Diethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (equivalent or similar to OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA/pKM101 with and without metabolic activation

In vitro hprt test (OECD 476): negative for mutations at the hprt locus with and without metabolic activation

RA from diethylamine (CAS 109-89-7)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

Range finder: 22.86; 45.71; 91.43; 182.9; 365.7; 731.4 µg/ml (with and without S-9 mix)
Experiment 1: 100, 200, 300, 350, 400, 450, 500, 550, 650, 731.4 (without S-9 mix)
100, 200, 300, 400, 500, 550, 600, 650, 700, 731.4 (with S-9 mix)
Experiment 2: 50, 100, 200, 300, 350, 400, 450, 500, 600, 731.4 (without S-9 mix)
100, 200, 300, 400, 450, 500, 550, 600, 650, 731.4 (with S-9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 4-nitroquinoline 1-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days

SELECTION AGENT (mutation assays): 6GT, 15 µg/mL

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
There was a significant concentration relationship as indicated by the linear trend analysis (p≤0.05)
The effects described above were reproducible.
Results that only partially satisfy the assessment criteria described above are considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 731.4 µg/mL, were tested in the absence and presence of S 9. Following the treatment incubation period, the highest two concentrations in the absence of S-9 and the highest three concentrations in the presence of S-9 (650 to 731.4 µg/mL in each case) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest two remaining concentrations in the absence of S-9 (500 and 550 µg/mL) and the highest remaining concentration in the presence of S 9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. The highest concentrations selected were 450 µg/mL in the absence of S 9 and 550 µg/mL in the presence of S 9, which gave 10% and 7% relative survival (RS), respectively. In the presence of S-9, no concentration gave 10 and 20% RS. Cultures treated at 500 and 550 µg/mL gave 21% and 7% RS, respectively, therefore both concentrations were analysed.

In Experiment 2, ten concentrations, ranging from 50 to 731.4 µg/mL in the absence of S-9 and from 100 to 731.4 µg/mL in the presence of S 9, were tested. Following the treatment incubation period, the highest two concentrations in the absence of S-9 (600 and 731.4 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentration in the absence of S 9 (500 µg/mL) and the highest four concentrations in the presence of S 9 (550 to 731.4 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. However, the concentration of 300 µg/mL in the presence of S-9 was later rejected from analysis due to extreme heterogeneity for viability. Marked heterogeneity (also for viability) was observed at concentrations of 50 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9, but these were included for comparative purposes. The highest concentrations analysed were 450 µg/mL in the absence of S 9 and 500 µg/mL in the presence of S 9, which gave 10% and 13% RS, respectively.

Experiment 1 (3 hours treatment in the absence and presence of S-9 mix)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS	MF§					% RS	MF§
0		100	4.80			0		100	3.57
100		72	3.25		NS	100		96	3.14		NS
200		74	4.91		NS	200		69	2.75		NS
300		67	2.03		NS	300		62	3.91		NS
350		43	5.24		NS	400		62	4.65		NS
400		29	7.07		NS	500		21	2.87		NS
450		10	6.85		NS	550		7	6.33		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		44	44.33			2		52	21.21
0.15		51	56.73			3		23	56.04

§: 6TG resistant mutants/10⁶ viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

Experiment 2 (3 hours treatment in the absence and presence of S-9 mix)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS		MF§				% RS		MF§
0		100		2.59		0		100		2.49
50	$$	80		1.85		100		127		3.84	NS
100		71		2.38	NS	200		76		3.36	NS
200		68		3.50	NS	400		45		2.70	NS
300		48		2.28	NS	450	$$	30		2.96
350		27		4.48	NS	500		13		5.28	NS
400		18		3.32	NS
450		10		10.10	*
Linear trend				*		Linear trend				NS
NQO						B[a]P
0.1		61		24.70		2		58		39.09
0.15		41		21.20		3		32		69.84

§: 6TG resistant mutants/ 10⁶ viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

* : Comparison of each treatment with control: Dunnett#s test (one-sided), significant at 5% level

*,**,*** : Test for linear trend: χ² (one-sided), significant at 5%, 1% and 0.1% level respectively

$$ : Treatment has marked heterogeneity for viability but is included for comparative purposes

