levels 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate of 1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”) both in the absence and presence of the metabolic activation system (10% v/v S9 mix). Volumes of 100 µL of relevant stock solutions A (5000 µg/plate) – F (156.25 µg/plate) for TA1537, TA1535, TA98, TA100, and TA102 were used to obtain the required test concentrations for treatment in the absence and presence of the metabolic activation system (10% v/v S9 mix), respectively.
Plates were maintained in triplicates for each test concentration of test item, negative and positive controls. The numbers of revertant colonies were recorded after 48 h incubation period at 37 ± 1 ºC.
Treatment with 2-aminoanthracene in the absence of the metabolic activation system was also performed for tester strain TA100 in both initial toxicity-mutation test and confirmatory mutation test to verify the efficiency of the S9 fraction used in the study.

Evaluation criteria:

Cytotoxicity Evaluation Criteria
Six analysable doses were available to evaluate assay data. Cytotoxicity is detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn (Teo et al., 2003).
Assay Evaluation Criteria
A result is considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Biological relevance of the result was considered first.
Strains TA1535, TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
Strains TA98, TA100 and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
Statistical analysis was used as an aid in the evaluation of dose response.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

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

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Negative Control:
Results of this study indicate that values of the negative control in all tester strains were within historical range of respective strains (APPENDIX 6).
The mean count of histidine revertant colonies in the negative control is provided in TABLES 1 and 2 for the initial toxicity-mutation test and in TABLES 3 and 4 for the confirmatory mutation test.
Positive Controls:
2-Aminoanthracene was used as the positive control, in the presence of the metabolic activation system for all tester strains, during the initial toxicity and the confirmatory mutation test. Large historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control, in the presence of the metabolic activation system. The batch of S9 used in this study was characterised by supplier with benzo(a)pyrene which required metabolic activation system by microsomal enzymes. Positive controls exhibited a clear increase in the number of revertant colonies when compared with that of the concurrent negative controls. This demonstrated the efficiency of the test system and suitability of procedures employed in the assay.
An increase in revertants was not observed in TA100 (initial toxicity-mutation test and confirmatory mutation test), treated with 2-aminoanthracene, in the absence of the metabolic activation; but a clear increase was observed, in the presence of the metabolic activation. This demonstrated the efficiency of the S9, fraction used in this assay.
Initial Toxicity-Mutation Test:
Cytotoxicity was characterised by inhibition of the bacterial background lawn and/or reduction in the number of revertant colonies.
A normal bacterial background lawn, without reduction in the number of revertant colonies was observed up to 5000 µg/plate, in the absence and presence of the metabolic activation (5% v/v S9 mix) in TA1537, TA1535, TA98, TA100, and TA102. No increase in the number of revertant colonies was observed, either in the absence or presence of the metabolic activation (5% v/v S9 mix), at any tested concentration level, in any tester strain. Results revealed that there was no mutagenic effect in TA1537, TA1535, TA98, TA100, and TA102. Statistical analysis did not reveal any significant effect.
Based on results of initial toxicity mutation test, a concentration of 156.25, 312.5, 625, 1250, 2500 and 5000 µg 1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”)/plate, was selected for the confirmatory mutation test, in the absence and presence of the metabolic activation system (10% v/v S9 mix) for TA1537, TA1535, TA98, TA100, and TA102.
Confirmatory Mutation Test:
A normal bacterial background lawn without reduction in the number of revertant colonies was observed up to 5000 µg/plate in TA1537, TA1535, TA98, TA100, and TA102, in the absence and presence of the metabolic activation system (10% v/v S9 mix).
No increase in the number of revertant colonies was observed, either in the absence or presence of the metabolic activation system (10% v/v S9 mix) in any tester strain.
Results revealed that there was no positive mutagenic effect in TA1537, TA1535, TA98, TA100, and TA102, at 5000, 2500, 1250, 625, 312.5, 156.25 µg/plate of 1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”) either, in the absence and presence of the metabolic activation system (10% v/v S9 mix), when compared with that of the concurrent negative control. Statistical analysis did not reveal any significant effect.

