Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information


Short description of key information:

- In vitro Mammalian cell genen mutation test using hprt and xprt genes (OECD 476) (1 OECD-conform study according to TG 476): negative

- Ames test: negative (5 studies)
- in vitro Chromosome aberration test: negative without metabolic activation, positive with metabolic activation (2 studies, restricted reliability)
- in vivo Chromosome Aberration study in rats: negative (1 study)
- Dominant Lethal Assay: negative (1 study)

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sep-2018 to Feb-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted on 29 th July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Sodium phenobarbitone and ß-Naphthoflavone induced rat liver homogenate
Test concentrations with justification for top dose:
The test item was found soluble in DMSO at 200 mg/mL. Since the test item showed slight precipitation and no changes in pH up to 2 mg /mL, same concentration was considered as the highest concentration in the initial cytotoxicity test.
There was no excessive cytotoxicity up to 2 mg/mL, therefore 2 mg/mL was selcted as the highest concentration for testing in the gene mutation test.
For concentrations i.e. 0.25, 0.50, 1 and 2 mg/ml were selcted for gene mutation test based on initial cytotoxicity test.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
The derivative of the CHO-K1, CHO AA8 cells were used as the test system as recommended in teh OECD TG 476. CHO AA8 cells, Batch No. 5000062 procured fromAmerican Type Culture Collection (ATCC) was used for the test.

Benozo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation test.

Cytotoxicity was assessed by determining the adjusted cloning efficiency and relative survival in the test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Based on the results obtained, the test item is considerd as non-mutagenic and and up to the concentration of 2 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.
Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells, according to OECD TG 476 " In vitro Mammalian cell genen mutation test using hprt and xprt genes" adopted on 29th july 2016.

The test item was found soluble in DMSO at 200 mg/mL. Slight precipitation was observed at 2 mg/ml. No change in pH and precipitate at and up to 2 mg/mL in culure medium. Based on the result of solubility, pH and precipitation test an initial cytotoxicity test was conducted at concentrations of 0.0625, 0.125, 0.25, 0.5 1 and 2 mg/mL using DMSO as vehicle in the presence and absence of metabolic activation (3 to 6 hours). The result of the initial cytotoxicity test indicated that the test item was not cytotoxic to CHO AA8 cells, as the relative survival of the treated CHO AA8 cellsat the test concentrations up to 2 mg/mL was in a range of 71.91 to 86.21 % when compared with the respective vehicle control, both in the presence and absence of metabolic activation. The gene mutation test was conducted at concentrations of 0.25, 0.5, 1 nd 2 mg/mL in for plates/groups in the presence and absence of metabolic activation (3 to 6 hours) Benozo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls for the gene mutation test. Cytotoxicity was assessed by determining the adjusted cloning efficiency and relative survival in the test.

There was no statistically significant increase in number of mutant colonies at any of concentrations tested when compared with the vehicle control. Positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control.

Based on the results obtained, the test item is considerd as non-mutagenic and and up to the concentration of 2 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1975 to 1980
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Study performance in line with current OECD guideline. Reporting restricted.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Remarks:
performed before GLP guidelines
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
no data
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
other: N-ethyl-N´-nitro-N-nitrosoguanidine, Benzo(a)pyrene
Details on test system and experimental conditions:
Testing procedure according to
B.N. Ames, W.E. Durston, E. Yamazaki, F.D. Lee; Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection, Proc. Natl. Acad. Sci. 70, 2281-2285 (1973)
except a pre-incubation for 20 min. before cell plating.
Evaluation criteria:
The final judgement was done as positve when the numbers of revertants exceeded 200, 100 and 50 for TA100, TA98, TA1537.
Statistics:
no data
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Methylparaben did not cause any mutagenicity in S. thyphimurium TA98, TA100 and TA1537.
Executive summary:

Methylparaben was tested for mutagenicity with the strains TA98, TA100 and TA1537 of Salmonella thyphimurim with and without metabolic activation and did not show any mutagenicity.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1973 to 1978
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Summary
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
no
Remarks:
performed before GLP guidelines
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Metabolic activation system:
no data
Test concentrations with justification for top dose:
no data
Vehicle / solvent:
no data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 7,12-Dimethylbenzanthracene, Benzo[a]pyrene
Details on test system and experimental conditions:
no data
Evaluation criteria:
no data
Statistics:
no data
Species / strain:
S. typhimurium TA 98
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Methylparaben did not cause any mutagenicity in S. thyphimurium TA98 and TA100.
Executive summary:

Methylparaben was tested for mutagenicity with the strains TA98 and TA100 of Salmonella thyphimurium and did not show any mutagenicity.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Date of receiving: 24. Nov. 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic aspects of study design (according to Ames et al., 1975) are in line with the current OECD guideline, but the study is restricted because no repeat experiment was done.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only one concentration tested, negative result not confirmed
Principles of method if other than guideline:
bacterial reverse mutation assay according to Ames
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA1538, TA1537, TA1535, TA100, TA98
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50 µg/plate
Vehicle / solvent:
sterile, double distilled water
Untreated negative controls:
yes
Remarks:
spontaneous mutations
Negative solvent / vehicle controls:
yes
Remarks:
water, ethanol, dimethyl sulfoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, 4-Nitro-o-phenylene diamine, Sodium azide, 9-Aminoacridine
Details on test system and experimental conditions:
Preparation of Methylparaben solution:
Methylparaben was weighed out in a sterile glass vial and enough solvent (water) was added to make a 50 mg/mL solution.

Solvents for positive controls:
- 2-Amino-anthracene: Dimethylsulfoxide
- 4-Nitro-o-phenylene: Dimethylsulfoxide
- 9-Aminoacridine: Ethanol (absolut)
- Sodium azide: double distilled water

Test procedure:
Following the Methylparaben treatment of the individual Salmonella strains according to Ames et al. (1975), the strains were plated on Vogel Bonner Medium E minimal agar plates and incubated for 48 hours at 37°C. The toxicity of the test item was evidenced by either a clearing of the bacterial lawn (the reduction of colony counts below the range of spontaneous revertants) or the appearance of the pinpoint his colonies. In addition, prior to and following Methylparaben exposure, plate counts were done to determine percentage survival of colony-forming units per complete growth medium plates.
Spot tests according to Ames et al. were used. Samonella/microsomal assays were carried out by making post-mitochondrial preparations (S9) from livers of male Sprague-Dawley rats (150-200 g; Charles River Laboratories), which had been induced with Aroclor 1254 (500 mg/kg bw in corn oil) administered in one dose on day 1. Animals were killed on day 6, and liver extracts were prepared according to Ames et al. (1975). The liver homogenate supernatant was stored as 1 mL aliquots at -70°C. The S9 mix was checked for bacterial contamination.
Reversion of all strains by 5 µg/plate of the promutagen 2-Aminoanthracene was included in the assay as a check on the S9 activation system. Methylparaben was spot tested (50 µg/plate) in all strains with and without the prepared S9 mix (50 µL/plate). The plates were incubated for 2 days at 37°C in the dark.
Controls were run in each test and included negative controls, positive controls, and sterility controls. The negative controls were made with and without S9 mix and included plates containing only bacteria and plates containing bacteria plus the solvent used in the test. Positive controls were prepared with known mutagens. Plates were prepared as in the spot tests but with 0.1 mL solution containing 50 µg of a known mutagen being added over the surface of the agar. Sterility control plates were made for each chemical, solvent, top agar, and S9. All control, experimental, and sterility plates were prepared in at least duplicate.
Evaluation criteria:
The test substance was considered mutagenic if there was a significant increase in the number of colonies on the plate compared with both the non-treated and Methylparaben-treated plates.Colonies with a diameter of more than 0.2 mm were counted. Periodically, mutagenic colonies were restreaked on media lacking hisitidine to ensure that they were indeed revertants.
Statistics:
no data
Species / strain:
other: TA1538, TA1537, TA1535, TA100, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Cytotoxicity test conducted, but result not stated
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

