Diethylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative

Mammalian cell gene mutation assay/ HPRT test: negative

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

Study period:

Dec 2009 - Feb 2010

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)

Version / remarks:

July 2016

GLP compliance:

yes (incl. QA statement)

Remarks:

Quality Assurance statement, 4 May 2010

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.: DEJ30990H0SOURCE OF TEST MATERIAL
- Analytical purity: >= 99.9 %
- Expiration date of the lot/batch: 30 Oct. 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 25°C
- Solubility and stability of the test substance in the solvent/vehicle: preliminary solubility tests revealed limit concentration of 906.8 µg/mL in culture medium, solubility in DMSO of at least 90.68 mg/mL

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: solution preperation under subdued lighting by formulating the test article in DMSOn(vortex mixing if required); The solutions were protected from light and used within 2 hours

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:

Type and composition of metabolic activation system:
- source of S9 : Molecular Toxicology Incorporated, USA; Sprague Dawley rats induced with Aroclor 1254
- method of preparation of S9 mix : G6P (180 mg/mL), NADP (25 mg/mL), KCl (150mM) and S9 were mixed in ratio 1:1:1:2
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of S9-mix to each cell culture (19 mL)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility test, protein content analysis, test for presence of adventitious agents, promutagen activation

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:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

4-nitroquinoline 1-oxide: 0.10/0.15 µg/ml (without S-9 mix); Benzo[a]pyrene: 2.00/3.00 µg/ml (with S-9 mix)

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 (single cultures used for the positive control treatments)

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.

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% RS, respectively. In the presence of S-9, no concentration gave 10 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.

Tab. 1 Summary of mutation data experiment 1

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

Tab. 2 Summary of mutation data experiment 2

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:

The test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells under the tested conditions.

Executive summary:

The test substance was investigated for mutation at the hprt locus in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment followed by two independent experiments (with and without rat S9 metabolic activation). The test article was formulated in DMSO. In the range-finding study concentrations of 22.86 - 731.4 µg/mL were tested. The highest concentration to provide more than 10% survival was 365.7 µg/mL. In experiment 1 ten concentrations (100 - 731.4µg/mL) were tested in the absence and presence of metabolic activation. Seven days after treatment a concentration of 450 µg/mL without S9-mix and 550 µg/mL with S9-mix gave 10% and 7% relative survival, respectively. In experiment 2, concentrations of 50 - 731.4 µg/mL without S9 -mix and 100 - 731.4 µg/mL with S9 mix were tested. Relative survival of 10% and 13% were determined for 450 µg/mL with S9-mix and 500 µg/mL without S9-mix, respectively. Positive and negative control data indicated the study validity. Testing with concentrations up to toxicity (450 µg/mL) in the abscence of S9 in experiment 2 revealed a significant increase in mutant frequency. However, the result was not reproducable with experiment 1and according to the acceptance criteria not of biological relevance. In conclusion, the test substance did not induce mutation at the hprt locus under the test conditions employed in this study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Acceptable, well documented NTP study, which meets general standards.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

yes

Remarks:

Only Salmonella strains tested, 4 tested bacteria strains, 2-Aminoanthracene as sole indicator of efficacy of the S9-mix

Principles of method if other than guideline:

Testing was performed as reported by Mortelmans et al. (1986).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Analytical purity: 99 %

Target gene:

Histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333, 10000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: without S9: 4-nitro-o-phenylenediamine (TA98 ); with S9: 2-aminoanthracene

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.

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 in all strains tested

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Tab. 1 Mutagenicity in bacterial tester strains

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

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:

Under the conditions tested, no mutagenic activity was observed in any strain/activation combination in the bacterial AMES-Test. The positive controls showed the expected values.

Executive summary:

The test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. Doses of 0, 33, 100, 333, 1000, 3333, and 10000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 3333 µg/plate.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: NTP standard protocol - contains 5th strain for Ames test Key-study ( E. coli WP2 uvr A pKM 101)

Principles of method if other than guideline:

Testing was performed as reported by Mortelmans et al. (1986)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Analytical purity: 99 %

Target gene:

Histidine

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

S. typhimurium TA 98, TA 100: 0, 50, 100, 250, 500, 750, 1000, 2000 µg/plate
E. coli pKM 101: 0, 50, 100, 500, 1000, 2000, 3000, 4000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: TA98: 4-nitro-o-phenylenediamine, E.coli WP2 uvrA: methyl methanesulfonate, all strains: 2-aminoanthracene with metabolic activation

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 .