TABLE 1:Mean Count of His⁺Revertant Colonies in NegativeControl, Positive Controls

and Treatment Plates in the Absence ofMetabolic Activation(Initial Toxicity-Mutation Test)

Concentration of Test item (µg/plate)	His^+Revertant Colonies/Plate (Absence of Metabolic Activation)
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	9.00	1.41	17.50	0.71	23.00	2.83	128.00	12.73	238.50	16.26
1.5	9.50	2.12	17.50	4.95	25.50	2.12	125.00	19.80	241.00	9.90
5	9.00	2.83	18.50	2.12	21.00	1.41	133.00	8.49	235.00	19.80
15	8.00	1.41	17.00	1.41	24.00	2.83	126.00	0.00	224.50	3.54
50	7.50	0.71	18.50	3.54	23.50	3.54	127.00	5.66	239.00	8.49
150	9.50	2.12	19.00	2.83	26.50	0.71	127.00	8.49	224.50	21.92
500	7.50	0.71	16.00	1.41	24.50	2.12	133.00	1.41	233.50	30.41
1500	9.50	0.71	18.00	1.41	22.00	1.41	127.50	12.02	226.50	12.02
5000	10.50	0.71	19.50	3.54	23.00	2.83	124.50	10.61	231.00	4.24
PC	389.00	43.84	552.00	1.41	613.50	10.61	746.00	25.46	957.00	11.31
2Aa	-	-	-	-	-	-	130.00	21.21	-	-

Key: SD = Standard Deviation,Test item=1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”),NC = Negative Control,DMSO =Dimethyl sulfoxide, PC = Positive Control {TA1537 = 9-Aminoacridine Hydrochloride Monohydrate (75 µg/plate), TA1535 = Sodium Azide (0.5 µg/plate), TA98 = 2-Nitrofluorene (7.5 µg/plate), TA100 = Sodium Azide (5 µg/plate), TA102 = Mitomycin-C (0.5 µg/plate)}, 2-Aa = 2-Aminoanthracene (5 µg/plate for TA100), - = Not Applicable.

TABLE 2: Mean Count of His⁺Revertant Colonies in Negative Control, Positive Control

and Treatment Plates in the Presence ofMetabolic Activation(Initial Toxicity-Mutation Test)

Concentration of Test item (µg/plate)	His^+Revertant Colonies/Plate [Presence of Metabolic Activation (5% v/v S9 mix)]
	TA1537		TA1535		TA98		TA100		TA102
	Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
NC (DMSO)	10.00	1.41	18.00	1.41	23.50	4.95	147.00	5.66	248.50	2.12
1.5	9.50	2.12	21.50	7.78	24.50	3.54	114.00	8.49	238.50	14.85
5	9.00	1.41	14.00	1.41	22.50	4.95	125.50	4.95	232.50	12.02
15	9.50	0.71	19.50	4.95	30.00	2.83	129.00	11.31	223.50	14.85
50	11.50	2.12	20.50	3.54	25.00	9.90	123.00	11.31	238.00	4.24
150	10.50	3.54	17.50	0.71	24.00	2.83	149.00	1.41	217.00	0.00
500	10.00	1.41	19.00	4.24	24.00	2.83	119.00	5.66	235.50	13.44
1500	9.50	0.71	18.50	0.71	28.00	4.24	125.50	4.95	223.50	6.36
5000	8.00	1.41	18.50	2.12	24.00	2.83	123.50	2.12	220.00	5.66
PC	315.00	62.23	530.50	28.99	646.50	23.33	794.00	41.01	877.50	9.19

Key: SD = Standard Deviation,Test item=1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”),NC = Negative Control,DMSO =Dimethyl sulfoxide, PC = Positive control {2Aa = 2-Aminoanthracene (10 µg/plate for TA1537, TA1535, TA102 and 5 µg/plate for TA98 and TA100)}.

Conclusions:

From results of this study, it is concluded that 1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”) is non-mutagenic to any strain of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102, when tested under specified experimental conditions. The test result is in line with Moertelmanns 1980 test also on five strains of Salmonella typhimurium. By this test the non mutagenic result was proven also for strain TA102.

Executive summary:

This study was performed to evaluate mutagenic activity of 1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacycl[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”)bythe bacterial reverse mutation test, using five histidine deficient mutant tester strains ofSalmonella typhimurium(i.e., TA1537, TA1535, TA98, TA100 and TA102).

1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene

(“Dechlorane Plus”) was tested in two independent experiments, in the absence and presence of the metabolic activation. Bacterial cultures were exposed to the test item at 8 concentration levels (two plates/concentration) from1.5 to 5000mg/plate in initial toxicity-mutation test. A normal bacterial background lawn was observed up to 5000 µg/plate in all tester strains, in the absence and presence of the metabolic activation (5% v/v S9 mix). No increase in the number of revertant colonies (no mutagenic effect) was observed, either in the absence or presence of the metabolic activation system (5% v/v S9 mix) in any tester strain. To confirm negative results obtained in the initial toxicity mutation test, a confirmatory mutation test was conducted with an increased S9 concentration, i.e., 10% v/v S9 mix andmodified concentration spacing. Based on results of the initial toxicity mutation test, bacterial cultures were exposed to1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”) at 6 concentration levels (three plates/concentration) from 156.25 to 5000 µg/plate for TA1537, TA1535, TA98, TA100, and TA102, in the absence and presence (10% v/v S9 mix) of the metabolic activation. After 48 hours of incubation at 37 ± 1°C, the revertant colonies were enumerated.

