Sodium 4-(methoxycarbonyl)phenolate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-14 to 2012-01-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

His

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:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Experiment I (plate incorporation test):
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II (pre-incubation test):
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: A.dest.

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 111102, 111125, 111201, 111219

Negative solvent / vehicle controls:

yes

Remarks:

A. dest., BSL Lot No. 111102, 111125, 111201, 111219

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates per experiment were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxic effects of the test item were noted in all tester strains except tester strain TA 98 in experiment I and in all tester strains evaluated in experiment II.

In experiment I toxic effects of the test item were observed in tester strains TA 100, TA 1535 and TA 1537 at a concentration of 5000 µg/plate (without metabolic activation). In tester strain TA 102 toxic effects of the test item were noted at a concentration of 5000 µg/plate (with and without metabolic activation).

In experiment II toxic effects of the test item were noted in tester strains TA 98 and TA 102 at concentrations of 2500 µg/plate and higher (with and without metabolic activation). In tester strains TA 100, TA 1535 and TA 1537 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.5 found in experiment II in tester strain TA 1535 at a concentration of 100 µg/plate (with metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship.

Table 1. Test results of experiment 1 (plate incorporation test).

Bacterial Reverse Mutation Assay, mean revertants colonies/plate ± standard deviation (mutation factor)
EXPERIMENT 1 (plate incorporation test)
S9-Mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Aqua dest.	18 ± 3.5 (1.0)	106 ± 7.1 (1.0)	8 ± 4.2 (1.0)	6 ± 0.6 (1.0)	189 ± 13.5 (1.0)
31.6	13± 3.5 (0.7)	106± 9.5 (1.0)	11 ± 2.0 (1.3)	7 ± 5.1 (1.1)	231 ± 22.1 (1.2)
100	21 ± 2.5 (1.1)	102 ± 7.1 (1.0)	8 ± 0.6 (1.0)	6 ± 0.6 (0.9)	206 ± 5.1 (1.1)
316	16 ± 4.7 (0.9)	103 ± 7.2 (1.0)	11 ± 3.2 (1.4)	6 ± 2.0 (0.9)	189 ± 14.2 (1.0)
1000	19 ± 2.1 (1.1)	105 ± 8.2 (1.0)	7 ± 5.0 (0.9)	9 ± 1.5 (1.5)	162 ± 33.5 (0.9)
2500	16 ± 2.6 (0.9)	76 ± 7.5 (0.7)	10 ± 3.1 (1.2)	10 ± 2.9 (1.6)	123 ± 21.2 (0.7)
5000	12 ± 2.6 (0.7)	32 ± 10.1 B (0.3)	2 ± 1.5 (0.2)	3 ± 3.5 (0.5)	50 ± 4.2 (0.3)
4-NOPD	322 ± 32.3 (17.5)	---	---	86 ± 9.5 (13.5)	---
NaN3	---	793 ± 200.3 (7.5)	838 ± 118.5 (100.6)	---	---
MMS	---	---	---	---	1387 ± 171.7 (7.3)
S9-Mix	With
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Aqua dest.	28± 2.3 (1.0)	114± 1.2 (1.0)	10± 1.5 (1.0)	6± 1.5 (1.0)	282± 3.8 (1.0)
31.6	24± 3.5 (0.9)	122± 11.6 (1.1)	8 ± 0 (0.8)	5 ± 1.5 (0.9)	290 ± 7.0 (1.0)
100	21 ± 1.5 (0.8)	110 ± 3.2 (1.0)	6 ± 0.6 (0.7)	4 ± 0.6 (0.8)	285 ± 5.9 (1.0)
316	28 ± 1.2 (1.0)	103 ± 5.5 (0.9)	8 ± 0.6 (0.8)	8 ± 3.2 (1.5)	244 ± 18.0 (0.9)
1000	22 ± 2.5 (0.8)	117 ± 19.3 (1.0)	6 ± 1.5 (0.6)	6 ± 1.4 (1.1)	233 ± 28.4 (0.8)
2500	24 ± 4.2 (0.9)	103 ± 15.9 (0.9)	6 ± 1.7 (0.6)	10 ± 2.5 (1.7)	216 ± 15.3 (0.8)
5000	19 ± 5.0 (0.7)	73 ± 4.0 (0.6)	6 ± 3.2 (0.7)	11 ± 3.6 (1.9)	131 ± 25.7 (0.5)
2-AA	1909 ± 64.1 (69.0)	2180 ± 178.4 (19.1)	161 ± 21.0 (16.7)	208 ± 74.4 (36.6)	595 ± 9.6 (2.1)
B = background lawn reduced 4-NOPD = 4-nitro-o-phenylene-diamine NaN3= sodium azide MMS = methylmethanesulfonate 2-AA = 2-Aminoanthracene

Table 2. Test results of experiment 2 (pre-incubation test).