AmesMethylparaben (Belvins and Taylor)

 

 

Table 1:Summary of mean number of revertants (mean of at least 6 plates) inSalmonella typhimuriumwith and without metabolic activation (negative and positive controls)

Control

TA 1538

TA 1537

TA1535

TA 100

 

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

Negative:

 

 

 

 

 

 

 

 

0

5±3

5±3

3±3

3±2

11±7

7±4

100±26

70±24

100 µL (Water)

4±2

4±2

6±2

3±1

12±6

7±1

95±19

84±22

100 µL(Ethanol)

6±3

4±3

5±2

4±3

9±6

10±6

88±17

65±5

100 µL (DMSO1)

4±4

3±1

5±1

3±2

6±4

5±1

72±6

70±1

 

 

 

 

 

 

 

 

 

Positive:

 

 

 

 

 

 

 

 

5 µg (2-Aminoanthracene)

7±3

110±45

10±3

100±31

16±10

440±340

101±36

4000±500

50 µg (4-Nitro-o-phenylene diamine)

160±34

 

 

 

 

 

 

 

50 µg (Sodium azide)

 

 

 

 

2500±1200

 

3500±800

 

50 µg (9-Aminoacridine)

 

 

3600±1000

 

 

 

 

 

 

Control

TA 98

 

 

 

 

- S9 mix

+ S9 mix

 

 

 

 

 

 

Negative:

 

 

 

 

 

 

 

 

0

17±5

22±11

 

 

 

 

 

 

100 µL (Water)

14±5

26±9

 

 

 

 

 

 

100 µL (Ethanol)

21±10

37±2

 

 

 

 

 

 

100 µL (DMSO1)

16±3

25±2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive:

 

 

 

 

 

 

 

 

5 µg (2-Aminoanthracene)

11±9

2900±1300

 

 

 

 

 

 

50 µg (4-Nitro-o-phenylene diamine)

4700±900

 

 

 

 

 

 

 

50 µg (Sodium azide)

 

 

 

 

 

 

 

 

50 µg (9-Aminoacridine)

 

 

 

 

 

 

 

 

1         Dimethyl sulfoxide

Table 2: Spot test Results of Methylparaben using the Salmonella/Microsome System

S9 mix

TA1538

TA1537

TA1535

TA100

TA98

 

 

 

 

 

 

-

-

-

-

-

-

+

-

-

-

-

-

(-): negative result of 50 µg of the test item in the spot test 

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Methylparaben did not cause any increase in the number of revertant colonies with any of the tester strains in absence or presence of S9 mix.
Executive summary:

Methylparaben was tested for mutagenicity with the strains TA1538, TA1537, TA1535, TA100 and TA98 of Salmonella thyphimurium.

The mutagenicity study was conducted in the absence and presence of a metabolising system derived from rat liver homogenate. A concentration of 50 µg Methylparaben/plate was used.

Control plates without mutagen but with Water, Ethanol or Dimethylsulfoxide did not show an increase in the number of revertant colonies compared to the spontaneous negative control. All positive control compounds gave the expected increase in the number of revertant colonies.

Methylparaben did not show an increase in the number of revertants in any of the bacterial strains in the presence as well as in the absence of metabolic activation.