Species / strain:

other: S. typhimurium TA 98, TA 100, E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1000 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Tab. 1 Mutagenicity in bacterial tester strains

Strain: eColi pKM101

Dose	NA (Negative)		10% RLI (Negative)		NA (Negative)		10% RLI (Negative)
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	132	9.1	157	2.4	122	6.7	139	3.3
50	186	13.9			132	9.1	155	5.5
100	152	14.2			158	12.5	172	10.5
500	136	4.6	201	11.9	140	3.3	151	5
1000	105	4.8	199	30.6	67	0.3	161	7.2
1500	150	12.2
2000	18	8.6	182	4.6	0	0	178	14.4
3000			110	8.5
4000			0	0
Positive Control	1101	54.8	734	6.9	812	14.4	711	30.7

 

Strain: TA100

Dose	10% RLI (Negative)		NA (Negative)		10% RLI (Negative)		NA (Negative)
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	84	2.6	61	4	56	4.4	51	6
50	90	6.7	63	6.6	51	6.4	65	2.1
100	105	10.3	55	2.1	66	0.9	63	7
250							64	1.9
500	90	4.3	54	3.3	57	4	48	4
750							55	4.7
1000	76	0.9	t		47	5.8
2000	83	7.5	t		63	8.5
Positive Control	768	36.1	553	13.8	609	29.4	609	28.8

 

Strain: TA98

Dose	NA (Negative)		10% RLI (Negative)		NA (Negative)		10% RLI (Negative)
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	20	0.7	30	0.9	20	2.4	25	2.7
50	17	1.2	40	2.4	17	1.2	27	1.9
100	19	0.3	29	2	15	1	22	1.5
250					12	3.7
500	24	1.2	38	2.1	13	2.3	29	3.8
750					13	0.9
1000	t		36	4			19	2.1
2000	t		27	0.9			25	3.5
Positive Control	417	14.8	752	58.3	572	8.4	1137	18.8

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:

Under the conditions tested, no mutagenic activity was observed in any strain/activation combination in the bacterial AMES-Test. The positive controls showed the expected values.

Executive summary:

In a second Ames test conducted under NTP standard conditions (NTP, 2005), S. typhimurium TA 98 and TA 100 strains were treated with doses of 0, 50, 100, 250, 500, 750, 1000, 2000 µg/plate and E. coli WP2 uvr A pKM 101 with 0, 50, 100, 500, 1000, 2000, 3000, 4000 µg/plate. The results confirm the negative mutagenic potential of the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus Test: negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2002 - Mar 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Peripheral blood samples were obtained from male and female B6C3F1 mice at the end of a 14-week toxicity study. Smears were immediately prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y and coded. Slides were scanned at 630 or 1000 times magnification using a semi-automated image analysis system to determine the frequency of micronuclei in 10000 normochromatic erythrocytes (NCEs) in each of 10 animals per dose group. A detailed discussion of this assay can be found in MacGregor et al. (1990).

GLP compliance:

no

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Physical state: colorless liquid with a strong ammonia odor
- Analytical purity: approximately 99.9%
- Lot/batch No.: BE/07/01
- Stability under test conditions: no degradation of the bulk chemical was detected
- Storage condition of test material: at controlled room temperature

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


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C (72°±3 °F)
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: vapour

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation chamber (Harvord Systems Division of Lab Products, Inc. Aberdeen, MD)
- Method of conditioning air: glass beads in a heated glass coloum for vapourization
- Air flow rate: 15 air changes /h

Duration of treatment / exposure:

14 weeks (93 days exposure)

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

8 ppm (nominal)

Remarks:

analytical concentration 8 ± 0.3 ppm

Dose / conc.:

16 ppm (nominal)

Remarks:

analytical concentration 15.9 ± 0.6 ppm

Dose / conc.:

32 ppm (nominal)

Remarks:

analytical concentration 32 ± 1.3 ppm

Dose / conc.:

62 ppm (nominal)

Remarks:

analytical concentration 62.2 ± 2.3 ppm

Dose / conc.:

125 ppm (nominal)

Remarks:

analytical concentration 126 ± 5 ppm

No. of animals per sex per dose:

5

Control animals:

yes

Tissues and cell types examined:

Peripheral blood
Justification: According to the OECD 474, the use of peripheral blood is a valid alternative in mice, if the treatment time exceeds 4 weeks. Since the animals were treated for 90 days, peripheral blood can be used instead of bone marrow.