Bacterial Reverse Mutation Assay, mean revertants colonies/plate ± standard deviation (mutation factor)
EXPERIMENT 1 (plate incorporation test)
S9-Mix	Without
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Aqua dest.	18 ± 9.3 (1.0)	114 ± 12.5 (1.0)	12 ± 4.9 (1.0)	5 ± 2.3 (1.0)	205 ± 12.9 (1.0)
10.0	22 ± 5.1 (1.2)	114 ± 9.5 (1.0)	10 ± 3.2 (0.8)	4 ± 0.6 (0.7)	230 ± 12.1 (1.1)
31.6	21 ± 7.9 (1.2)	126 ± 23.5 (1.1)	16 ± 4.2 (1.3)	7 ± 2.6 (1.3)	240 ± 16.1 (1.2)
100	22 ± 5.5 (1.3)	120 ± 21.6 (1.1)	10 ± 1.2 (0.9)	6 ± 0 (1.1)	217 ± 6.8 (1.1)
316	23 ± 3.6 (1.3)	112 ± 15.9 (1.0)	10 ± 3.5 (0.9)	7 ± 4.4 (1.3)	202 ± 7.2 (1.0)
1000	23 ± 3.6 (1.3)	109 ± 1.5 B (1.0)	6 ± 4.8 (0.5)	7 ± 1.7 B (1.3)	175 ± 11.3 (0.9)
2500	7 ± 6.4 B (0.4)	0 ± 0 B (0)	1 ± 0.6 B (0.1)	0 ± 0 B (0)	37 ± 38.2 B (0.2)
5000	3 ± 2.6 B (0.2)	0 ± 0 B (0)	0 ± 0 B (0)	0 ± 0 N (0)	0 ± 0 B (0)
4 -NOPD	1111 ± 127.2 (62.9)	---	---	117 ± 25.4 (21.9)	---
NaN3	---	903 ± 61.7 (7.9)	753 ± 50.0 (64.5)	---	---
MMS	---	---	---	---	1184 ± 57.2 (5.8)
S9-Mix	With
Test item (µg/plate)	TA 98	TA 100	TA 1535	TA 1537	TA 102
Aqua dest.	27 ± 1.5 (1.0)	120 ± 6.6 (1.0)	12 ± 6.5 (1.0)	8 ± 1.0 (1.0)	326 ± 15.2 (1.0)
10.0	27 ± 4.4 (1.0)	113 ± 10.7 (0.9)	11 ± 5.0 (0.9)	6 ± 2.0 (0.8)	295 ± 42.1 (0.9)
31.6	28 ± 1.5 (1.0)	124 ± 6.0 (1.0)	11 ± 5.7 (0.9)	8 ± 2.5 (1.0)	324 ± 21.8 (1.0)
100	24 ± 3.8 (0.9)	132 ± 13.7 (1.1)	7 ± 1.5 (0.5)	7 ± 1.0 (0.9)	249 ± 11.3 (0.8)
316	29 ± 9.5 (1.1)	135 ± 4.6 (1.1)	8 ± 1.0 (0.6)	9 ± 2.5 (1.1)	268 ± 22.9 (0.8)
1000	33 ± 6.7 (1.2)	147 ± 16.8 (1.2)	10 ± 2.5 (0.8)	8 ± 1.0 (1.0)	277 ± 9.8 (0.8)
2500	26 ± 1.2 B (1.0)	36 ± 10.8 B (0.3)	9 ± 0.6 B (0.7)	7 ± 5.5 B (0.9)	158 ± 16.1 (0.5)
5000	0 ± 0 N (0)	0 ± 0 B (0)	0 ± 0 N (0)	0 ± 0 N (0)	8 ± 3.5 B (0)
2-AA	2027 ± 277.7 (74.1)	1466 ± 326.5 (12.2)	112 ± 23.3 (9.1)	170 ± 26.3 (21.3)	659 ± 31.5 (2.0)
B = background lawn reduced N = no background lawn 4-NOPD = 4-nitro-o-phenylene-diamine NaN3= sodium azide MMS = methylmethanesulfonate 2-AA = 2-Aminoanthracene