Therefore, it can be stated that Methylparaben is not mutagenic in these bacterial test systems either with or without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Date of receiving: 5. Jan. 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic aspects of the studydesign (according to Ames et al., 1975) are in line with the current OECD guideline, but the quality of reporting is restricted .
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only TA1535, TA1537, TA1598 and TA98 tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA98, TA100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The first mutagenesis test was performed using 4 to 6 doses separated by factors of 10, the highest dose being 10 mg/plate. The test was then repeated with 4 to 6 doses separated by approximately half-log intervals, the highest dose being 3 mg or a minimal toxic dose determined in the first experiment.
Vehicle / solvent:
Dimethylsulphoxide
Untreated negative controls:
yes
Remarks:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulphonate, 9-Aminoacridine, 2-Nitrofluorene, Nitrofurantoin, Sodium azide, 2-Fluorenylacetamide, 2-Anthramine
Details on test system and experimental conditions:
Plate incorporation assay was performed as described by Ames et al (please refer to "Any other information on materials and methods incl. tables" below) except the the base agar contained only 0.5% glucose. 100 µL Aroclor 1254-induced rat liver S9 mix was used.
A mutagenesis test was performed using 4 to 6 doses separated by factors of 10, the highest dose being 10 mg/plate. The tests were performed with and without metabolic activation. The test was then repeated with 4 to 6 doses separated by approximately half-log interval.

Positive control compounds:
without metabolic activation
- Ethylmethane sulphonate: TA1535
- 9-Aminoacridine: TA1537
- 2-Nitrofluorene: TA98
- Nitrofurantoin: TA100
- Sodium azide: TA1535, TA100

with metabolic activation
- 2-Fluorenylacetamide: TA98
- 2-Anthramine: TA100, TA1535, TA1537

All plating were performed at least in duplicate at all doses in each experiment. Each culture was checked for sensitivty to crystal violet and, where appropriate, resistence to 10 µg Ampicillin on discs.

The procedures used for preparation of S9 fractions from Aroclor 1254-induced male Sprague-Dawley rats were according to
M.J. Prival, V.D. King and A.T. Sheldon, Envir. Mutagen. 1, 95 (1979)
Evaluation criteria:
no data
Statistics:
no data
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
other: only data shown for Sodium azide and Nitrofurantoin: valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Methylparaben did not show any mutagenic properties in any of the tester strains in absence or presence of metabolic activity.
Executive summary:

Methylparaben was tested for mutagenicity with the strains TA98, TA100, TA1535 and TA1537 of Salmonella thyphimurium.

The mutagenicity study was conducted in the presence and absence of S9 mix. Two independent experiments were conducted (all platings were performed at least in duplicate). The first was performed using 4 to 6 doses, the highest concentration being 10 mg/plate. The test was repeated with 4 to 6 doses, the highest concentration being 3 mg/plate.

Positve and negative controls were performed with Ethylmethane sulphonate, 9 -Aminoacridine, 2 -Nitrofluoren, Nitrofurantoin, Sodium azide, 2 -Fluorenylacetamide and 2 -Anthramine being the positive control compounds. The experimental data shown for Sodium azide and Nitrofurantoin gave the expected increase in the number of revertant colonies.

Methylparaben did not show an increase in the number of revertants in any of the bacterial strains in the presence or absence of metabolic activation.

Therefore, Methylparaben is considered to be not mutagenic in this Ames test either with or without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
accepted: 23. Jan. 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study design (according to Ames et al., 1975) is in line with current OECD guideline, but study reporting is restricted.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 0.033, 0.10, 0.33, 1.00, 3.30, 10 mg/plate
Vehicle / solvent:
Dimethyl sulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Diemthyl sulfoxide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene, Sodium azide, 9-Aminoacridine, Furylfuramide, N-methyl-N´-nitro-N-nitrosoguanidine, 2-Anthramine
Details on test system and experimental conditions:
The standard plate incorporation assay (S. thyphimurium, E. coli) was performed according to Ames el a., 1975. The S9 mix used as an in vitro metabolic activation system contained 10% Aroclor 1254-induced liver S9 from male Sprague-Dawley rats. Methylparaben was dissolved in Dimethyl sulfoxide and tested using doses of 0.033, 0.10, 0.33, 1.00, 3.30 and 10 mg/plate.
All platings were performed in duplicate and all tests were repeated.
Concurrent positive controls were run with each test, both with direct-acting mutagens and with mutagens requiring S9 activation.
Positive control compounds:
without S9 mix
- 2-Nitrofluorene (5 or 10 µg): TA98, TA1538
- Sodium azide (0.5 or 1 µg): TA100, TA1535
- 9-aminoacridine (50 or 100 µg) : TA537
- Furylfuramide or N-methyl-N´-nitro-N-nitrosoguanidine (10 µg): WP2
with S9 mix
- 2-Anthramine (1-10 µg)
Evaluation criteria:
The test results were considered valid only if the positive control compounds induced increases in mutant counts to at least twice background.
Statistics:
no data
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Methylparaben did not show any mutagenic properties in any of the tester strains in absence or presence of metabolic activity.
Executive summary:

Methylparaben was tested for mutagenicity with the S. thyphimurium strains TA98, TA100, TA1535, TA1537, TA1538 and the E. coli strain WP2. The mutagenicity study was conductedd in the presence and absence of S9 mix. Two independent experiments were conducted (all platings were performed in duplicate). Used doses were 0, 0.033, 0.10, 0.33, 1.00, 3.3 and 10 mg/plate.

Positive and negative controls were performed with 2 -Nitrofluorene, Sodium azide, 9 -Aminoacridine, Furylfuramide, N-methyl-N´-nitro-N-nitrosoguanidine, 2 -Anthramine and Dimethyl sulfoxide.

Methylparaben did not show an increase in the number of revertants in any of the bacterial strains in the presence or absence of metabolic activation.

Therefore, Methylparaben is considered to be not mutagenic in this Ames test either with or without metabolic activation.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not stated
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented guideline study.
Qualifier:
according to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Deviations:
no
GLP compliance:
no
Remarks:
study performed before
Type of assay:
rodent dominant lethal assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
10 to 12 week old, male, albino rats obtained from a closed colony (random-bred) were used.
Body weight: 280 to 350 g (400 g in case of high dose group rats)
The animals were fed a commercial 4% fat diet water ad libitum until they were put on experiment. Periodic tests to verify the absence of coliforms, Salmonella and Pseudomonas sp. were performed.
Acclimatisation period: 4 to 11 days
Housing: 5/cage
Identification: Ear punch
Sanitary cages and bedding used, and changed 2 times per week, at which time water containers were cleaned, sanitised and filled. Once a week, cages were repositioned on racks; racks were repositioned within rooms monthly.
Route of administration:
oral: gavage
Vehicle:
0.85% saline
Details on exposure:
Dose levels employed for Methylparaben:
- acute (single dose): 5, 50, 500, 5,000 mg/kg bw
- subacute (5 doses on 5 consecutive days): 5, 50, 500, 5,000 mg/kg bw
Duration of treatment / exposure:
n.a. (gavage study)
Frequency of treatment:
acute: 1 single oral administration
subcaute: once a day for 5 consecutive days (24 hours apart)
Post exposure period:
8 weeks (sequential matings)
Remarks:
Doses / Concentrations:
5
Basis:
actual ingested
mg/kg body weight
Remarks:
Doses / Concentrations:
50
Basis:
actual ingested
mg/kg body weight
Remarks:
Doses / Concentrations:
500
Basis:
actual ingested
mg/kg body weight
Remarks:
Doses / Concentrations:
5000
Basis:
actual ingested
mg/kg body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
0.3 mg/kg bw Triethylene Melamine intraperitoneally
Tissues and cell types examined:
determination of fertility index
Necropsy of the uteri of mated females
- early deaths (deciduomata)
- absorptions
- dead implatations
- total implantations
- Number of Corpora lutea
Details of tissue and slide preparation:
Following treatment, the males were sequentially mated to 2 females per week for 8 weeks (7 weeks in the subacute study). Females were killed using CO2 at 14 days after separating from the male, and at necropsy the uterus was examined for deciduodimata, late fetal deaths and total implantations.
Corpora lutea, early fetal deaths, late fetal deaths and total implantations per uterine horn were recorded.
Evaluation criteria:
Each male was mated with 2 females per week, and this provided for an adequate number of implantations per group per week (200 minimum) for negative controls, even if there was a 4-fold reduction in fertility of implantations.
Statistics:
yes, please refer to "any other information..." below
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
100 and 500 mg/kg: no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxicity:
Test I
1000 mg/kg: 1 of 5 animals dead; reddened stomach lining, lung congested (day 3)
2000 mg/kg: 2 of 5 animals dead, reddened stomach lining, lung congested (day 2)
3000 mg/kg: 4 of 5 animals dead, reddened stomach lining, lung congested (day 1: 3 animals, day 2: 1 animal)
4000 mg/kg: 4 of 5 animals dead, reddened stomach lining, lung congested (day 1: 4 animals)
5000 mg/kg: 10 of 10 animals dead, reddened stomach lining, lung congested (day 1: 10 animals)
=> LD50 2,100 mg/kg (Litchfield-Wilcoxon method)