Statistics:

The frequency of micronucleated cells among normochromatic cells was analysed by 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.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Tab. 1 Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes

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.

 

The results from the vehicle control group as well as all treated groups were within the negative control range of micronucleated cells obtained by the lab (1.8 - 5.3 for males, 0.7 - 5.1 for females). The data have been compiled in the table below.

 

Tab. 2: Results for the vehicle control groups obtained by ILS Inc.

Year	Report	Male	Female	RoE
2011	TR564	2 +/- 0.61	1.8 +/- 0.25	Inhalation
2014	TR581	2.4 +/-0.33	2.5 +/-0.35	Inhalation
2008	TR552	3.4 +/-0.54	4 +/- 0.47	Inhalation
2009	TR 542	2.4 +/- 0.69	2.3 +/- 0.4	Inhalation
2007	TR 543	5.3 +/- 0.5	5.1 +/- 0.46	Inhalation
2011	TR 567	2.8 +/- 0.2	1.7 +/- 0.2	gavage
2011	TR 570	1.9 +/-0.19	0.7 +/- 0.2	gavage
2011	TR565	4.6 +/- 0.58	3.7 +/- 0.46	feed
2011	tox076	1.8 +/- 0.29	2 +/- 0.26	gavage

 

No concurrent positive control was included, but positive results have been reported by the laboratory. E.g, inhalation exposure for 90-days with α-Methylstyrene results in 9.1 micronucleated NCEs / 1000 NCEs in peripheral blood (TR 543). 

Conclusions:

There was no increase in the percentage of micronucleated cells compared to the vehicle control group. Additionally, all results were within the negative control range. Consequently, the substance did not cause chromosome damage in this study.

Executive summary:

In a micronucleus assay with B6C3F1 mice, peripheral blood samples were obtained from 5 male/female animals at the end of a 93 days inhalation toxicity study (6 hours per day, 5 days per week; doses of 0, 8, 16, 32, 62, 125 ppm). No significant increase in micronucleated normochromatic erythrocytes was observed in males or females and all tested dose groups.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies:

The test substance was evaluated for mutagenicity in the Salmonella/microsome preincubation assay (NTP 1987) using a standard protocol approved by the National Toxicology Program. Doses of 0, 33, 100, 333, 1000, 3333, and 10000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 3333 µg/plate. In a second Ames test (NTP 2005) performed under NTP standard conditions, S. typhimurium TA 98 and TA 100 strains were treated with doses of 0, 50, 100, 250, 500, 750, 1000, 2000 µg/plate and E. coli WP2 uvr A pKM 101 with 0, 50, 100, 500, 1000, 2000, 3000, 4000 µg/plate confirmed these negative results. Further standard Ames tests with two strains or three strains revealed no evidence of mutagenicity in the bacterium Salmonella typhimurium either in the presence or absence of a mammalian liver metabolic activation system.

One negative GLP-compliant in vitro Mammalian Cell Gene Mutation Test according to OECD TG 476 is also available. The study was conducted in mammalian cells (L5178Y cells) at a test substance concentration range from 45.71 to 731.4 µg/ml with or without S-9 mix (2010, reliability 1).

 

In vivo study:

In a micronucleus assay with B6C3F1 mice, peripheral blood samples were obtained from 5 male/female animals at the end of a 13-week inhalation toxicity study (6 hours per day, 5 days per week; doses of 0, 8, 16, 32, 62, 125 ppm). No significant increase in micronucleated erythrocytes was observed in males or females and all tested dose groups.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity was observed in in-vitro or in-vivo studies. Additionally, no increase in neoplastic lesions was observed in rats or mice in two cancer studies equivalent to OECD 451 (NTP, 2005). This further supports the conclusion that DEA does not cause DNA damage.

As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480