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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It is concluded that TCPD (Distilled Tricyclopentadiene) is non-mutagenic to five strains of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102, when tested under the specified experimental conditions.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation (4 January 2019) to Experimental Completion (21 January 2019)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Materia inc.
- Lot/batch No.of test material: RP229-0714
- Expiration date of the lot/batch: 28 April 2019
- Purity test date: 28 March 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in orginial container as supplied by the Sponsor. Stored after a brief sweep with Nitrogen gas in a sealed bottle.
- Stability under test conditions: Assumed stable for the duration of the test.
- Solubility and stability of the test substance in the solvent/vehicle: In DMSO, the test item was soluble after 40 minutes sonication with heating (between 35 to 45°C), however this formulation crystalized when at room temperature. The test item was therefore maintained at a temperature between 35 to 45°C so the test item would remain soluble in the vehicle throughout its use in this test system.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The test item in the vehicle (DMSO) crystallised when at room temperature, however as the test item stock was maintained at a temperature between 35 to 45°C within in this test system, the use of the vehicle was considered to have had no adverse affect on the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared as a solution in DMSO (after sonication at heating between 35 to 45°C). The formulation was thereafter maintained at a temperature between 35 to 45°C throughout the study.





Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: S9 fraction procured from Dr. G.P Meshram, Nagpur, and (Lot N ° MWR/ARI/S9F/01/18) was used in this study.

Composition of Co-factor Mix and S9 Mix:

Co-factor Mix
D- Glucose –6- phosphate : 0.80 g
 Nicotinamide adenine dinucleotide
Phosphate (NADP) : 1.75 g
Magnesium chloride : 0.90 g
Potassium chloride : 1.35 g
Sodium phosphate, dibasic: 6.40 g
Sodium phosphate, monobasic: 1.40 g
Distilled water : 450 mL
The prepared co-factor mix was dispensed into suitable volumes and stored below 0 °C.

S9 Mix (10 mL)
Constituent 5% v/v S9 mix (10 mL) 10% v/v S9 mix (10 mL)

Co-factor mix 9.5 mL 9.0 mL
S9 fraction 0.5 mL 1.0 mL
The S9 mix was prepared fresh by adding the required quantity of S9 fraction to thawed co-factors and maintained in an ice bath. Any remaining portions of S9 mix were discarded.

- source of S9 : S9 fraction (Aroclor 1254-induced (Analab, USA) 500 mg/kg bw) was procured from Dr. G.P Meshram, Nagpur, and (Lot N ° MWR/ARI/S9F/01/18) was used in this study.
- method of preparation of S9 mix: The S9 fraction is buffered and supplemented with the essential co-factors β-NADP and Glucose-6-phosphate to form “S9 mix”. This mix was then added to the top agar in this activated assay.
- concentration or volume of S9 mix and S9 in the final culture medium: A volume of 0.1 mL of S9 mix (5% v/v S9 mix for the initial toxicity-mutation test and 10% v/v S9 mix for the confirmatory mutation test) was prepared for treatment and added aseptically to 2 mL of top agar, mixed well and poured onto MGA plates.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability). yes, an efficiency check report (as a CoA) was issued from the vendor for the batch used in this study. This confirmed acceptable metabolic activation of this batch to tester strians prior to use on this study.
Test concentrations with justification for top dose:
Initial toxicity mutation test concentrations:
All strains, TA1537, TA1535, TA98, TA100 and TA102 (+/- S9 mix, 5% v/v S9 mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/ plate.

As the test item crystalized at room temperature in DMSO, the test item stock (50000 µg/mL) was maintained at a temperature between 35 to 45°C so the test item would remain soluble. Once treated, cultures were incubated at 37 ± 1°C.
In the initial toxicity mutation test the OECD 471 limit concentration of 5000 µg/plate was acheived based on solubility in DMSO. Normal growth was observed up to the guideline limit concentration of 5000 µg/plate in tester strain TA100 in the absence of metabolic activation, and in tester strain TA102 in the absence and presence of metabolic activation (5% v/v S9 mix); with partial and complete inhibition and up to 50 µg/plate in tester strains TA1537, TA1535 and TA98 in the absence of metabolic activation and up to 150 µg/plate in tester strains TA1537, TA1535, TA98, and TA100 in the presence of metabolic activation (5% v/v S9 mix).
No increase in the number of revertant colonies was observed either in the absence or presence of metabolic activation (5% v/v S9 mix) at any tested concentration, in any tester strain. Results revealed that there was no mutagenic effect in tester strains TA1537, TA1535, TA98, TA100 and TA102.

Based on these observations the below concentrations were selected for the confirmatory mutation test:

TA100 (-S9, 10% v/v S9 mix): 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ plate.
TA102 (+/-S9, 10% v/v S9 mix): 156.25, 312.5, 625, 1250, 2500 and 500 µg/ plate.
TA1537, TA1535 and TA98 (-S9, 10% v/v S9 mix): 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/ plate.
TA1537, TA1535, TA98 and TA100 (+S9, 10% v/v S9 mix): 15.63, 31.25, 62.5, 125, 250 and 500 µg/ plate.







Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO for the test item.

- Justification for choice of solvent/vehicle: DMSO was selected as the vehicle for the study. The test item crystalized at room temperature in DMSO, therefore to maintain test item solubility the test item stock was maintained at a temperature between 35 to 45°C. DMSO was therefore selected as the vehicle for the study. Once treated, cultures were incubated at 37 ± 1°C under standard test conditions.

