Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The submission substance was not mutagenic in a guidline conform Ames test using the five Salmonella typhimurium tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation (S9-mix from induced rat liver).

Likewise, the submission substance was also not mutagenic in a guideline compliant mammalian cell gene (HPRT) mutation assay in V79 Chinese hamster cells and did not induce chromosome aberrations or clastogenic effects in a guideline conform cytogenetic study in V79 cells in vitro with and without metabolic activation. Furthermore in a guidline conform micronucleus test the submission substance was non clastogenic and/or non aneugenic.

Link to relevant study records

Referenceopen allclose all

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
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
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
Target gene:
Histidine Locus
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
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
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


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           

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 August 2018 to 07 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: external sample from China
- 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
Target gene:
Human lymphocytes
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human
- Cell cycle length, doubling time or proliferation index: 44 to 48 hours
- Sex, age and number of blood donors if applicable: male and female donors (21 and 33 years of age)
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Modal number of chromosomes:46±2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640, 5 PERCENT
- Properly maintained: yes
Cytokinesis block (if used):
cytochalasin B
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:
As no cytotoxicity or precipitation observed, the highest test concentration correspond to 2 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test item was found soluble in dimethyl sulphoxide at 200 mg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 to 6 hours and 20 to 22 hours


STAIN (for cytogenetic assays): Acridine orange

NUMBER OF REPLICATIONS: duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Pellet were mixed with 3 mL of freshly prepared 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cells were incubated for 10 minutes at room temperature and later suspension was centrifuged at 2000 rpm for 10 minutes. The procedure was repeated twice by adding 3 mL of cold acetic acid: methanol fixative (1:3) and slides were prepared.
Clean slides were stored in a container with distilled and kept in the refrigerator for 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension was aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation(-S9), treatment/group and slide number. The slides were air dried. Smear was stained using Acridine Orange stain by allowing the stain to retain for 5 minutes

NUMBER OF CELLS EVALUATED: 1000 per replicate


CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Micronucleus in binucleates

DETERMINATION OF CYTOTOXICITY
- Method: Measurement of the relative frequencies of mononucleate, binucleate and multi-nucleate cells in the culture was made to determine cell proliferation and the cytostatic activity
Evaluation criteria:
Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures was determined from each treatment.
The CBPI was calculated for all treated and control cultures as measurement of cell cycle delay.
Slides was evaluated for the presence of Micronucleus in at least 2000 Binucleates (at least 1000 per culture).
Statistics:
Data was analyzed using SPSS software version 22 for differences among vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p≤0.05) and the statistical significance was designated by the superscripts thoughout the study report as stated below.
*: Statistically significant (p≤0.05).
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- pH: no change
- Water solubility: insolublle
- Precipitation: no


RANGE-FINDING/SCREENING STUDIES:

CYTOKINESIS BLOCK (if used) : Cytochalasin B
- Distribution of mono-, bi- and multi-nucleated cells: yes

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 1000 per culture
- Indication whether binucleate or mononucleate where appropriate: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used:CBPI
Remarks on result:
other: .
Remarks:
Results are summerized in the next part

TABLE 1.      SUMMARY OF MICRONUCLEI INCIDENCE WITH METABOLIC ACTIVATION FOR 3 TO 6 HOURS

Treatment

Concentration

mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.62

0.0

2039

3

0.15

2

Propylparaben

0.5

1

1.58

6.45

2099

4

0.19

2

1

1

1.56

9.68

2030

3

0.15

2

2

1

1.54

12.90

2035

5

0.25

2

Cyclophosphamide Monohydrate

10 µg/mL

1

1.61

1.61

2007

26

1.30*

2

TABLE 2.      SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 3 TO 6 HOURS

Treatment

Concentration      mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.63

0.0

2016

3

0.15

2

Propylparaben

0.5

1

1.61

3.17

2015

3

0.15

2

1

1

1.57

9.52

2071

3

0.15

2

2

1

1.54

14.29

2020

2

0.10

2

Colchicine

0.03 µg/mL

1

1.61

3.17

2053

26

1.27*

2

Table 3 SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 20 TO 24 HOURS

Treatment

Concentration      mg/mL

Replicate No.