Conclusions:

It is concluded that Diethylamine, anhydrous did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

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:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented NTP study, which meets scientific standards.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Species / strain / cell type:

other: S. typhimurium TA989, TA100 and E. Coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

rat and hamster S9-Mix (10% Aroclor 1254-induced)

Test concentrations with justification for top dose:

Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses of diethylamine. In the first study, doses up to 10,000 µg/plate were tested; toxicity was observed at 3,333 µg/plate and higher doses. In the second study, 4,000 µg/plate was the highest dose tested. All trials were repeated, and those that were conducted with S9 activation enzymes were repeated using the same concentrations of S9.

Salmonella strains: 0, 33, 100, 333, 1000, 3333, 10000 µg/plate
E. coli WP2 uvr A / pKM 101: 0, 50, 100, 500, 1000, 2000, 3000, 4000 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: water
E. coli: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine, 2-aminoanthracene (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days
NUMBER OF REPLICATIONS: Each trial consisted of triplicate plates and was done in a replicate.

Evaluation criteria:

Positive, if a reproducible dose related response over the solvent control was obtained.

Key result

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:

from 3333 µg/plate onwards; all strains

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA98, TA100 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 3333 µg/plate onwards; all Salmonella strains; from 2000 µg/plate onwards for the E.coli strain strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

 Strain: TA1535

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	31	0.7	37	6.4	42	3.6	14	2.3	41	5.5	23	3.8
33			26	4.0			12	2.1			17	1.2
100	22	2.1	25	0.9	47	1.2	13	0.7	40	6.1	12	1.8
333	21	3.4	25	0.3	41	7.5	7	1.3	36	2.2	13	1.9
1000	31	3.3	19	0.7	55	2.6	10	2.2	42	6.2	17	5.5
3333	0s	0	20	4.0	30s	15.3	7	0.7	31	3.5	14	4.7
10000	0s	0			0t	0			0s	0
Positive Control	443	29.1	399	18.0	645	25.2	563	28	331	13.7	266	33.5

 Strain: TA100

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	108	3.2	101	8.7	134	8.3	117	10.4	116	7.8	95	1.5
33			122	9.3			112	2.5			106	3
100	158	14.7	109	3.7	160	9.1	119	4.7	152	4.3	111	3.3
333	144	6.9	121	6.7	142	9	116	13.5	141	4.2	119	15.2
1000	153	4.8	121	10.4	146	8	123	13.3	141	2.9	104	11.4
3333	0s	0	111	8.4	104s	52.1	97	12.5	116	17.9	102	3.7
10000	0t	0			t				0t	0
Positive Control	467	18	477	6.4	2355	34.5	1511	49.9	846	26.4	820	36.8

 Strain: TA98

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	28	2.2	21	3.8	48	5.8	28	2.6	37	2.3	36	3.2
33			20	2.3			23	2.4			31	4.9
100	19	0.7	15	1.5	46	7	30	4.2	43	1.2	30	1.7
333	16	1.5	17	2.3	52	0.7	24	2.6	34	4.7	31	5.2
1000	15	0.6	20	2.1	57	2.6	22	2.7	37	6.1	35	4.1
3333	1s	0.7	17	0.9	0s	0	23	1.5	43	4.2	26	0.3
10000	0s	0.3			t				0s	0
Positive Control	758	14.2	722	8.2	1856	19.6	1102	66.6	436	5.1	591	44.4

 Strain: TA1537

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	11	1.8	7	1.5	19	4.7	9	1.3	14	1.2	11	1
33			7	1.2			6	1.5			9	0
100	5	0.9	7	0.3	23	2.8	7	0.9	10	2.7	17	1.2
333	7	2	9	1.5	27	1	5	0.6	13	2.9	13	3.5
1000	6	0	10	1	21	6.7	10	2.1	13	0.7	15	1.8
3333	0s	0	7	1.2	0s	0	5s	0.9	6	1.5	12	0.3
10000	0s	0			t				0s	0
Positive Control	388	33.5	205	40.4	591	16.8	465	15.2	266	9.8	241	15.6

Note: E. coli table is in the next text box

Abbreviations: RLI = induced male Sprague Dawley rat liver S9 HLI = induced male Syrian hamster liver S9

s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Conclusions:

Interpretation of results
negative

Diethylamine (CAS: 109-89-7) was thus not mutagenic in this assay under conditions where the positive control showed the expected clear mutagenic responses.