1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene

(“Dechlorane Plus”) did not induce any significant increase in the number of revertant colonies in trials, with or without S9 mix, in any tester strain. All values for the negative control were within historical control ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, demonstrating sensitivity and the efficiency of the test system.

All criteria for a valid study were met, as described in the study plan. From results of this study, under the specified experimental conditions,1,6,7,8,9,14,15,16,17,17,18,18- Dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene (“Dechlorane Plus”) is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.

COMPLIANCE:Signed and dated GLP, Quality Assurance and Data Confidentiality statements are provided. There was no deviation from regulatory requirements.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

37 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20190519024
- Expiration date of the lot/batch: May 18,2020
- Analysed Purity: 99.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
Storage Temperature : Room temperature
Storage Container : Stored in original Container as supplied by the
Sponsor
Storage Location : Test Item Control Office. JRF

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

Human Peripheral Blood Lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

A metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction was used. Total Volume of media: 100 mL (2% S9 v/v)

Test concentrations with justification for top dose:

125, 250, 500, 625 and 750 µg test item/mL in culture medium in the absence and presence (2% v/v S9) of metabolic activation.
The criteria for the selection ofthe highest concentration should be met i.e. 55 ± 5% reduction in RI compared to the vehicle control, and if cytotoxicity is not observed, 2000 µg/mL or 2 µL/mL, 10 mM or limit of solubility, whichever is lower, should be tested as the maximumconcentration.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSOcontrols

True negative controls:

yes

Remarks:

RPMI-1640 controls

Positive controls:

yes

Positive control substance:

cyclophosphamide

vinblastine

Details on test system and experimental conditions:

Cytotoxicity Test
Before conducting the main study, test item was evaluated for cytotoxicity in the absence and presence of metabolic activation (2% v/v S9 mix).
The cytotoxicity test was performed at test concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg test item/mL in culture medium. A concurrent negative control (RPMI-1640) and vehicle control (DMSO) were also maintained. A quantity of 403.25 mg of test iem was made up to 2 mL with DMSO to attain the stock solution concentration of 200012 µg/mL (stock A), considering its purity (99.2%). A volume of 1 mL of stock A was added to 1 mL DMSO to obtain 100006 µg/mL (stock B). Further stock solutions of 50003 µg/mL (stock C), 12500.75 µg/mL (stock D), 6250.375 µg/mL (stock E) and 3125.18 µg/mL (stock F) were prepared by serial dilution. A 180 µL volume of relevant stock solution were added into respective tubes containing 17.82 mL of medium to obtain concentrations of 31.25, 62.5, 125, 250, 500 and 1000 µg test item/mL in culture medium for treatment in the absence and presence ofmetabolic activation. To each culture tube 8 mL of treatment medium was added. Serum free medium with KCl was used in the absence of metabolic activation while medium with S9 mix was used for treatment in the presence of metabolic activation (2% v/v S9). The cultures were exposed for 4 h and 05 minutes both in the absence and presence of metabolic activation.