Average CBPI

 Average % of Cytotoxicity

Total No. of Binucleates

Total No. of Micronucleus

Average Percentage of Micronucleus

Vehicle Control

0

1

1.63

0.0

2031

3

0.15

2

Propylparaben

0.5

1

1.59

6.35

2031

3

0.15

2

1

1

1.54

14.29

2018

3

0.15

2

2

1

1.44

30.16

2060

4

0.19

2

Mitomycin C

0.075 µg/mL

1

1.60

4.76

2077

26

1.25*

2

Conclusions:
Based on the results obtained in this study, it is concluded that the test item Propylparaben is non clastogenic and/or non aneugenic in cultured human lymphocytes at and up to 2 mg/mL both in short term and long term treatment, both in presence and absence of metabolic activation as it showed no evidence of increase in the induction of micronuclei under the test conditions.
Executive summary:

The test item was evaluated for the formation of micronuclei in the cytoplasm of interphase cells according to OECD TG 487  “In vitro Mammalian Cell Micronucleus Test” .Test item was found soluble in dimethyl sulphoxide at 200 mg/mL. Moderate and mild precipitation was observed at 2 and 1 mg/mL respectively. No precipitation was observed in any other concentration tested. No change in pH was observed in any of the concentrations tested. Hence, 2 mg/mL was selected as highest concentration for initial cytotoxicity test and other concentrations tested were 0.125, 0.25, 0.5, 1 and 2 mg/mL using DMSO as a vehicle. The experiment was conducted with and without metabolic activation. The test item was assessed in proliferated lymphocytes in duplicates by exposing for a short term (3 to 6 hours, with and without metabolic activation) and a long term (20 to 24 hours, without metabolic activation).

Cytokinesis was blocked using Cytochalasin B, the cells were harvested and slides were prepared. In order to assess the cytotoxicity of the test item, the Cytokinesis-Block Proliferation Index (CBPI) was calculated for cultures treated with the test item and vehicle control.  

In initial cytotoxicity test, the test item at the concentrations of 0.125, 0.25, 0.50, 1 and 2 mg/mL resulted in a percentage reduction of average Cytokinesis-Block Proliferation Index (CBPI) was in the range of 1.67 to 30.00 at the tested concentrations in both short term and long term treatment. The percentage reduction of average Cytokinesis-Block Proliferation Index (CBPI) was not greater than 45±5% at 2 mg/mL. Hence 2 mg/mL was selected as highest concentration for the micronucleus test. Other concentrations tested were 0.5 and 1 mg/mL.

In micronucleus test, the test item was tested at the concentrations of 0.5, 1 and 2 mg/mL for short term in presence and absence of metabolic activation and long term treatment in the absence of metabolic activation system. The test item did not induced cytotoxicity up to 2 mg/mL when compared to vehicle control. No statistical significant increase in the percentage of micronuclei in binucleated cells observed in any of the tested concentration when compared to the vehicle control. The positive controls resulted increase in the micronuclei frequency with statistical significance at 95% level of confidence (p<0.05) under identical conditions, when compared with the vehicle control. This demonstrated the sensitivity of test system towards positive control and confirmed that the test conditions were adequate and within the range of historical control.

Conclusion

Based on the results obtained, it is concluded that the test item Propylparaben is non clastogenic and/or non aneugenic in cultured human lymphocytes at and up to 2 mg/mL both in short term and long term treatment, both in presence and absence of metabolic activation as it showed no evidence of increase in the induction of micronuclei under the test conditions.