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Sodium methyl-4-hydroxybenzoate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Sodium methyl-4-hydroxybenzoate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to Sodium methyl-4-hydroxybenzoate at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I) and 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II).

Sodium methyl-4-hydroxybenzoate was tested up to the limit concentration of 5000 µg/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Reference 2

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:

2012-01-03 to 2012-05-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from livers of male Wistar rats treated with Phenobarbital and β-Naphthoflavone.

Test concentrations with justification for top dose:

Pre-experiment for experiment I, short term exposure (with and without metabolic activation):
10, 25, 50, 100, 250, 500, 750, 1000, 2500, 5000 µg/mL

Experiment I
without metabolic activation:
50, 100, 200, 400, 600, 700, 900, 950, 1000 and 1100 µg/mL
and with metabolic activation:
100, 150, 200, 300, 400, 450, 600 and 700 µg/mL

Experiment II
without metabolic activation:
5, 10, 25, 50, 100, 200, 400 and 600 µg/mL
and with metabolic activation:
130, 160, 190, 220, 250, 280, 310, 340, 400 and 500 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: The test item was dissolved in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: not required, cell culture medium is used as vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Migrated to IUCLID6: 300 µg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: not required, cell culture medium is used as vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Migrated to IUCLID6: 1.0 and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure in Experiment I with and without metabolic activation and in Experiment II with metabolic activation), 20 h (long-term exposure in Experiment II without metabolic activation)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥ 900 μg/mL; experiment I with S9: ≥ 150 μg/mL; Experiment II without S9: ≥ 200 μg/mL; Experiment II with S9:≥ 220 μg/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (DMSO)	88	100	24.57	---
0 (DMSO)	87		20.06
100	93	86.8	46.49	2.08
150	8	68.3	38.32	1.72
200	87	38.0	31.70	1.42
300	96	24.0	17.71	0.79
400	79	16.0	24.20	1.08
450	88	11.9	40.34	1.81
600	82	11.3	53.66	2.40
650	74	10.8	41.89	1.88
DMBA (1.0)	77	111.0	583.12	26.13
DMBA (1.5)	66	118.4	866.41	38.83

DMBA 7,12-Dimethylbenz(a)anthracene

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (DMSO)	72	100	13.89	---
0 (DMSO)	74		20.88
50	73	103.3	24.05	1.38
100	71	98.3	24.03	1.38
200	71	69.7	16.96	0.98
400	70	71.4	23.49	1.35
600	73	70.8	17.06	0.98
700	77	76.1	12.38	0.71
900	61	55.6	39.51	2.27
950	55	36.0	21.92	1.26
1000	57	29.0	21.15	1.22
1100	52	10.4	25.12	1.45
EMS (300)	68	100.0	217.58	12.52
EMS (300)	66	114.0	202.28	11.64

EMS  Ethylmethanesulphonate

Table 3: Experiment II - 4 h Exposure - With Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (DMSO)	63	100	25.40	---
0 (DMSO)	70		20.79
130	61	96.2	52.46	2.27
160	73	83.8	26.21	1.13
190	69	76.3	41.16	1.78
220	64	46.0	28.35	1.23
250	72	36.2	44.60	1.93
280	66	34.9	28.14	1.22
310	67	32.2	26.32	1.14
340	69	26.0	18.18	0.79
400	57	26.2	25.66	1.11
500	71	18.2	17.54	0.76
DMBA (1.0)	64	101.7	219.53	9.51
DMBA (1.5)	72	107.2	481.25	20.84