Main Study
Based on the cytotoxicity test results, test item was evaluated for its potential to induce micronuclei at tested concentrations of 125, 250, 500, 625 and 750 µg test item/mL in culture medium in the absence and presence (2% v/v S9) of metabolic activation. The main study was conducted in two phases with lymphocytes exposed for 4 h with and without metabolic activation (2% v/v S9) as Phase 1, and for 24 h and 05 minutes exposure without S9 as
Phase II. There were two replicates per treatment concentration including positive, vehicle (DMSO) and negative (RPMI-1640) controls.
Phase 1 (Absence and presence (2% v/v S9 mix) of metabolic activation and 4 h exposure) In Phase 1, cultures were exposed to concentrations of 125, 250, 500, 625 and 750 µg test item/mL in culture medium in the absence and presence (2% v/v S9) of metabolic activation. For treatment, the first test item stock solution (stock F) were prepared by diluting 151.21 mg of test item in DMSO and then made up to 2 mL (75000.16 µg/mL) considering its purity (99.2%), and 126.01 mg made up to 2 ml to achieve 62500.96 µg/mL (stock G) were prepared. For treatment, the first test Item stock solution (stock A) was prepared by diluting 403.25 mg of test item in DMSO and then made up to 2 mL (200012 µg/mL) considering its purity (99.2%). A
volume of 1 mL of stock A was added to 1 mL DMSO to obtain 100006 µg/mL (stock B). Further stock solutions of 50003 µg/mL (stock C), 25001.5 µg/mL (stock D) and 12500.75 µg/mL (stockE).
A concurrent negative (untreated) control and vehicle control (DMSO) were also maintained. A 180 pL volume of relevant stock solution C - G was added into 17.82 mL of medium (total volume 18 mL) to obtain the required test concentrations in the absence and presence of metabolic activation.
8 mL of treatment medium was used for treatment of respective replicate cultures.
Similar procedure was followed for treatment in all phases of the main study. Serum free medium with KCl was used in the absence of metabolic activation while medium with S9 mix was used in the presence of metabolic activation. The cultures were exposed for 4 h both in the absence and presence of metabolic activation. At the end of treatment, cultures were centrifuged at approximately 1500 rpm for 8 minutes and the supernatant replaced with fresh complete medium containing cytochalasin B (6 µg/mL) and incubated at 37 ± I °C in a 5% CO2 incubator utill harvesting. The cells were harvested and processed for slide preparation approximately 24 h from the beginning of treatment.

Phase II (Absence of metabolic activation and 24 h exposure)
In Phase II, cultures were exposed to concentrations of 125, 250, 500, 625 and 750 µg test item/mL in culture medium (supplemented with 10% FBS), for approximately 24 h and 05 minutes without S9 (in the absence of metabolic activation) with cytochalasin B (6 µg/mL). The cells were harvested and processed for preparation of slides after the 24 h and 05 minutes exposure period.

As the phase II experiment was performed to confirm the negative results obtained in the absence of metabolic activation in Phase 1, slide scoring data was evaluated after data from the phase I experiment.

Controls
A concurrent negative control (RPMI-1640), vehicle control (DMSO) and positive controls were maintained, in duplicate, for each phase of the experiment (except for a positive control in phase I in the absence of S9), both in the absence and presence of metabolic activation. This demonstrated the activity of the metabolic activation system and the responsiveness of the test system. Cyclophosphamide (30 µg/mL) was used as the positive control in the presence of metabolic activation In Phase I and Vinblastine (0.008 µg/mL) was used as the positive control in the absence of metabolic activation (Phase II).

Harvesting of Cells
Each culture was harvested and processed separately for micronuclei frequency evaluation. Cells were hypotonically treated with cold (2-8 °C) Potassium Chloride solution (0.075 M KCI) and centrifuged immediately for 8 minutes. The supernatant was removed and replaced with chilled Camoy's fixative and centrifuged for 8 minutes. Cells were given a further two changes of chilled Camoy's fixative washing.

Slide Preparation
Slides were prepared from each culture tube by pouring approximately 0.5 mL of fixed cell
suspension, drop by drop on two, pre-cleaned, ice-chilled slides. The slides were dried over a slide warmer and labelled with the study number, treatment code, absence/presence of metabolic activation and slide number. Two slides were prepared per replicate culture per test concentration (i.e. four slides per test concentration). Out of these two slides, one was used for scoring and the other served as a reserve or used for additional scoring, if required. The dried slides were stained with 5% Giemsa in phosphate buffer for 10 minutes. The slides were made permanent by mounting a cover slip with DPX mountant. To prevent bias in the scoring procedure for micronuclei, the slide numbers were masked with coded numbers provided by Department of Bio-statistics and Systems Information, JRF. All slides were coded before microscopic analysis and decoded after scoring was completed. All slides, including those of positive, negative control (RPMI-1640), treatment group and vehicle control (DMSO), were independently coded prior to microscopic analysis for replicative
index (during the cytotoxicity test and main study), and for concentrations selected for micronuclei analysis (during the main study). The slides were examined under a light microscope and a minimum of 500 cells per slide (culture) were counted, the number of binucleated cells, multi nucleated cells and mono nucleated cells were recorded in different fields of view to determine the replicative index.

Micronucleus Frequency
Two replicate slides per concentration were used to record the number of micronucleated binucleated cells whereas the other two slides were kept in reserve, for scoring if required (i.e. if insufficient binucleated cells were observed on two slides). The slides were examined for the presence of micronuclei in binucleated cells under a light microscope. A minimum of 1000 binucleated cells per replicate were screened per concentration to evaluate the incidence of micronuclei. The masked labels were removed and all slides were decoded after scoring.

