1,4-dimethoxybenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames tests with negative response are available.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP conditions

Qualifier:

according to guideline

Guideline:

other: NTP protocols

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Induced S9 mix with Aroclor 1254, from rat or hamster liver cells.

Test concentrations with justification for top dose:

0; 10; 33; 100; 333; 666; 750; 900; 1000; 2000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of the test material

Untreated negative controls:

yes

Remarks:

choline chloride, glycerol, glycine, mannitol and sodium phosphate.

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene on all strains with rat (1.5 µg/plate) and hamster (0.75 µg/plate) S9; 4-Nitro-o-phenylenediamine on TA98 without S9 (12µg/plate); sodium azide on TA100 and TA1535 without S9 (2.5 µg/plate); 9-aminoacridine on TA1537 (80 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation with S9 mix or buffer
DURATION
- pre-incubation period: 20 min at 37°C
- Exposure duration of incubation: 48 h at 37°C

Evaluation criteria:

Although procedures for the statistical analysis of Salmonella plate test data have been developed [Margolin et al, 1981], they were not incorporated
into the initial data evaluations. The data were evaluated in an ad hoc manner by each testing laboratory and by NTP personnel. Prior to statistical
analysis no formal rules were used; however, a positive response was indicated by a reproducible, dose-related increase, whether it be twofold over
background or not. The matrix of test strains and activation systems used allowed the investigators to detect trends or patterns that might not be
as evident if only one strain and activation system were examined. In addition to the standard "positive" and "negative" categories, there is also
"questionable" (or "inconclusive"). This applied to low-level responses that were not reproducible within the laboratory or to results that showed a
definite trend but with which the investigator did not feel comfortable in making a " +" or " -" decision. It also included tests in which an elevated
revertant colony yield occurred at only a single dose level. After a decision on the mutagenicity of a sample was made, a request to decode the
sample was sent to the repository, and the code was broken.

Statistics:

The data were subsequently evaluated using an analysis based on the models presented by Margolin et al [1981]. As a result of these statistical
analyses, a number of calls were changed from the original "negative" to "equivocal." The statistical analysis did not result in any "positive" or
"equivocal" calls being called "negative".
Because the criteria for "positive" or "questionable" decisions have evolved during the course of the study and because the recent use of statistical
analysis, some of the results differ from the initial evaluations published in the NTP Technical Bulletins (1980 to 1983).

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

yes, slight to toxic for all strains at high concentrations

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'.

 Strain: TA1535

Dose	No Activation (Negative)		No Activation (Positive)		No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
Dose units	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	23	2	13	1.8	26	1.2	11	2.6	9	1.8	7	1.2	11	0.9	11	0.9
10			20	1.7							6	0.9			6	0.6
33	33	1.7	21	2.6	21	4.2	17	1	13	1.3	7	2	10	1	9	2.5
100	27	2	23	0.7	18	3.8	10	0.7	11	0.6	7	1.2	14	3.5	9	2.7
333	24	6.4	41	1.5	21	1.8	15	1.8	9	2	8	1.5	10	1.8	6	1.2
666			106	3	23s	0.6	14s	1			7	1.2			7	1.2
750					18s	1.5	13s	1.9
900					14s	5.8	8s	4.1
1000	T								11s	0.5			5s	1
2000	T								T				T
Positive Control	1408	20.8	1446	11.7	2002	67.1	1413	91.8	148	6.8	85	5.9	77	1	53	8.

Strain: TA100

Dose	No Activation (Equivocal)		No Activation (Weak Positive)		No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
Dose units	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	97	2.1	82	5	103	11.9	108	6.7	85	6.9	82	6.1	92	13.2	81	6.2
10			88	12.9							83	8.6			70	8.1
33	108	9.8	91	4.3	99	4.5	103	15.9	91	3.8	75	2.2	98	6.2	74	2.9
100	124	2	99	9.5	105	8.5	107	7.6	84	6.5	106	8.4	98	4.3	82	8.4
333	109	5.4	141	2.3	96	4.7	94	8.7	92	5.2	83	0.9	99	9	81	8.9
666			246	17.2	101	3.8	114s	12.7			75	5			84	3.5
750					105s	8.1	116s	10.1
900					74s	33.5	62s	27
1000	T								69s	9.5			T
2000	T								T				T
Positive Control	1919	39.3	2152	99.5	2173	58.7	1590	94.7	1634	59.6	861	16.2	1303	116.4	457	35.9

Strain: TA98

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
Dose units	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	19	2.5	18	5.1	29	3.3	23	0.9	20	3.8	22	2.9
10			16	3.6			16	1.7			21	2.4
33	14	0.9	17	3.8	27	2.3	22	3.2	22	1.2	24	0.7
100	17	2.3	20	4.1	29	2.4	22	3	19	4.1	22	3.1
333	16	1.5	15	3.3	23	0.9	15	1.9	33	1.7	21	1.2
666			14	3.3			21	1.5			20	1.2
1000	T				27s	6.5			T
2000	T				T				T
Positive Control	1174	17.5	1352	55.2	983	53.9	808	31.9	529	73.4	532	18.2

  Strain: TA1537

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
Dose units	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	8	1.9	3	0.6	10	1.7	5	0.7	10	1	6	2.6
10			5	1.2			6	0.3			5	0.6
33	6	0.7	4	1.2	9	0.3	4	1.2	7	1.5	3	0.3
100	6	0.9	3	0.9	7	0.7	3	0.7	10	3.7	4	0.6
333	8	1.3	4	1.2	10	2.3	5	0.7	9	0.9	8	2.5
666			5	0.7			5	1.2			7	3.7
1000	T				8s	1			9s	2.6
2000	T				T				T
Positive Control	383	200.3	518	52.7	206	29.5	105	14.5	73	4.7	58	3.5

Conclusions:

Negative with and without metabolic acivation.