Test II
A single dose of 5000 mg/kg bw Methylparaben was administered to 10 male rats (average body weight: 262 g). No signs of toxicity or abnormal behavior were observed in the 7-day observation period. No deaths occured. At termination all animals were killed and on necropsy no gross findings were observed.
=> LD50 > 5000 mg/kg
Conclusions:
Interpretation of results (migrated information): negative
Methylparaben is considered to be non-mutagenic in rats in the Dominant Lethal Assy applying dosages up to 5000 mg/kg bw (at single dose or 5 dosages on 5 consecutive days).
Executive summary:

Methylparaben was tested in the dominant lethal assay in rats. The test item was suspended in 0.85% saline and dosed at 5, 50, 500 and 5000 mg/kg bodyweight to male rats (acute: single dose; subacute: 5 doses at 5 consecutive days), upon the results of the previously conducted dose range finding study. According to the test procedure the animals were sequentially mated to 2 females per week for 8 weeks (7 weeks in the subacute study). Females were killed at 14 days after mating and at necropsy the uterus was examined for the number of Corpora lutea, early deaths, late fetal deaths and total implantations.

Triethylene Melamine (TEM) was used as positive control substance and administered intraperitoneally at a dose of 0.3 mg/kg bodyweight. TEM caused significant preimplantation loss and embryo resorption during the first 5 weeks.Saline was used as negative control.

Methylparaben is considered to be non-mutagenic in rats in this dominant lethal assay when using dosages of 5, 50, 500 and 5000 mg/kg bw/d since no dose response or time trend patterns, which would suggest an effect, were observed.

Additional information

Based on the results obtained from the In vitro Mammalian cell genen mutation test using hprt and xprt genes

, tmethyl paraben is considerd as non-mutagenic and and up to the concentration of 2 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

A number of bacterial reverse mutation assays (Ames test) have been performed on Methylparaben covering the strains Salmonella typhimurium TA98, TA100, TA1535, TA1537, and TA1538 and all are consistently negative.

 

In two mammalian cytogenicity studies in which the genotoxic potential of Methylparaben was examined in vitro using Chinese hamster cells for the detection of chromosomal aberrations, negative results were obtained in the absence of metabolic activation. A slight increase in chromosome aberrations was observed in the presence of the S9 mix. However, Methylparaben did not induce chromosome aberrations up to 500 mg/kg bw in an in vivo Mammalian Bone Marrow Chromosome Aberration test performed with rats according to OECD475. Furthermore, Methylparaben is considered to be non-mutagenic in rats in the Dominant-Lethal assay applying dosages up to 5000 mg/kg d (at single dose or 5 dosages on 5 consecutive days).


Short description of key information:

- In vitro Mammalian cell genen mutation test using hprt and xprt genes (OECD 476) (1 OECD-conform study according to TG 476): negative

- Ames test: negative (5 studies)
- in vitro Chromosome aberration test: negative without metabolic activation, positive with metabolic activation (2 studies, restricted reliability)
- in vivo Chromosome Aberration study in rats: negative (1 study)
- Dominant Lethal Assay: negative (1 study)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

It can be concluded from the data available that mutagenicity and genotoxicity are not caused by Methylparaben and therefore classification for this endpoint is not warranted.