N,N-diethylaniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 1, 3, 10, 33, 100 or 333 µg/plate in lab using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration study:

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and with and without S9-induced metabolic activation for 3 hours. The results showed that there was evidence of cytotoxicity after treatment with 5 mM or above. Independently of tested concentration with S9 metabolic activation system, the results showed no evidence of gene toxicity. However, treatment with 5 mM of test chemical showed evidence of potential gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation. The test chemical however in the concentrations of 2.5 mM or below caused no genetic mutation(s), while concentrations at 5mM or above may, when CHO cells are exposed to the test chemical in the absence of metabolic activation.

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:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Preincubation method was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA97 and TA98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

10% and 30% HLI and RLI S-9 (9,000 g supernatant) fractions were prepared from Aroclor 1254-induced, male Sprague- Dawley rat and male Syrian hamster livers

Test concentrations with justification for top dose:

0, 1, 3, 10, 33, 100, 333 µg/plate

Vehicle / solvent:

Dimethyl Sulfoxide- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was dissolved in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl Sulfoxide

True negative controls:

not specified

Positive controls:

yes

Remarks:

in the absence of S9 activation

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98, with S9) ; The positive control for metabolic activation with all strains was 2-aminoanthracene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

Evaluation criteria:

Evaluations were made at both the individual trial and overall chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable
(?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “ +W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a nonmutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two fold over background for a chemical to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related reponse over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials.
Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity.

Statistics:

Mean ± SD

Species / strain:

S. typhimurium, other: TA100, TA1535, TA97 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Additional information on results
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: No mutagenic potential

Table: Mutagenicity of the test chemical

 

Dose (µg/plate)	TA100
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	127	7.0	98	3.3	129	3.6	93	1.2	147	6.2
1	119	6.6
3	116	11.7	96	2.6	134	7.9	103	6.7	119	8.3
10	96	7.9	96	10.2	124	4.3	103	6.1	132	10.5
33	123	10.0	97	6.5	143	4.4	104	5.0	123	7.1
100	127	14.3	97	5.0	131	8.5	97	7.0	123	8.1
333			52	226.2	136	10.6	80	4.5	112	6.4
Positive control	375	12.3	1828	130.2	873	46.0	725	24.1	449	7.0

 

Dose (µg/plate)	TA1535
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	24	3.4	11	3.3	12	2.4	10	4.0	12	6.0
1	23	4.4
3	26	3.8	14	5.0	10	2.9	10	1.5	12	3.5
10	18	1.2	9	1.8	9	3.2	7	1.7	22	1.5
33	22	4.3	10	2.2	13	0.6	11	2.7	14	2.0
100	12	0.7	7	1.9	7	1.5	10	1.5	12	1.5
333			4	0.3	15	1.9	7	2.7	15	2.8
Positive control	418	23.1	557	10.3	616	69.9	205	12.4	162	27.5

 

Dose (µg/plate)	TA97
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	121	8.1	117	5.5	234	15.2	193	8.2	236	9.4
1	134	0.3
3	108	7.8	133	4.4	204	12.1	169	10.3	207	21.5
10	129	637	143	5.8	206	15.2	156	10.5	211	4.6
33	132	11.2	138	6.4	207	10.7	139	7.1	223	13.0
100	108	2.0	144	6.1	197	10.2	123	9.0	186	23.8
333			113	6.0	186	9.2	129	18.0	222	14.5
Positive control	1156	22.0	1885	76.9	61.9	1452	80.7	480	60.6	7.0

 

Dose (µg/plate)	TA1535
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	20	2.6	26	4.7	28	2.5	28	2.9	33	2.8
1	16	5.0
3	16	2.6	26	3.2	27	1.7	24	3.6	25	3.5
10	17	2.9	31	4.5	31	3.8	26	1.9	23	3.8
33	22	3.6	35	3.2	27	1.9	30	0.3	25	2.6
100	12	1.7	31	9.0	27	3.3	32	2.5	26	2.4
333			30	0.9	29	1.5	23	3.5	28	3.9
Positive control	845	69.2	1187	29.6	576	116.6	408	20.3	246	18.1

 

Conclusions:

The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium.

 

The test compound was dissolved in DMSO and was tested at concentration of 0, 1, 3, 10, 33, 100 or 333 µg/plate in lab using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study.

The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0.1, 2.5, 5 or 10 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Details on test system and experimental conditions:

Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
STAIN (for cytogenetic assays): Crystal violet
NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 0.5 x 10 E5 cells

Rationale for test conditions:

No data

Evaluation criteria:

The cell line were observed for gene mutation at the HGPRT locus

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

genotoxicity was ambiguous without S9 metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation

N,N-diethylaniline in the concentration of 0.1, 2.5, 5 or 10 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical.

Without S9 metabolic activation the interpretation of results = Ambiguous without metabolic activation
Cells treated with N,N-diethylaniline in the concentration of 0, 1, 2.5, 5 or 10 mM showed evidence of potential gene toxicity in CHO cells when exposed to the test chemical at 5 mM.

Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and with and without S9-induced metabolic activation for 3 hours. The results showed that there was evidence of cytotoxicity after treatment with 5 mM or above. Independently of tested concentration with S9 metabolic activation system, the results showed no evidence of gene toxicity. However, treatment with 5 mM of test chemical showed evidence of potential gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation. The test chemical however in the concentrations of 2.5 mM or below caused no genetic mutation(s), while concentrations at 5mM or above may, when CHO cells are exposed to the test chemical in the absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Bone Marrow Chromosomal Aberration was performed to determine the mutagenic nature of the test chemical in vivo. The study was performed using male and female Bor: NMRI (SPF Han). The test chemical was tested at dose concerntration of 0 or 600 mg/Kg/day. The animals were given a single repeated administration of the test chemical. There was an altered ratio between polychromatic and normochromatic erythrocytes. However the test chemical did not induce clastogenic effects on the cells. Based on the observations made, thetest chemical did not induce chromosome aberrations in the bone marrow cells in vivo isolated from the test chemical treated male and female NMRI mice and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

data is from secondary literature

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Bone Marrow Chromosomal Aberration was performed to determine the mutagenic nature of the test chemical in vivo.

GLP compliance:

not specified

Type of assay:

other: Bone Marrow Chromosomal Aberration

Species:

mouse

Strain:

NMRI

Remarks:

Bor: NMRI (SPF Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

intraperitoneal

Vehicle:

No data

Details on exposure:

No data

Duration of treatment / exposure:

No data

Frequency of treatment:

Single administration

Post exposure period:

No data

Remarks:

0 or 600 mg/Kg/day

No. of animals per sex per dose:

No data

Control animals:

yes, concurrent vehicle

Positive control(s):

No data

Tissues and cell types examined:

Bone marrow polychromatic and normochromatic erythrocytes

Details of tissue and slide preparation:

No data

Evaluation criteria:

The cells were observed for chromosome aberrations

Statistics:

No data

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: No mutagenic potential

Additional information on results:

No data

Conclusions:

The test chemical did not induce chromosome aberrations in the bone marrow cells in vivo isolated from the test chemical treated male and female NMRI mice and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Bone Marrow Chromosomal Aberration was performed to determine the mutagenic nature of the test chemical in vivo. The study was performed using male and female Bor: NMRI (SPF Han). The test chemical was tested at dose concerntration of 0 or 600 mg/Kg/day. The animals were given a single repeated administration of the test chemical. There was an altered ratio between polychromatic and normochromatic erythrocytes. However the test chemical did not induce clastogenic effects on the cells. Based on the observations made, thetest chemical did not induce chromosome aberrations in the bone marrow cells in vivo isolated from the test chemical treated male and female NMRI mice and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Ames assay:

The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 1, 3, 10, 33, 100 or 333 µg/plate in lab using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and with and without S9-induced metabolic activation for 3 hours. The results showed that there was evidence of cytotoxicity after treatment with 5 mM or above. Independently of tested concentration with S9 metabolic activation system, the results showed no evidence of gene toxicity. However, treatment with 5 mM of test chemical showed evidence of potential gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation. The test chemical however in the concentrations of 2.5 mM or below caused no genetic mutation(s), while concentrations at 5mM or above may, when CHO cells are exposed to the test chemical in the absence of metabolic activation.

DNA damage/repair assay:

The hepatocyte/DNA repair test which measures unscheduled DNA synthesis (UDS) is known to be sensitive to various classes of DNA-reactive carcinogens and is regarded as a reliable short-term test for the detection of chemical carcinogens. In this study, the genotoxicity of the test chemical was examined by a DNA repair test with rat hepatocytes. The test was performed basically in accordance with the method of Williams et al. The test material was dissolved in DMSO and used at dose level of 10^-3, 10^-4, 10^-5, 10^-6M and the positive control used was N-2-fluorenylacetamide. The test chemical did not induce mutation in ACI rat hepatocytes and hence is negative in the rat hepatocyte/ DNA repair test.

Gene mutation in vivo:

Bone Marrow Chromosomal Aberration was performed to determine the mutagenic nature of the test chemical in vivo. The study was performed using male and female Bor: NMRI (SPF Han). The test chemical was tested at dose concerntration of 0 or 600 mg/Kg/day. The animals were given a single repeated administration of the test chemical. There was an altered ratio between polychromatic and normochromatic erythrocytes. However the test chemical did not induce clastogenic effects on the cells. Based on the observations made, thetest chemical did not induce chromosome aberrations in the bone marrow cells in vivo isolated from the test chemical treated male and female NMRI mice and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical does not exhibit gene mutation in vitro, The negative results are supported by the negative results observed the in vivo study. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

The test chemical does not exhibit gene mutation in vitro, The negative results are supported by the negative results observed the in vivo study. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.