Executive summary:

The results and data from the testing of 255 chemicals for mutagenicity in Salmonella are presented. All chemicals were tested under code using a preincubation modification of the Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters.

From the data presented it can be concluded that for diethylamine (CAS: 109-89-7) under the conditions tested, no mutagenic activity was observed in any strain/activation combination in the bacterial AMES-Test. The positive and negative controls showed the expected values.

Diethylamine (CAS: 109-89-7) was thus not mutagenic in this assay under conditions where the positive control showed the expected clear mutagenic responses.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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:

other: Salmonella typhimurium: TA 98, TA 100, TA1535 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 3333 µg/plate onwards; all strains

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: Salmonella typhimurium TA 98 and TA 100; Escherichia coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 3333 µg/plate onwards; all Salmonella strains; from 2000 µg/plate for the E.coli strain strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

 Strain: TA1535

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	31	0.7	37	6.4	42	3.6	14	2.3	41	5.5	23	3.8
33			26	4			12	2.1			17	1.2
100	22	2.1	25	0.9	47	1.2	13	0.7	40	6.1	12	1.8
333	21	3.4	25	0.3	41	7.5	7	1.3	36	2.2	13	1.9
1000	31	3.3	19	0.7	55	2.6	10	2.2	42	6.2	17	5.5
3333	0s	0	20	4	30s	15.3	7	0.7	31	3.5	14	4.7
10000	0s	0			0t	0			0s	0
Positive Control	443	29.1	399	18	645	25.2	563	28	331	13.7	266	33.5

 Strain: TA100

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	108	3.2	101	8.7	134	8.3	117	10.4	116	7.8	95	1.5
33			122	9.3			112	2.5			106	3
100	158	14.7	109	3.7	160	9.1	119	4.7	152	4.3	111	3.3
333	144	6.9	121	6.7	142	9	116	13.5	141	4.2	119	15.2
1000	153	4.8	121	10.4	146	8	123	13.3	141	2.9	104	11.4
3333	0s	0	111	8.4	104s	52.1	97	12.5	116	17.9	102	3.7
10000	0t	0			t				0t	0
Positive Control	467	18	477	6.4	2355	34.5	1511	49.9	846	26.4	820	36.8

 Strain: TA98

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	28	2.2	21	3.8	48	5.8	28	2.6	37	2.3	36	3.2
33			20	2.3			23	2.4			31	4.9
100	19	0.7	15	1.5	46	7	30	4.2	43	1.2	30	1.7
333	16	1.5	17	2.3	52	0.7	24	2.6	34	4.7	31	5.2
1000	15	0.6	20	2.1	57	2.6	22	2.7	37	6.1	35	4.1
3333	1s	0.7	17	0.9	0s	0	23	1.5	43	4.2	26	0.3
10000	0s	0.3			t				0s	0
Positive Control	758	14.2	722	8.2	1856	19.6	1102	66.6	436	5.1	591	44.4

 Strain: TA1537

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	11	1.8	7	1.5	19	4.7	9	1.3	14	1.2	11	1
33			7	1.2			6	1.5			9	0
100	5	0.9	7	0.3	23	2.8	7	0.9	10	2.7	17	1.2
333	7	2	9	1.5	27	1	5	0.6	13	2.9	13	3.5
1000	6	0	10	1	21	6.7	10	2.1	13	0.7	15	1.8
3333	0s	0	7	1.2	0s	0	5s	0.9	6	1.5	12	0.3
10000	0s	0			t				0s	0
Positive Control	388	33.5	205	40.4	591	16.8	465	15.2	266	9.8	241	15.6

Note: E. coli table is in the next text box

Abbreviations: RLI = induced male Sprague Dawley rat liver S9 HLI = induced male Syrian hamster liver S9

s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Conclusions:

Diethylamine (CAS: 109 -89 -7) was not mutagenic in this assay under conditions where the positive control showed the expected clear mutagenic responses. Applying the RA approach, similar results are expected for the target substance.