Rationale for test conditions:

Assay Acceptance Criteria
The following acceptance criteria were used to determine a valid assay:
• The negative (untreated) control and vehicle (DMSO) control is considered acceptable for addition to the laboratory historical negative control database.
• The positive controls should induce responses that are compatible with the historical positive control data base and produce a statistically significant increase when compared to the concurrent vehicle control.
• Cell proliferation (Replicative Index) in the negative and vehicle control should be used to demonstrate that a sufficient number of treated cells have reached mitosis during the test and that the treatments are conducted at appropriate levels of cytotoxicity.

Evaluation criteria:

Assay Evaluation Criteria
The test item was considered to have ciastogenic activity in this study if the following criteria were
met:
• At least one of the test concentrations exhibits a statistically significant increase in micronucleus frequency when compared with the concurrent vehicle control.
• The increase micronucleus frequency is concentration-related (biologically significant) when evaluated with an appropriate trend test e.g. Chi-square trend analysis.
• Increases in micronuclei are not associated with large changes in pH or osmclality of the treated cultures.
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria were met, the test item was considered as able to induce the formation of micronuclei in this test system.
The test item was considered clearly negative in this study if the following criteria were met:
• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
• There is no concentration-related increase when evaluated with an appropriate trend test (e.g. Chi-square trend analysis).
• All results were inside the distribution of the historical control data,

When all of these criteria were met, the test item was considered as unable to induce micronuclei
formation i.e. chromosome breaks and/or the gain or loss of chromosomes in this test system.

Statistics:

Statistical Analysis
Micronuclei data containing binucleated cells was statistically analysed for normality using Shapiro-Wilk's test, where results of normality test were significant, non- parametric test (Kruskal- Wallis test) and Mann Whitney test was performed and for homogeneity of variance using the Bartlett test before conducting appropriate parametric test (ANOVA, t-test).

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

From results of this study, it is concluded that 1,6,7,8,9,14,15,16,17,17,18,18-Dodecachloropentacyclo[12.2.1.16,9.02,13.05,I0]Octadeca-7,15-Diene ("Dechlorane Plus") did not show any potential to induce micronuclei or clastogenic or aneugenic potential in isolated cultured human peripheral blood lymphocytes, in the absence or presence (2% v/v S9 mix) of the metabolic activation, under the described experimental conditions.

Executive summary:

In a mammalian cell micronucleus test, human peripheral blood lymphocytes, cultured in vitro, were exposed to the test item (1,6,7,8,9,14,15,16,17,17,18,18-Dodecachloropentacyclo [12.2.1.16,9.02,13.05,10]Octadeca-7,15-Diene("Dechlorane Plus") at different concentrations, in the absence and presence of the metabolic activation (2% v/v S9 mix).

Hereafter the test item's name 1,6,7,8,9,14,15,16,17,17,18,18-DodecachloropentacycIo[12.2.1.16,9.02,13.05,10]Octadeca7,15-Diene ("Dechlorane Plus") is represented as the test item, in this experiment.

The test item was tested in two independent experiments, in the absence and presence of the metabolic activation (2% v/v S9 mix). In the main study, human peripheral blood lymphocyte cultures were exposed to the test item at 5 concentrations (two cultures/concentration level) from 125 µg/mL to 750 µg/mL, in a culture medium, in the absence and presence of the metabolic activation system in phase-I and in the absence of the metabolic activation system in phase-II.

Concentrations were selected based on results from a cytotoxicity test.

The test item did not induce either a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei, in the absence and presence of the metabolic activation, in either the independently conducted experiments.

All criteria of a valid study were met. From results of this study, it is concluded that the test item did not show any potential to induce micronuclei or clastogenic or aneugenic potential in the isolated cultured human peripheral blood lymphocytes, in the absence or presence (2% v/v S9 mix) of the metabolic activation, under the described experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

No mutagenic and no toxic effect of Dechlorane Plus was observed in a standard Ames plate incorporation test in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 with and without metabolic activation by rat liver S9-mix at concentrations up to 10,000 µg/plate.

An additional test (Patil 2019) was performed accoding to OECD 471 with the same results also for strain TA 102 and the former assessed strains TA1537, TA1535, TA98, TA100.

The in vitro test on mamalian cells also did not show any potential to induce micronuclei or clastogenic or aneugenic potential in isolated cultured human peripheral blood lymphocytes.

Justification for selection of genetic toxicity endpoint

Standard test system, high concentrations used, with and without metabolic activation.

Justification for classification or non-classification

No positive response in five in-vitro and one in-vivo study for mutagenicity were seen and thus the substance is not subject to classification for mutagenicity according to CLP (Regulation EC No 1272/2008).