- Justification for percentage of solvent in the final culture medium: 5% v/v (0.1 mL of DMSO, used as vehicle was added aseptically to 2 mL of top agar, mixed well and poured onto MGA plates).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate in the initial toxicity mutation test, Triplicate in the main confirmatory mutation assay
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cell densities (OD at 660 nm) of all tester strains were within the required range to produce cultures with approximately 1 - 2 x 109 bacteria/mL, demonstrating that appropriate numbers of bacteria were plated.
- Test substance added in medium; Treatments were performed using the plate incorporation technique.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: None
- Exposure duration/duration of treatment: Plates were maintained in triplicate for each test item concentration, negative and positive controls. The numbers of revertant colonies were recorded after 48 h incubation at 37 ± 1 ºC.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, 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).
- Any supplementary information relevant to cytotoxicity: In the initial toxicity mutation test, partial and complete inhibition in bacterial background lawn pattern with a reduction in the number of revertant colonies was observed at the higher tested concentrations in some tester strains.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : A result was considered positive if a concentration-related increase over the range tested and/or a reproducible increase in the number of revertant colonies per plate at one or more test concentration in at least one strain in the absence or presence of metabolic activation was observed.
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.
Statistics:
A simple linear regression analysis was performed for strains: 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 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Not specifed
- Data on osmolality: Not specified
- Possibility of evaporation from medium: None, non-volatile substance
- Water solubility: Non water soluble
- Precipitation and time of the determination: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES (if applicable): In an initial toxicity-mutation test, bacterial cultures were exposed to the test item at 8 concentrations (two plates/concentration) from 1.5 to 5000 ug/plate. Based on the results, a confirmatory mutation test (three plates/concentration) was then performed a selected test concentrations.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : yes, all values for the negative control were within laboratory historical control ranges. Positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

Ames test:
- Signs of toxicity : Cytotoxicity observed in some tester strains
- Individual plate counts: yes (see below tables)
- Mean number of revertant colonies per plate and standard deviation : yes (see below tables)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Individual Plate Count (Confirmatory Mutation Test)
Absence of Metabolic Activation

Concentration of

TCPD (Distilled Tricyclopentadiene) (µg/plate)

Number of Revertant Colonies

TA1537

TA1535

TA98

TA100#

TA102#

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

NC (DMSO)

11

13

5

12

11

15

26

19

29

116

125

113

268

220

243

3.91

156.25#

9

13

12

12

14

19

30

18

25

122

123

145

247

234

232

7.81

312.5#

8

8

10

12

10

13

33

25

23

147

131

105

243

236

221

15.63

625#

4

8

9

13

21

9

22

26

33

123

147

98

249

229

237

31.25

1250#

8

7

13

9

11

13

28

30

14

133

142

135

221

237

227

62.5

2500#

5

10

4

9

10

7

21

12

20

128

140

142

231

260

250

125

5000#

2

0

1

6

6

4

12

10

16

99

129

137

238

215

244

PC

242

285

424

217

224

221

321

375

378

662

627

664

905

1016

895

2Aa

-

-

-

-

-

-

-

-

-

120

139

133

-

-

-

Presence of Metabolic Activation (10% v/v S9 mix)

Concentration of

TCPD (Distilled Tricyclopentadiene) (µg/plate)

Number of Revertant Colonies

TA1537

TA1535

TA98

TA100

TA102#

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

R1

R2

R3

NC (DMSO)

9

6

9

15

13

10

17

23

31

153

108

116

232

206

231

15.63

156.25#

13

6

2

19

11

15

19

25

21

128

105

110

227

240

227

31.25

312.5#

10

6

4

12

14

13

25

24

17

108

146

123

221

255

227

62.5

625#

5

9

11

12

17

11

33

23

20

116

130

102

250

238

246

125

1250#

19

4

5

22

12

8

23

22

26

144

150

124

215

218

240

250

2500#

4

5

5

7

7

6

16

19

14

123

48

58

255

248

217

500

5000#

0

0

0

5

2

3

14

9

10

29

21

47

217

232

249

PC

291

284

284

262

262

489

688

688

684

868

732

818

810

896

1036

The mean number of histidine revertant colonies (Confirmatory test)

Concentration of

TCPD (Distilled Tricyclopentadiene)  (µg/plate)

Reduction in Number of Revertant Colonies in the Absence of Metabolic Activation

TA1537

TA1535

TA98

TA100*

TA102*

NC (DMSO)

NA

NA

NA

NA

NA

3.91

-17.17

-18.39

1.38

-10.17

2.46

7.81

10.34

7.89

-9.44

-8.19

4.24

15.63

27.61

-13.10

-9.44

-3.96

2.19

31.25

3.52

13.18

2.72

-15.82

6.30

62.5

34.54

31.57

28.37

-15.82

-1.37

125

84.49

57.93

48.64

-3.11

4.65

PC

NA

NA

NA

NA

NA

2AA

NA

NA

NA

-10.74

NA

Concentration of

TCPD (Distilled Tricyclopentadiene)  (µg/plate)

Reduction in Number of Revertant Colonies in the Absence of Metabolic Activation (10% v/v S9 mix)

TA1537

TA1535

TA98

TA100

TA102*

NC (DMSO)

NA

NA

NA

NA

NA

15.63

12.50

-18.39

8.45

9.02

-3.74

31.25

16.63

-2.60

7.06

0.00

-5.08

62.5

-4.13

-5.21

-7.01

7.69

-9.72

125

-16.63

-10.50

0.00

-10.87

-0.60

250

41.63

47.36

31.01

39.26

-7.62

500

100.00

73.72

53.53

74.27

-4.34

PC

NA

NA

NA

NA

NA

Conclusions:
It is concluded that TCPD (Distilled Tricyclopentadiene) is non-mutagenic to five strains of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102, when tested under the specified experimental conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

It is concluded that TCPD (Distilled Tricyclopentadiene) is non-mutagenic to five strains of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102, when tested under the specified experimental conditions. Therefore it has not been classified.