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
Study period:
2012-02-16 to 2012-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I / without metabolic activation / 4 hours treatment:
7.0, 14.0, 28.0, 56.0, 93.0, 112.0 µg/mL

Experiment I / with metabolic activation / 4 hours treatment:
28.0, 56.0, 112.0, 224.0, 336.0, 448.0 µg/mL

Experiment II / without metabolic activation / 24 hours treatement:
14.0, 28.0, 56.0, 112.0, 168.0, 224.0 µg/mL

Experiment II / with metabolic activation / 4 hours treatment:
28.0, 56.0, 112.0, 224.0, 336.0, 448.0 µg/mL

In experiment I with and without metabolic activation and in experiment II with metabolic activation the cultures at the maximum concentration were not continued due to exceed-ingly severe cytotoxicity. In the second experiment without metabolic activation the cultures at the two highest concentrations were not continued for the same reason.
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the corresponding solvent control data.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.35 measured in the solvent control versus pH 7.34 measured at 1800 µg test item/mL
- Effects of osmolality: Not increased (383 in the solvent control versus 362 measured at 1800 µg test item/mL
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation: In the main experiments I and II no precipitation occurred.

- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
In the range finding pre-experiment test item concentrations between 14.1 and 1800 µg/mL (≈ 10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Relevant cytotoxic effects indicated by a relative suspension growth below 50% were noted at 112.5 µg/mL and above without metabolic activation following 4 hours treatment. In the presence of metabolic activation cytotoxic effects were observed at 450 µg/mL and above. After 24 hours treatment the cell growth was completely inhibited at 225 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treat-ment period prior to removal to the test item. Precipitation occurred at 900 µg/mL in the pres-ence and absence of metabolic activation following 4 and 24 hours treatment.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were generally spaced by a factor of 2. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Summary Table
  relative relative relative mutant   relative relative relative mutant  
conc. S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
µg/mL mix efficiency I density efficiency II 106cells factor efficiency I density efficiency II 106cells factor
      % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12
Experiment I / 4 h treatment     culture I          culture II
Solvent control with DMSO - 100.0 100.0 100.0 10.1 1.0 100.0 100.0 100.0 20.7 1.0
Positive control (EMS) 150.0 - 99.2 90.3 80.1 145.3 14.4 95.1 101.2 94.4 179.5 8.7
Test item 7.0 - 108.9 107.0 106.0 9.5 0.9 98.8 87.3 107.9 24.0 1.2
Test item 14.0 - 100.2 94.0 113.4 11.5 1.1 102.5 86.5 120.5 6.2 0.3
Test item 28.0 - 101.7 108.1 96.6 27.3 2.7 97.5 83.4 114.1 10.5 0.5
Test item 56.0 - 77.9 82.9 113.9 6.3 0.6 74.0 71.6 106.8 2.5 0.1
Test item 93.0 - 18.1 26.7 116.6 6.0 0.6 20.4 22.3 101.8 2.2 0.1
Test item 112.0 - 0.0 culture was not continued# 0.0 culture was not continued#
Solvent control with DMSO + 100.0 100.0 100.0 14.0 1.0 100.0 100.0 100.0 11.8 1.0
Positive control (DMBA) 1.1 + 24.1 93.3 87.7 1105.5 79.2 25.7 74.5 85.4 751.9 63.6
Test item 28.0 + 82.5 133.1 90.6 12.4 0.9 89.5 118.4 107.2 9.3 0.8
Test item 56.0 + 95.2 125.9 98.5 14.2 1.0 100.8 103.9 106.9 11.2 0.9
Test item 112.0 + 86.9 130.6 95.8 13.2 0.9 94.4 103.4 94.8 7.3 0.6
Test item 224.0 + 79.3 118.9 100.8 4.9 0.4 97.1 90.8 93.4 4.2 0.4
Test item 336.0 + 14.3 117.8 95.7 7.8 0.6 13.0 95.1 100.7 6.8 0.6
Test item 448.0 + 0.2 culture was not continued# 0.3 culture was not continued#
Experiment II / 24 h treatment     culture I          culture II
Solvent control with DMSO - 100.0 100.0 100.0 15.8 1.0 100.0 100.0 100.0 9.5 1.0
Positive control (EMS) 150.0 - 107.9 86.8 89.2 315.0 19.9 98.1 87.3 85.6 309.2 32.5
Test item 14.0 - 88.3 89.9 102.5 15.3 1.0 90.8 107.2 95.5 16.6 1.7
Test item 28.0 - 86.8 78.0 98.5 0.0 0.0 98.2 90.5 102.9 13.7 1.4
Test item 56.0 - 90.7 77.5 92.9 9.0 0.6 78.8 76.1 91.9 16.8 1.8
Test item 112.0 - 75.0 51.9 88.2 18.1 1.1 67.1 50.4 93.7 21.3 2.2
Test item 168.0 - 0.0 culture was not continued# 0.0 culture was not continued#
Test item 224.0 - 0.0 culture was not continued# 0.0 culture was not continued#
Experiment II / 4 h treatment        
Solvent control with DMSO + 100.0 100.0 100.0 18.5 1.0 100.0 100.0 100.0 22.0 1.0
Positive control (DMBA) 1.1 + 29.6 65.4 68.4 987.0 53.4 39.5 56.9 74.3 1056.5 48.1
Test item 28.0 + 102.3 115.9 89.9 6.5 0.4 92.2 92.0 75.5 16.7 0.8
Test item 56.0 + 103.2 115.5 78.1 15.6 0.8 101.7 101.3 121.6 10.9 0.5
Test item 112.0 + 101.8 126.2 90.8 23.4 1.3 98.2 96.8 127.8 11.0 0.5
Test item 224.0 + 100.5 103.0 99.5 17.5 0.9 96.8 91.0 93.5 14.8 0.7
Test item 336.0 + 24.6 66.3 97.8 21.7 1.2 17.3 35.9 99.1 3.2 0.1
Test item 448.0 + 0.0 culture was not continued# 0.0 culture was not continued#

