Propyl 4-hydroxybenzoate

Genetic toxicity: in vitro

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted on 21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Propyl 4-hydroxybenzoate

Cas Number:

94-13-3

IUPAC Name:

Propyl 4-hydroxybenzoate

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.