2,2'-[(4-methylphenyl)imino]bisethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Oct - 17 Nov 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

(non-standard positive controls were used, which are not recommended in the guideline; single plates were used per concentration instead of triplicate plating)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

yes

Remarks:

(non-standard positive controls were used, which are not recommended in the guideline; single plates were used per concentration instead of triplicate plating)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(4-methylphenyl)imino]bisethanol

EC Number:

221-359-1

EC Name:

2,2'-[(4-methylphenyl)imino]bisethanol

Cas Number:

3077-12-1

Molecular formula:

C11H17NO2

IUPAC Name:

2,2'-[(4-methylphenyl)imino]diethanol

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Pre-test for toxicity/Experiment I (plate incorporation): 16, 50, 160, 500, 1600 and 5000 µg/plate with and without metabolic activation
Experiment II (preincubation method): 16, 50, 160, 500, 1600 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was soluble in DMSO and formed a clear colorless solution.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: 2-aminoanthracene: +S9, 3 µg/plate for all strains; nitrofurantoin: - S9, 0.2 µg/plate for TA100; 4-nitro-1,2-phenylene diamine: -S9, 0.5 and 10 µg/plate for TA1537 and TA98, respectively

Remarks:

Each batch of S9 mix was checked for its metabolizing capacity by using reference mutagenes (not further specified); appropriate activity was demonstrated.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 20 min (Experiment II)
- Exposure duration: 48 h (Experiment I and II)

NUMBER OF REPLICATIONS: Single plates per concentration each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: background growth, marked and dose-dependent reduction in mutant count, titre count

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100 and TA98 this increase should be about twice that of negative controls, whereas for TA1537, at least a threefold increase should be reached. For TA102 an increase about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 160 and 500 µg/plate with and without metabolic activation, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was not observed.

RANGE-FINDING/SCREENING STUDIES: Experiment I (plate incorporation) served as pre-test for toxicity.

HISTORICAL CONTROL DATA
Only single plates were counted in the present study for each control and concentration level. Moreover only mean values without standard deviation were given in the historical control data, therefore it cannot be confirmed if the positive or negative control values are within the range of the historical control data. However, the positive control values from the study and from the historical control data increased mutant counts well over those of the negative controls.

Any other information on results incl. tables

Table 1 Results of Experiment I (plate incorporation)

With or without S9-Mix	Test substance concentration (μg/plate)	Revertant colonies per plate
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
–	DMSO	64	11	115	9	9
–	16	63	10	139	9	8
–	50	75	11	131	16	14
–	160	71	9	162	11	6
–	500	68	9	109	9	8
–	1600	77	12	93B	11	5
	5000	77	14	60B	8	4
Positive controls, –S9	Name	NF	SA	Cumene	4-NPDA	4-NPDA
	Concentrations (μg/plate)	0.2	10	50	0.5	10
	Revertant colonies per plate	293	714	277	148	129
+	DMSO	141	9	203	31	12
+	16	96	C	195	14	16
+	50	104	7	198	27	17
+	160	104	10	151	35	9
+	500	119	6	161B	28	8
+	1600	145	11	117B	31	5
+	5000	145	5	51B	18	2
Positive controls, –S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	3	3	3	3	3
	Revertant colonies per plate	1658	379	492	1214	382

NF = Nitrofurantoin

SA = Sodium azide

Cumene = Cumene hydroperoxide

4-NPDA = 4-Nitro-1,2-phenylene diamine

2AA = 2-Aminoanthracene

C = contaminated

B = reduced background lawn

Table 2: Results of Experiment II (preincubation method)

With or without S9-Mix	Test substance concentration (μg/plate)	Revertant colonies per plate
		Base-pair substitution type			Frameshift type
		TA100	TA1535	TA102	TA98	TA1537
–	DMSO	73	14	178	16	7
–	16	96	14	216	22	7
–	50	100	8	211	18	11
–	160	72	6	215	16	7
–	500	114	8	202	19	9
–	1600	106	9	179	18	4
	5000	83B	5B	75B	5B	2B
Positive controls, –S9	Name	NF	SA	Cumene	4-NPDA	4-NPDA
	Concentrations (μg/plate)	0.2	10	50	0.5	10
	Revertant colonies per plate	347	650	523	152	146
+	DMSO	125	9	239	27	9
+	16	94	12	266	22	13
+	50	101	12	237	19	9
+	160	112	10	266	22	11
+	500	133	8	252	16	11
+	1600	155	13	168	9	8
+	5000	151B	10B	79B	17B	3B
Positive controls, –S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	3	3	3	3	3
	Revertant colonies per plate	1798	198	439	1587	184

NF = Nitrofurantoin

SA = Sodium azide

Cumene = Cumene hydroperoxide

4-NPDA = 4-Nitro-1,2-phenylene diamine

2AA = 2-Aminoanthracene

C = contaminated

B = reduced background lawn

Table 3: Historical control values (January - December 1996): plate incorporation

With or without S9-Mix	Compound	TA100	TA1535	TA102	TA98	TA1537
–	DMSO	86	9	292	20	8
–	SA	--	739	--	--	--
–	NF	253	--	--	--	--
–	4-NPDA	--	--	--	155	139
–	Cumene	--	--	624	--	--
+	DMSO	103	11	340	30	10
+	2-AA	1540	204	829	1461	281

NF = Nitrofurantoin

SA = Sodium azide

Cumene = Cumene hydroperoxide

4-NPDA = 4-Nitro-1,2-phenylene diamine

2AA = 2-Aminoanthracene

Table 4: Historical control values (January - December 1996): preincubation method

With or without S9-Mix	Compound	TA100	TA1535	TA102	TA98	TA1537
–	DMSO	80	9	271	22	8
–	SA	--	747	--	--	--
–	NF	278	--	--	--	--
–	4-NPDA	--	--	--	183	127
–	Cumene	--	--	602	--	--
+	DMSO	94	11	337	28	8
+	2-AA	1318	172	601	1239	164

NF = Nitrofurantoin

SA = Sodium azide

Cumene = Cumene hydroperoxide

4-NPDA = 4-Nitro-1,2-phenylene diamine

2AA = 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:

Based on the results of the conducted study, the test substance was not considered to reveal mutagenic properties in Salmonella typhimurium strains TA1535, TA1537, TA89, TA100 and TA102 with and without metabolic activation, respectively.