# culture was not continued due to exceedingly severe cytotoxic effects

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, NIPASOL M (= Propylparaben) is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The experimental part without metabolic activation was prematurely terminated due to microbial contamination. This part of the experiment was repeated as experiment IA. The data of experiment IA are reported as experiment I without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

Relevant cytotoxic effects defined as a reduction of the relative cloning efficiency I and/or relative cell density to values below 50% in both parallel cultures were noted in the first experiment at 336.0 µg/mL with and at 93.0 µg/mL without metabolic activation. In the second experiment cytotoxic effects as described above occurred at 336.0 µg/mL with metabolic activation. In the experimental part of the second experiment without metabolic activation a very steep cytotoxic gradient was observed. Borderline cytotoxicity was noted at 112.0 µg/mL (relative cell density of 51.9 and 50.4%). Evaluation of any data on mutagenicity was impossible at the next higher concentration of 168.0 µg/mL as exceedingly severe cytotoxic effects completely inhibited the cell growth. The recommended cytotoxic range of approximately 10- 20% relative cloning efficiency I or relative cell density

was covered with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency remained within the historical range of solvent controls. The induction factor did not reach or exceed the threshold of 3 time the mutation frequency of the corresponding solvent control.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the first culture of the first experiment with metabolic activation. This trend however, was judged as irrelevant since it actually was reciprocal, going down versus increasing concentrations.In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.5 up to 22.0 mutants per 106 cells; the range of the groups treated with the test item was from 0.0 up to 27.3 mutant colonies per 106 cells. EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information


Short description of key information:
In vitro Bacterial reverse mutation assay, Ames test: negative
In vitro Mammalian Cell Gene Mutation Test, HPRT test: negative

In vitro mammalian micronucleus Test: negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data from three independent mutagenicity assays, a respective mutagenic potential of the test item can most probably be excluded. Thus, the submission substance do not have to be classified for mutagenicity in accordance with the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) as well as in the EU Classification, Labellling and Packaging Regulation (1272/2008/EC).