Executive summary:

The results and data from the testing of 255 chemicals for mutagenicity in Salmonella are presented. All chemicals were tested under code using a preincubation modification of the Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters.

From the data presented it can be concluded that for diethylamine (CAS: 109-89-7) under the conditions tested, no mutagenic activity was observed in any strain/activation combination in the bacterial AMES-Test. The positive and negative controls showed the expected values.

Diethylamine (CAS: 109 -89 -7) was thus not mutagenic in this assay under conditions where the positive control showed the expected clear mutagenic responses.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

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:

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:

Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 731.4 µg/mL, were tested in the absence and presence of S 9. Following the treatment incubation period, the highest two concentrations in the absence of S-9 and the highest three concentrations in the presence of S-9 (650 to 731.4 µg/mL in each case) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest two remaining concentrations in the absence of S-9 (500 and 550 µg/mL) and the highest remaining concentration in the presence of S 9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. The highest concentrations selected were 450 µg/mL in the absence of S 9 and 550 µg/mL in the presence of S 9, which gave 10% and 7% relative survival (RS), respectively. In the presence of S-9, no concentration gave 10 and 20% RS. Cultures treated at 500 and 550 µg/mL gave 21% and 7% RS, respectively, therefore both concentrations were analysed.

In Experiment 2, ten concentrations, ranging from 50 to 731.4 µg/mL in the absence of S-9 and from 100 to 731.4 µg/mL in the presence of S 9, were tested. Following the treatment incubation period, the highest two concentrations in the absence of S-9 (600 and 731.4 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentration in the absence of S 9 (500 µg/mL) and the highest four concentrations in the presence of S 9 (550 to 731.4 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. However, the concentration of 300 µg/mL in the presence of S-9 was later rejected from analysis due to extreme heterogeneity for viability. Marked heterogeneity (also for viability) was observed at concentrations of 50 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9, but these were included for comparative purposes. The highest concentrations analysed were 450 µg/mL in the absence of S 9 and 500 µg/mL in the presence of S 9, which gave 10% and 13% RS, respectively.

Experiment 1 (3 hours treatment in the absence and presence of S-9 mix)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS	MF§					% RS	MF§
0		100	4.80			0		100	3.57
100		72	3.25		NS	100		96	3.14		NS
200		74	4.91		NS	200		69	2.75		NS
300		67	2.03		NS	300		62	3.91		NS
350		43	5.24		NS	400		62	4.65		NS
400		29	7.07		NS	500		21	2.87		NS
450		10	6.85		NS	550		7	6.33		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		44	44.33			2		52	21.21
0.15		51	56.73			3		23	56.04

§: 6TG resistant mutants/10⁶ viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

Experiment 2 (3 hours treatment in the absence and presence of S-9 mix)

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS		MF§				% RS		MF§
0		100		2.59		0		100		2.49
50	$$	80		1.85		100		127		3.84	NS
100		71		2.38	NS	200		76		3.36	NS
200		68		3.50	NS	400		45		2.70	NS
300		48		2.28	NS	450	$$	30		2.96
350		27		4.48	NS	500		13		5.28	NS
400		18		3.32	NS
450		10		10.10	*
Linear trend				*		Linear trend				NS
NQO						B[a]P
0.1		61		24.70		2		58		39.09
0.15		41		21.20		3		32		69.84

§: 6TG resistant mutants/ 10⁶ viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

* : Comparison of each treatment with control: Dunnett#s test (one-sided), significant at 5% level

*,**,*** : Test for linear trend: χ² (one-sided), significant at 5%, 1% and 0.1% level respectively

$$ : Treatment has marked heterogeneity for viability but is included for comparative purposes

Conclusions:

It is concluded that diethylamine, anhydrous did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).
Applying the RA approach, similar results are expected for the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test, mouse (equivalent or similar to OECD 474): negative