Executive summary:

In a reverse gene mutation assay in bacteria (Haworth, 1983), strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to paradimethoxybenzene at concentrations of 0 to 2000 µg/plate in the presence and absence of mammalian metabolic activation (rat or hamster liver cells).

 

Paradimethoxybenzene was tested up to cytotoxic concentrations.

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  OECD 471 for in vitro mutagenicity bacterial reverse gene mutation data.

Reference 2

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:

from 22 feb 1988 to 20 june 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat liver cells, induced with Aroclor 1254

Test concentrations with justification for top dose:

0, 10, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: usually used in this type of test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details below

Details on test system and experimental conditions:

Positive controls:
- Without metabolic activation: Sodium azide (3 µg/plate) for TA100 and TA1535; 9-Aminoacridine for TA1537; 2-Nitrofluorene (0.5 µg/plate) for TA98 and TA1538.
- With metabolic activation: 2-Aminofluorene (5 µg/plate) for TA98 and TA1538.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- incubation period: 48 hours at 37°C
- Exposure duration: 48 hours

NUMBER OF REPLICATES: 3

Evaluation criteria:

- in TA 98 and TA 100: a test article is considered as positive if it produce at least a 2-fold increase in the mean revertant per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control.
This increase in the number of revertant must be accompanied by a dose response with increasing concentrations of the test article
- in TA 1535, TA 1537 and TA 1538: idem, but a 3-fold increase is required

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Tables of results (2 studies):

 

	TA 98			TA 100			TA 1535
Conc. [unit]	-S9	+S9	Cytotoxic ity	-S9	+S9	Cytotoxic ity	-S9	+S9	Cytotoxic ity
0*	25/21	28/28	no	94/94	92	no	15/22	11/14	no
10	25/20	29/28	no	95/85	87	no	13/14	12/10	no
100	20/19	34/27	no	96/98	85	no	21/18	13/15	no
500	18/19	28/32	no	105/96	84	no	18/18	15/12	no
1000	20/24	21/33	no	98/95	93	no	18/27	9/17	no
5000	14/14	10/1/	no	84/83	79	no	15/25	8/9	no
Positive control	182/216	2012/1556	no	1036/889	/	no	938/697	/	no

 

	TA 1537			TA 1538
Conc. [unit]	-S9	+S9	Cytotoxic ity	-S9	+S9	Cytotoxic ity
0*	6/7	7/6	no	10/12	22/21	no
10	7/7	7/6	no	11/11	28/22	no
100	7/5	8/5	no	14/13	25/25	no
500	9/6	7/9	no	8/9	21/24	no
1000	6/6	9/8	no	13/14	21/24	no
5000	7/6	7/4	no	14/13	22/17	no
Positive control	1419/1563	/	no	283/239	1947/1435	no

*solvent control with DMSO

Conclusions:

Negative in Ames test, with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (Hoechst, 1988), strains TA98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Paradimethoxybenzene (purity unknown), in DMSO at concentrations of 0, 10, 100, 500, 1000 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (plate incorporation method).

Paradimethoxybenzene was tested up to the limit concentration (5000 µg/plate). It was not mutagenic in Ames test.

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 OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genotoxicity in vitro:

Two studies were available: one with relaibilty 1 (GLP study, Hoechst, 1988) and another one with reliability 2 (Haworth, 1983). They were selected as key studies.

In these reverse gene mutation assays in bacteria, strains TA98, TA100, TA1535, TA1537 and TA1538 of S. typhimurium were exposed to paradimethoxybenzene at concentrations of 0 to 5000 µg/plate in the presence and absence of mammalian metabolic activation (rat or hamster liver cells).

Paradimethoxybenzene was tested up to cytotoxic concentrations.

The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background in these two studies.

Genotoxicity in vivo:

Only one study (Hoechst, 1996) was available and was selected as key study.

The micronucleus test was performed with Paradimethoxybenzene, according to the OECD 474 guideline under GLP conditions.

The test compound was suspended in starch mucilage and was given once as an orally dose of 2000 mg/kg bw to male and female NMRI mice, based on the results of a preliminary study.

According to the test procedure, the animals were killed 12, 24 or 48 hours after administration.

Endoxan (cyclophosphamide in distilled water) was used as positive control substance and was administered once orally at a dose of 50 mg/kg bw.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Paradimethoxybenzene and was statistically not different from the control values.

Positive control induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

Under the test conditions, the results indicate that Paradimethoxybenzene is not mutagenic in the micronucleus test.