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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacterial cells (Ames test): negative in Salmonella typhimurium strains TA100, TA1535, TA102, TA98 and TA1537 with and without metabolic activation

Cytogenicity (chromosome aberration test): negative in cultured peripheral human lymphocytes with and without metabolic activation (based on read-across)

Gene mutation in mammalian cells (mouse lymphoma assay): positive in L5178Y mouse lymphoma cells with and without metabolic activation (based on read-across)

Link to relevant study records

Reference

Reference 1

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)

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

Target gene:

his operon

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.

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.

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

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

Positive controls validity:

valid

Conclusions:

A chromosome aberration study with the source substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]- (EC 911-490-9) was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the source substance is not clastogenic in human lymphocytes. Applying the read-across approach, similar results are expected for the target substance N,N-Dihydroxyethyl-p-toluidine (CAS 3077-12-1).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 3330 μg/mL (3 h treatment); at and above 250 μg/mL (24 h treatment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1650 μg/mL (3 h treatment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The mutagenic activity of the source substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]- (EC 911-490-9) was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, performed according to OECD 476 and following GLP principles.The test substance induced mutations in the absence of metabolic activation, but not with metabolic activation, which was confirmed in an independent repeat experiment.
It is concluded that the test substance is positive in the in vitro mammalian cell gene mutation test. Applying the read-across approach, similar results are expected for the target substance N,N-Dihydroxyethyl-p-toluidine (CAS 3077-12-1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Gene mutation in vivo (Comet assay): negative in liver, duodenum and glandular stomach tissues of male rats (based on read-across)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

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

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: Source: EC 911-490-9

Conclusions:

It is concluded that the comet assay in liver, duodenum and glandular stomach was valid and the source substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]- (EC 911-490-9) does not provoke DNA damage in the Comet assay up to and including a dose of 750 mg/kg bw (the maximum tolerated dose in accordance with current regulatory guidelines). Applying the read-across approach, similar results are expected for the target substance N,N-Dihydroxyethyl-p-toluidine (CAS 3077-12-1).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation in bacterial cells: Ames test

Reliable data are available regarding genetic toxicity in bacterial cells for N,N-Dihydroxyethyl-p-toluidine (CAS 3077-12-1).

An Ames test was performed similar to OECD 471 with Salmonella typhimurium strains TA100, TA1535, TA102, TA98 and TA1537, with and without metabolic activation (Bayer AG, 1998). No test substance precipitation was observed when tested up to 5000 μg/plate. The pre-test for cytotoxicity served as Experiment 1. Cytotoxicity was observed at 5000 µg/plate with and without metabolic activation for S. typhimurium strains TA100, TA1535, TA98 and TA1537, respectively. In S. typhimurium TA102 the test substance showed cytotoxic properties at 160 and 500 µg/plate with and without metabolic activation, respectively. The positive controls showed the expected increase in mutant colonies when compared to the corresponding negative control. Evidence of mutagenic activity of N,N-Dihydroxyethyl-p-toluidine (CAS 3077-12-1) was not seen, since no biologically relevant increase in the mutant count in comparison with the negative controls were observed. Therefore, based on these data it is concluded that N,N-Dihydroxyethyl-p-toluidine (CAS 3077-12-1) is not mutagenic in bacterial cells under the conditions of this test.

No data is available regarding gene mutation and cytogenicity in mammalian cells and genetic toxicity in vivo for N,N-Dihydroxyethyl-p-toluidine (CAS 3077-12-1). Therefore, read-across from an appropriate structural analogue substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.4. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13). Information from the analogue substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol (EC 911-490-9) will be taken into account to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.4.

Cytogenicity in mammalian cells: In vitro chromosome aberration test

A chromosome aberration study with the source substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol (EC 911-490-9) was performed according to OECD 473 and GLP principles, in cultured peripheral human lymphocytes in two independent experiments (WIL Research, 2013d). No precipitation of the substance was observed, but the test substance was tested up to and beyond cytotoxic concentrations. Both in the absence and presence of S9-mix, the source substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. It can be concluded that Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol (EC 911-490-9) does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

In vitro gene mutation test in mammalian cells: mouse lymphoma assay:

An in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells, performed according to OECD 476 and following GLP principles is available. The source substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol (EC 911-490-9) was tested up to cytotoxic concentrations (WIL Research, 2013e). The positive control data and the solvent control data validated the study. In the presence of S9-mix, the source substance did not induce a significant increase in the mutation frequency in two experiments. In the absence of S9-mix, the test substance induced a 5.3-fold increase in the mutation frequency at the prolonged treatment period of 24 hours. This was verified in a further experiment, in which the substance induced an up to 3.3-fold increase in the mutation frequency. The mutation frequency of both the small and large colonies was significantly increased. Based on these data, it is concluded that the test substance is positive in the in vitro mammalian cell gene mutation test.

