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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 July 2018 TO 24 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted on 21st July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Composition:
Propyl 4-hydroxybenzoate: 99,7 %
4-Hydroxy-benzoic acid: 0.1 %
unspecified impurity: 0.2 %
Ethanol: < 200 ppm
Propanol: < 200 ppm

melting point: 98 °C
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Clariant India Limited and BP16102712
- Expiration date of the batch:27.10.2019
- Purity test date:99,7%

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test substance in the solvent/vehicle:Dimethyl sulphoxide

OTHER SPECIFICS:
- CAS No. :94-13-3
- Physical Appearance (with colour) : White powder

Method

Target gene:
Histidine Locus
Species / strain
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:
1 mL of S9 homogenate was mixed with 9 mL co factor
Test concentrations with justification for top dose:
Based on the results of solubility, precipition and initial cytotoxicity test, concentrations of 0.01, 0.03, 0.10, 0.32 and 1 mg/plate were selected for testing in the mutation test by plate incorporation and pre incubation method.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The test item was soluble in dimethyl sulphoxide at 50 mg/mL
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoantracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 30 minutes
- Exposure duration:The plates were incubated at 37±1ºC for 71 hours and 15 minutes for initial cytoyoxicity
Plate incorporation method - Plates were incubated at 37±1°C for 48 hours and 5 minutes.
Preincubation method - Plates were incubated at 37±1°C for 64 hours and 30 minutes.

NUMBER OF REPLICATIONS: Triplicates

Rationale for test conditions:
Not applicable
Evaluation criteria:
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537
Statistics:
Not applicable

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
TA1537,TA102,TA100,TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Insoluble in water
- Precipitation:The test item resulted in minimal precipitation at 5 mg/plate and no precipitation at and up to 4 mg/plate tested concentration


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Vehicle historical control data:
Plate incorporation method:
TA 98 TA100 TA1535 TA1537 TA102 (With S9)
Mean 20.6 101.8 22.2 8.2 260.3
SD 1.6 1.7 1.6 1.1 5.2
Min 18 98 19 6 251
Max 25 106 26 13 272

TA 98 TA100 TA1535 TA1537 TA102 (Without S9)
Mean 20.9 101.7 22.2 8.6 261.8
SD 1.5 2.0 1.8 1.1 5.2
Min 19 94 18 7 254
Max 26 105 27 13 274

Pre incubation method:
TA 98 TA100 TA1535 TA1537 TA102 (With S9)
Mean 20.6 102.3 22.1 8.4 262.3
SD 1.5 1.9 2.0 1.0 4.6
Min 19 99 19 7 256
Max 25 108 28 11 275

TA 98 TA100 TA1535 TA1537 TA102 (Without S9)
Mean 20.8 101.8 22.4 8.4 263.8
SD 1.6 2.0 2.0 1.1 5.5
Min 18 99 20 7 251
Max 24 106 28 11 275

- Positive historical control data:

Plate incorporation method:

TA 98 TA100 TA1535 TA1537 TA102 (With S9)
Mean 412.0 385.5 143.2 118.2 606.2
SD 21.7 20.9 9.6 8.7 27.6
Min 280 306 117 100 510
Max 440 419 292 149 670

TA 98 TA100 TA1535 TA1537 TA102 (Without S9)
Mean 399.7 378.3 145.0 120.2 613.48
SD 36.2 23.9 12.6 8.5 22.6
Min 310 311 112 103 589
Max 428 446 190 158 694

Pre incubation method:
TA 98 TA100 TA1535 TA1537 TA102 (With S9)
Mean 410.1 386.0 144.2 117.1 606.5
SD 18.2 22.0 11.3 6.5 26.7
Min 290 298 103 102 508
Max 428 425 187 128 670

TA 98 TA100 TA1535 TA1537 TA102 (Without S9)
Mean 404.0 379.1 146.5 118.7 616.0
SD 29.0 23.7 14.5 5.5 25.9
Min 314 317 110 105 590
Max 428 450 200 134 699


Any other information on results incl. tables

Historical Data

Time Period: 2009 to 2017

Total No. of studies: 50 studies

Plate Incorporation Method

Metabolic Activation

With Metabolic Activation

 

 

 

 

 

 

 

 

 