RA from diethylamine (CAS 109-89-7)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Scientific publication that meets documentation requirements. Study was performed according to NTP standard protocol.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 23.3 g (mean), female: 19.8 g (mean)
- Housing: individually in stainless steel wire bottom (Lab Products, Inc., Seaford, DE), changed weekly and rotated daily
- Diet: NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, except during exposure periods
- Water: Tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum
- Acclimation period: 12 days

Route of administration:

inhalation: vapour

Duration of treatment / exposure:

93 days

Frequency of treatment:

6 h/d, 5 d/w

Dose / conc.:

8 ppm (analytical)

Remarks:

(corresponding to 24 mg/m3)

Dose / conc.:

16 ppm (analytical)

Remarks:

(corresponding to 49 mg/m3)

Dose / conc.:

32 ppm (analytical)

Remarks:

(corresponding to 97 mg/m3)

Dose / conc.:

62 ppm (analytical)

Remarks:

(corresponding to 188 mg/m3)

Dose / conc.:

125 ppm (analytical)

Remarks:

(corresponding to 379 mg/m3)

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Tissues and cell types examined:

Peripheral blood

Details of tissue and slide preparation:

Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs; mature erythrocytes) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs; reticulocytes) in a population of 1,000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity.

Evaluation criteria:

In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above).

Statistics:

The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Following Treatment with Diethylamine by Inhalation for 13 Weeks:

Dose (ppm)

Micronucleated Normochromatic Erythrocytes/1000 cells

Male

Female

0

2.80 ± 0.30

2.60 ± 0.29

8

4.60 ± 0.60

2.50 ± 0.61

16

4.10 ± 0.48

2.20 ± 0.25

32

3.30 ± 0.34

3.50 ± 0.57

62

4.00 ± 0.52

3.80 ± 0.60

125

2.60 ± 0.33

2.20 ± 0.25

No significant increase in micronucleated NCEs was observed in males or females and all tested dose groups.

Conclusions:

Interpretation of results: negative
No significant increases in the frequencies of micronucleated NCEs were seen in peripheral blood of male or female mice from the 3-month study. The percentage of reticulocytes (PCEs) in the peripheral blood of male and female mice was unaltered by diethylamine exposure, suggesting a lack of chemical-associated bone marrow toxicity.

Executive summary:

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice exposed to 8 to 125 ppm (corresponding to 24 to 379 mg/m3) diethylamine by inhalation. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs; mature erythrocytes) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs; reticulocytes) in a population of 1,000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity. The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

No significant increases in the frequencies of micronucleated NCEs were seen in peripheral blood of male or female mice from the 3-month study. The percentage of reticulocytes (PCEs) in the peripheral blood of male and female mice was unaltered by diethylamine exposure, suggesting a lack of chemical-associated bone marrow toxicity.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

2003

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Dose / conc.:

8 ppm (analytical)

Remarks:

(corresponding to 24 mg/m3)

Dose / conc.:

16 ppm (analytical)

Remarks:

(corresponding to 49 mg/m3)

Dose / conc.:

32 ppm (analytical)

Remarks:

(corresponding to 97 mg/m3)

Dose / conc.:

62 ppm (analytical)

Remarks:

(corresponding to 188 mg/m3)

Dose / conc.:

125 ppm (analytical)

Remarks:

(corresponding to 379 mg/m3)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Following Treatment with Diethylamine by Inhalation for 13 Weeks:

Dose (ppm)

Micronucleated Normochromatic Erythrocytes/1000 cells

Male

Female

0

2.80 ± 0.30

2.60 ± 0.29

8

4.60 ± 0.60

2.50 ± 0.61

16

4.10 ± 0.48

2.20 ± 0.25

32

3.30 ± 0.34

3.50 ± 0.57

62

4.00 ± 0.52

3.80 ± 0.60

125

2.60 ± 0.33

2.20 ± 0.25

No significant increase in micronucleated NCEs was observed in males or females and all tested dose groups.

Conclusions:

Interpretation of results: negative
no significant increases in the frequencies of micronucleated NCEs were seen in peripheral blood of male or female mice from the 3-month study. The percentage of reticulocytes (PCEs) in the peripheral blood of male and female mice was unaltered by diethylamine exposure, suggesting a lack of chemical-associated bone marrow toxicity.
Applying the RA approach, similar results are expected for the target substance.