DMBA 7,12-Dimethylbenz(a)anthracene

Table 4: Experiment II - 20 h exposure - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor
0 (DMSO)	67	100	23.05	---
0 (DMSO)	63		29.25
5	61	100.0	30.20	1.16
10	69	101.8	18.98	0.73
25	63	101.2	42.40	1.62
50	67	89.9	19.48	0.74
100	59	81.0	46.41	1.77
200	69	57.9	31.27	1.20
400	59	34.9	25.42	0.97
600	63	14.0	21.60	0.83
EMS (300)	65	73.2	596.93	22.83
EMS (300)	60	69.0	660.58	25.26

EMS Ethylmethanesulphonate

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Sodium methyl-4-hydroxybenzoate is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus),V79 cells cultured in vitro were exposed to Sodium methyl-4 -hydroxybenzoate dissolved in cell culture medium at concentrations of

Experiment I

without metabolic activation:

50, 100, 200, 400, 600, 700, 900, 950, 1000 and 1100 µg/mL

and with metabolic activation:

100, 150, 200, 300, 400, 450, 600 and 700 µg/mL

Experiment II

without metabolic activation:

5, 10, 25, 50, 100, 200, 400 and 600 µg/mL

and with metabolic activation:

130, 160, 190, 220, 250, 280, 310, 340, 400 and 500 µg/mL

Sodium methyl-4 -hydroxybenzoate

was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 10.4% for the highest concentration (1100 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 700 µg/mL with a relative growth of 10.8%. In experiment II without metabolic activation the relative growth was 14.0% for the highest concentration (600 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 500 µg/mL with a relative growth of 18.2%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 2.27 was found at a concentration of 900 µg/mL with a relative growth of 55.6%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.40 was found at a concentration of 600 µg/mL with a relative growth of 11.3%.
In experiment II without metabolic activation the highest mutation rate (compared to the negative controls values) of 1.77 was found at a concentration of 100 µg/mL with a relative growth of 81.0%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.27 was found at a concentration of 130 µg/mL with a relative growth of 96.2%.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive responseof induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 forin vitromutagenicity (mammalian forward gene mutation) data.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

See attached read-across justification according to RAAF (chapter 13)

Reason / purpose for cross-reference:

read-across: supporting information

Reference 2

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study, tested with the source substance Methylparaben. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

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 or test system 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

Key result

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.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well documented guideline conform study with the exception that only male animals are tested.

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

only male rats tested

GLP compliance:

no

Remarks:

study performed before GLP guidelines

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system 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
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:

Dosage levels employed for Methylparaben:
- 5 mg/kg bw
- 50 mg/kg bw
- 500 mg/kg bw

for group sizes see tables below ("Examination: any other information on methods")

Exposure route: gavage

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:

6, 24 and 48 hours

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

No. of animals per sex per dose:

please refer to tables 1 and 2 below ("Examination: any other information on methods")

Control animals:

yes, concurrent vehicle

Positive control(s):

0.3 mg/kg bw Triethylene Melamine intraperitoneally

Tissues and cell types examined:

Bone marrow cells:
50 metaphase spreads were scored per animal. Mitotic indices were obtained by counting at least 500 cells in duplicate and the ratio of the number of cells in mitosis/the number of cells observed was expressed as the mitotic index.

Details of tissue and slide preparation:

4 hours after the last administration and 2 hours prior to killing, each animal was given 4 mg/kg bw Colcemid intraperitoneally. Animals were killed by using CO2 and the adhering muscle and epiphysis of one femur were removed. The marrow "plug" was removed with a tuberculin syringe and an 18 gauge needle, aspirated into 5 mL of Hanks´balanced salt solution (BSS) in a test tube and capped. The specimens were centrifuged at 1,500 rpm for 5 min, decanted and 2 mL of hypotonic 0.5% KCl solution was added with gentle agitation to resuspend the cells. The specimens were placed in a 37°C water bath for 20 min in order to swell the cells. Following centrifugation for 5 min at 1,500 rpm, the supernatant was decanted and 2 mL of fixative (3:1 absolute Ethanol:glacial acetic acid) was added. The cells were resuspended in the fixative with gentle agitation, capped and placed at 4°C for 30 min. The specimens were again centrifuged, decanted, 2 mL of prepared fixative was added and the cells were resuspended and placed at 4°C overnight.
The following day the specimens were again centrifuged, decanted and 0.3-0.6 mL of freshly prepared fixative was added to obtain a suitable density. The cells were resuspended and 2-3 drops of the suspension were allowed to drop onto a clean, dry slide. The slides were dried at roomtemparature overnight. Duplicate slides were prepared. The slides were stained using 5% Giemsa solution for 20 min, rinsed in acetone, 1:1 acetone:xylene, and placed in fresh xylene for 30 min. These specismens were scanned with 10x and 24x objectives and suitable metaphase spreads that were countable were examined using 40x, 63x or 100x oil immersion flatfield apochromatic objectives. Oculars were either 12x or 16x widefield periplanatics and the tube magnification either 1x or 1.25x.