Alkaline in vivo Comet assay in liver, stomach and duodenum of rats:

The alkaline in vivo Comet assay with the source substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol (EC 911-490-9) was performed according to OECD 489 and GLP principles (Charles River, 2017a). One experiment was performed for liver and two experiments were performed for glandular stomach and duodenum since the first experiment did not pass the acceptance criteria for the control group. Groups of 5 male rats were dosed once daily via oral gavage with vehicle or with 187.5, 375 and 750 mg of the test substance per kg body weight for three consecutive days. A positive control group was included. The animals that were dosed with 750 mg/kg bw/day showed clinical signs including lethargy, tremors, hunched posture and ataxia. Approximately 3-4 hours after the second dose of ethylmethanesulphonate (EMS) and third dose of the vehicle or the test substance, liver, glandular stomach, and duodenum tissue were collected, and single cell suspensions were made followed by Comet slide preparation. No biologically relevant increase in the mean Tail Intensity (%) was observed in liver cells. No statistically significant increase in the mean Tail Intensity (%) was observed in glandular stomach and duodenum cells. As the acceptance criteria for the control group were not passed for glandular stomach and duodenum, no conclusion could be drawn about the potential DNA damaging properties of the test substance in these tissues. Therefore the in vivo alkaline Comet assay was repeated for the organs duodenum and glandular stomach, because the Tail Intensity of the negative control did not meet the acceptance criteria.

It is concluded that the Comet assay in liver is valid and the source substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol (EC 911-490-9) does not provoke DNA damage in the Comet assay up to and including a dose of 750 mg/kg bw (the maximum tolerated dose in accordance with current regulatory guidelines).

The alkaline in vivo Comet assay with the source substance Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol (EC 911-490-9), performed according to OECD 489 and GLP principles, was repeated for the organs duodenum and glandular stomach, because the Tail Intensity (%) of the vehicle control group determined in the initial experiment (Charles River, 2017a) was considered to be invalid. Groups of 5 male rats were dosed once daily via oral gavage with vehicle or with 187.5, 375 and 750 mg of the test substance per kg body weight for three consecutive days (Charles River, 2018). A positive control group and a vehicle control were included. All animals of the low (187.5 mg/kg bw) and mid dose group (375 mg/kg bw) were lethargic. After approximately 21 hour these animals showed no abnormalities. Within one hour after the second and third treatment the animals showed no treatment related effects with exception of 1/5 animal in the mid dose group and 2/5 animals in the low dose group, which were lethargic after the second and third dosing. In addition, 1/5 animals of the low dose group showed ataxia after the third dose. 1/5 animals of the high dose group were lethargic and showed ataxia after the first dosing. After approximately 21 hour the animal showed no abnormalities. The same animal was lethargic and showed ataxia after the second and third dosing as well. The effects in another animal of the high dose group were the same with the exception that the animal showed only lethargy after the first dosing. The remaining animals treated with 750 mg/kg bw showed mortality (3/5), one after the first dosing and two after the second dosing. No statistically significant increase in the mean Tail Intensity (%) was observed in stomach cells and in duodenum cells of test item-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity (%) of vehicle treated male rats was 0.91 ± 0.63 % in stomach cells and 2.47 ± 0.65 % in duodenum cells. The mean vehicle control Tail Intensity was within the 95% control limits of the distribution of the historical negative control database and the mean Tail Intensity (%) of the vehicle control was below 20% for both organs. The positive control EMS revealed an increase of the Tail Intensity, 52-fold induction in glandular stomach cells and 18-fold induction in duodenum cells, respectively. Thus all negative and positive control values in glandular stomach and duodenum met the acceptance criteria.

In conclusion, the test is valid and Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol (EC 911-490-9) does not cause DNA damage in the Comet assay in glandular stomach and duodenum cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for three consecutive days up to a dose of 750 mg/kg bw (the maximum tolerated dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Justification for classification or non-classification

Reliable data with the registration substance and from a structural analogue substance regarding in vitro and in vivo genetic toxicity indicate that the registration substance does not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.