Executive summary:

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice exposed to 8 to 125 ppm (corresponding to 24 to 379 mg/m³) diethylamine by inhalation. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs; mature erythrocytes) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs; reticulocytes) in a population of 1,000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity. The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

No significant increases in the frequencies of micronucleated NCEs were seen in peripheral blood of male or female mice from the 3-month study. The percentage of reticulocytes (PCEs) in the peripheral blood of male and female mice was unaltered by diethylamine exposure, suggesting a lack of chemical-associated bone marrow toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for read-across

There are no data available on the genetic toxicity of diethylammonium chloride (CAS 660-68-4). Thus, read-across from an appropriate structural analogue substance (diethylamine, CAS 109-89-7) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard information requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.4. Common functional groups and structural similarities combined with similar toxicokinetic properties 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).

Genetic toxicity (mutagenicity) in bacteria in vitro

A bacterial gene mutation assay (Ames test) was performed with diethylamine (CAS 109-89-7) equivalent or similar to OECD 471 (reference 7.6.1-1). The S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were tested conducting the preinccubation method in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat and/or hamster liver S9-mix). The experiment was conducted in 3 replicates each in two independent experiments up to concentrations of 10000 µg/plate (salmonella, vehicle: water) and 4000 µg/plate (E .coli, vehicle: DMSO). Cytotoxicity was observed in all Salmonella strains from 3333 µg/plate onwards. Appropriate positive and solvent controls were included into the test and showed the expected results. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Thus, under the conditions of the study, no mutagenic activity was observed in any strain/activation combination in the bacterial Ames-Test for diethylamine.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro hprt test was conducted with diethylamine (CAS 109-89-7) in accordance with OECD 476 under GLP conditions (reference 7.6.1-2). L5178Y mouse lymphoma cells were cultured and treated with the test material or vehicle (DMSO) in the absence or presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix) in duplicates at concentrations of 50 to 731.4 µg/mL for 3 h (with and without S9-mix). Fixation and staining of the cells were performed 7 days after start of exposure with the test material. Cytotoxicity was assessed by determination of the mitotic index. Appropriate solvent and positive controls were included in the test and gave the expected results. Cytotoxicity was observed at concentrations above 400 µg/mL in the absence and at concentrations above 450 µg/mL in the presence of metabolic activation. Diethylamine did not induce mutations at the hprt locus of L5178Y mouse lymphoma cells, when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9). Thus, the test substance was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

 

Genetic toxicity (cytogenicity) in mammals in vivo

An in vivo chromosome aberration test (micronucleus assay) with diethylamine (CAS 109-89-7) was performed equivalent or similar to OECD 474 (reference 7.6.2-1). The test was conducted as part of a 90-day repeated dose inhalation study. The test material was administered for 90 days via inhalation to 5 B6C3F1 mice of each sex at concentrations of 8, 16, 32, 62 and 125 ppm (corresponding to 24, 49, 97, 188, 379 mg/m³). At the end of the treatment period peripheral blood samples were obtained from animals of all groups, smears were prepared, fixed in methanol, and stained with acridine orange. Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs; mature erythrocytes) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs; reticulocytes) in a population of 1000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity. No statistically or biologically significant increases in the incidence of micronucleated normochromatic erythrocytes compared to the vehicle control values were seen in either sex. The percentage of reticulocytes (PCEs) in the peripheral blood of male and female mice was unaltered by diethylamine exposure, suggesting a lack of chemical-associated bone marrow toxicity.

Overall conclusion for genetic toxicity

The available experimental data from in vitro and in vivo genetic toxicity from the analogue substance diethylamine (CAS 109-89-7) do not indicate any mutagenic or clastogenic potential in vitro and in vivo. Therefore following the analogue approach, diethylammonium chloride is considered to have no potential for genetic toxicity.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to diethylammonium chloride, data will be generated from information on reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Based on the available data, there is no indication that the source substance diethylamine has any mutagenic or clastogenic potential in vitro and in vivo. Applying the RA-A approach, similar results are expected for the target substance diethylammonium chloride which accordingly does not meet the criteria for classification for genetic toxicity according to Regulation (EC) 1272/2008.