Evaluation criteria:

The chromosomes of each cell were counted and only diploid cells were analysed. They were scored for chromatid gaps and breaks, chromosome gaps and breaks, reunions, cells with greater than 10 aberrations, polyploidy, pulverisation, and any other chromosomal aberrations which were observed.

Statistics:

no data

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

Chromosome Aberration Study Results:
Methylparaben did not produce detectable significant aberration of the bone marrow metaphase chromosomes of rats when administered orally at 5, 50 and 500 mg/kg.

Table 3: Metaphase summary sheet (acute study; single application)

Dosage (mg/kg bw)	Time (h)	No. of cells	Mitotic index^3(%)	% Cells with Breaks	% Cells with Reunion	% Cells other Aberrations^4	% Cells other Aberrations^5
Saline	6	150	14	1	0	0	1
	24	150	9	2	0	0	2
	48	150	12	1	0	0	1
5^1	6	250	10	2	0	0	2
	24	250	10	0	0	0	0
	48	250	6	0	0	0	0
50^1	6	250	12	1	0	0	1
	24	250	8	2	0	0	2
	48	250	7	3	0	0	3
500^1	6	250	9	3	0	0	3
	24	250	9	3	0	0	3
	48	250	6	3	0	0	3
TEM^2: 0.3	48	250	3	31	12	6(a)	44

¹       Methylparaben

²       Triethylene Melamine

³       Percent of cells in mitosis: 500 cells observed/animal

⁴       Cells that have polyploidy, pulverization, fragments or greater than 10 aberrations (a)

⁵       Duplicate aberrations in a single cell will cause this to be less than a summation of the % aberration seen.

Table 4: Metaphase summary sheet (subacute study; 5 consecutive applications, each 24 hours apart)

Dosage (mg/kg bw)	No. of cells	Mitotic index^1(%)	% Cells with Breaks	% Cells with Reunion	% Cells other Aberrations^2	% Cells other Aberrations
Saline	150	8	2	0	0	2
5	250	6	1	0	0	1
50	250	5	2	0	0	2
500	250	9	4	0	0	4

¹       Percent of cells in mitosis: 500 cells observed/animal

²       Cells that have polyploidy, pulverization, fragments or greater than 10 aberrations

Conclusions:

Interpretation of results (migrated information): negative
Oral administration of Methylparaben did not lead to a substantial increase of chromosomal aberrations and therefore, was not mutagenic in the in vivo chromosome aberration study in rats.

Executive summary:

Methylparaben was tested in the chromosome aberration test in rats. The test item was suspended in 0.85% saline and dosed at 5, 50 and 500 mg/kg bodyweight to male rats, upon the results of the previously conducted dose range finding study. According to the test procedure the animals were killed 6, 24 and 48 hours after administration.

Triethylene Melamine was used as positive control substance and administered intraperitoneally at a dose of 0.3 mg/kg bodyweight.

The chromosomal abnormalities observed in the positive controls were significantly higher than the solvent control or Methylparaben. Methylparaben did not produce a detectable significant aberration of the bone marrow metaphase chromosomes of rats at the dosages employed in this study.

Reference 4

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification according to RAAF (chapter 13)

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Based on source substance methyl 4-hydroxybenzoate

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The in vitro genetic toxicity of sodium methyl 4 -hydroxybenzoate was investigated in a bacterial reverse mutation assay (Ames test) according to OECD 471 and GLP (Donath, 2012). The test was conducted with S. typhimurium strains TA 1535, TA 1537, TA 98, TA100 and TA 102 at concentrations up to 5000 µg/plate with and without metabolic activation. No reversion was noticed in any of the strains tested. Cytotoxic effects were observed in both experiments. In the first experiment, cytotoxicity was observed in the highest dose group in tester strains TA 100, TA 1535 and TA 1537 without metabolic activation and in TA 102 with and without metabolic activation. In the second experiment, cytotoxicity was noted in all strains treated with and without metabolic activation at concentrations of 1000 µg/plate and higher. Precipitation of the test substance was not observed.