Without Metabolic Activation

Tester strain

Salmonella typhimurium

Salmonella typhimurium

TA 98

TA 100

TA 1535

TA 1537

TA 102

TA 98

TA 100

TA 1535

TA 1537

TA 102

Vehicle Control (DMSO)

Mean

20.6

101.8

22.2

8.2

260.3

20.9

101.7

22.2

8.6

261.8

±SD

1.6

1.7

1.6

1.1

5.2

1.5

2.0

1.8

1.1

5.2

Min

18

98

19

6

251

19

94

18

7

254

Max

25

106

26

13

272

26

105

27

13

274

Positive Control

Mean

412.0

385.5

143.2

118.2

606.2

399.7

378.3

145.0

120.2

613.4

±SD

21.7

20.9

9.6

8.7

27.6

36.2

23.9

12.6

8.5

22.6

Min

280

306

117

100

510

310

311

112

103

589

Max

440

419

292

149

670

428

446

190

158

694

 

Preincubation Method

Metabolic Activation

With Metabolic Activation

 

 

 

 

 

 

 

 

 

Without Metabolic Activation

Tester strain

Salmonella typhimurium

Salmonella typhimurium

TA 98

TA 100

TA 1535

TA 1537

TA 102

TA 98

TA 100

TA 1535

TA 1537

TA 102

Vehicle Control (DMSO)

Mean

20.6

102.3

22.1

8.4

262.3

20.8

101.8

22.4

8.4

263.8

±SD

1.5

1.9

2.0

1.0

4.6

1.6

2.0

2.0

1.1

5.5

Min

19

99

19

7

256

18

99

20

7

251

Max

25

108

28

11

275

24

106

28

11

275

Positive Control

Mean

410.1

386.0

144.2

117.1

606.5

404.0

379.1

146.5

118.7

616.0

±SD

18.2

22.0

11.3

6.5

26.7

29.0

23.7

14.5

5.5

25.9

Min

290

298

103

102

508

314

317

110

105

590

Max

428

425

187

128

670

428

450

200

134

699

Min: Minimum no. of colonies, Max: Maximum no. of colonies SD: Standard deviation           

Applicant's summary and conclusion

Conclusions:
The test item, Propyl 4-hydroxybenzoate was evaluated for mutagenicity in Bacterial Reverse Mutation Test using Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 as per the OECD guideline for testing of chemicals No. 471. Based on initial cytotoxicity test the test item was tested at concentrations of 0.01, 0.03, 0.10, 0.32 and 1 mg/plate for mutagenecity in plate incorporation and preincubation method.
In both tests Propyl 4-hydroxybenzoate resulted in no appreciable increase in the number of revertant colonies in all five tester strains and in all tested concenrtrations over the vehicle control,while the positive controls tested simultaneously, resulted in 2.2 to 18.8 fold increasein the number of revertant colonies/plate under identical conditions. Based on the results obtained from the study, it is concluded that the test item, Propyl 4-hydroxybenzoate, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 1 mg/plate under the test conditions.
Executive summary:

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test using Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 according to OECD guideline 471 “Bacterial Reverse Mutation Test”. The test item was found to be soluble in dimethyl sulphoxide at a concentration of 50 mg/mL. The test item resulted in minimal precipitation at 5 mg/plate. Based on these results the highest concentration selected for initial cytotoxicity test was 5 mg/plate. Salmonella typhimurium TA100 tester strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate in the presence and absence of metabolic activation. This treatment resulted in cytotoxicity and those concentrations are graded as 0 (Absent lawn) for 5 mg/plate, 1+ (extremely reduced lawn) for 3 and 4 mg/plate, 2+(moderately reduced lawn) for 2 mg/plate and 3 + (Slightly reduced lawn) for 1 mg/platewhen compared to vehicle control. On the basis of cytotoxicity results, solubility and precipitation 1 mg/plate was considered as the highest test concentration for mutation assay. In mutation assay the test item was tested at the concentrations of 0.01, 0.03, 0.10, 0.32 and 1mg/plate. Two independent trials (trial 1 and 2) were conducted by plate incorporation method and pre incubation method in the presence and absence of metabolic activation system. Vehicle control (dimethyl sulphoxide) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The number of revertant colonies in the positive controls resulted in 2.3 to 18.8 fold increase under identical conditions. Based on the results obtained from the study, it is concluded that the test item, Propyl 4-hydroxybenzoate, is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 1 mg/plate under the test conditions.