 

The induction of gene mutations by sodium methyl 4 -hydroxybenzoate was assessed in a HPRT test according to OECD 476 and GLP (Wallner, 2012). The test was conducted in V79 cells with metabolic activation at concentrations up to 700 µg/mL and without metabolic activation at concentrations up to 1100 µg/mL. No genotoxicity was observed up to the highest dose tested. Cytotoxic effects were observed in both experiments conducted at concentrations of 200 µg/mL and higher without metabolic activation and at concentrations of 150 µg/mL and higher with metabolic activation. The used positive controls were valid.  

There are no data available on in vivo genetic toxicity for sodium methyl 4 -hydroxybenzoate. However, there are reliable data on methylparaben which is considered suitable for read-across using the analogue approach.

Sodium methylparaben (sodium methyl 4 -hydroxybenzoate) is the sodium salt of methylparaben (methyl 4-hydroxybenzoate). The substance is highly water soluble (418 g/L), dissociates in aqueous solutions and is hydrolysed to sodium hydroxide and the source substance methylparaben. Based on the assumption that upon oral and dermal administration, sodium methylparaben dissociates and is hydrolysed to methylparaben and sodium hydroxide, it was predicted that after exposure the primary effect is local irritation/corrosion at the site of contact due to sodium hydroxide.

Target and source substance are of low acute toxicity. No systemic or local effects were found up to single doses of 5000 mg/kg bw for sodium methylparaben and 2100 mg/kg bw for methylparaben. A dermal absorption of 80% was calculated for sodium methylparaben using QSAR and the available physico-chemical properties. For methylparaben, the dermal absorption was estimated in an in vitro test to be 84.7% in human skin (Fasano, 2004). Therefore, it can reasonably be deduced that no higher amounts than tested in the acute oral toxicity study will be systemically available via the intact skin barrier. Inhalation is of no concern for the target as well as the source substance because of the low vapour pressure.The results of several repeated dose toxicity studies indicate a low toxicological concern for methylparaben. Methylparaben caused no systemic toxicity and no effects on fertility or developmental toxicity (NOAEL > 1000 mg/kg be per day). Moreover, toxicokinetic data have shown that methylparaben is completely absorbed after oral and dermal administration, hydrolysed, conjugated and rapidly excreted in urine. Thus, there is no evidence of accumulation.

As bioavailability and metabolism and therefore mammalian toxicity of sodium methyl 4 -hydroxybenzoate were considered to be comparable to that of methylparaben, the assessment of in vivo genetic toxicity based on analogue approach can be considered as justified. 

The read across substance Methylparaben was tested in a dominant-lethal test in rats according to OECD 478 (Litton Bionetics Inc, 1974). Methylparaben was applied to male rats in doses up to 5000 mg/kg bw/d by oral gavage in a single treatment and daily on 5 consecutive days. During the post exposure period (8 week for the single treatment group and 7 weeks for the subacute group), the male rats were sequentially mated to 2 females per week. Two weeks after mating, the females were sacrificed and at necropsy the uteri were examined for corpora lutea, early deaths, late fetal deaths and number of total implantations.

No dose response relationship or time trend pattern was observed across the groups. Therefore, methylparaben was considered to be non-mutagenic in rats in the Dominant-Lethal assay applying dosages up to 5000 mg/kg bw/d.

 

In an in vivo Mammalian Bone Marrow Chromosome Aberration test performed in male rats according to OECD 475, Methylparaben did not induce chromosome aberrations up to 500 mg/kg bw/d.

Justification for selection of genetic toxicity endpoint
No single study was selected, since all available in vitro and in vivo genetic toxicity studies were negative.

Short description of key information:
Bacterial reverse mutation assay, Ames test (OECD 471): negative
Mammalian Cell Gene Mutation Test, HPRT test (OECD 476): negative
Rodent Dominant Lethal Test (OECD 478): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.