Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo, OECD 486

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.07.2006 - 29.05.2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

circa. 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Didecyldimethylammonium chloride

EC Number:

230-525-2

EC Name:

Didecyldimethylammonium chloride

Cas Number:

7173-51-5

Molecular formula:

C22H48N Cl

IUPAC Name:

N,N-Didecyl-N,N-dimethylammonium chloride

Test material form:

liquid: viscous

Details on test material:

- Didecyldimethylammonium chloride (DDAC)
- EC number: 230-525-2

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Hanlbm: WIST (SPF)

Details on species / strain selection:

The rat is an animal which has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the I-JDS test. In addition, the rat is an experimental animal in many physiological, pharmacological, and toxicological studies. Data from such experiments may also be useful for the design and the performance of the UDS test.

Sex:

male

Details on test animals or test system and environmental conditions:

Source: Harlan Winkelmann GmbH, D-33178 Borchen
Number of animals: 32 (males)
Acclimatisation and quarantine: minimum 5 days
Age of the animals: 6 - 10 weeks
Initial body weight at start of treatment: Mean value 179.4 g (SD* ± 14.8 g)
* SD = Standard deviation

According to the supplier's assurance the animals were in healthy condition. The animals underwent quarantine in the animal house of RCC-CCR for at least five days after their arrival. During this period the animals did not show signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage type: Makrolon Type Il, with wire mesh top (Ehret, D-79312 Emmendingen)
Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
Drinking water: tap water, ad libitum (Gemeindewerke Roßdorf, D-64380 Roßdorf)

Environment:
temperature 21 ± 30 C
relative humidity 30 - 72 %
artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

30% DMSO/70% PEG 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 30% DMSO /70% PEG 400 and heated to 37C. The vehicle was chosen to its non-toxicity for the animals. The animals received a single standar volume of 10 ml/kg bw orally.

Duration of treatment / exposure:

Test subsance administered in a single treatment.

Frequency of treatment:

Test subsance administered in a single treatment.

Post exposure period:

1, 2-4, 6 and 24 h after administrationof the test item

No. of animals per sex per dose:

Preliminary experiments: 2 animals per sex per dose group per dose per timepoint.
Main experiment: 4 male sex per dose per timepoint

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control 4hr: DMH; N,N'-dimethylhydrazinedihydrochloride 80 mg/kg bw
Positive control 16hr: 2-AAF; 2-acetylaminofluorene 100 mg/kg bw

Examinations

Tissues and cell types examined:

Primary Hepatocytes

Details of tissue and slide preparation:

Isolation of the Primary Hepatocytes
8.4.5.
After anaesthetising the rats with 46% Ketamin (Ketavet 100, Pharmacia GmbH, D-76139 Karlsruhe), 23% Xylazin (Rompun 2%, Bayer HealthCare, D-51368 Leverkusen) and 31% Midazolam (Dormicum, Hoffmann LaRoche, D-79639 Grenzach-Wyhlen) (approx. 2 mL/kg b.w.) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Invitrogen, D-76344 Eggenstein) supplemented with collagenase (0.05 % (w/v), Roche Diagnostics, D-68305 Mannheim) adjusted to pH 7.4 and maintained at 370 C (8).
The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh (94 pm) to yield a single cell suspension. The quality of the performed perfusion was determined by the trypan blue dye exclusion method for cell viability. In addition, the number of the cells was determined.

8.4.6.
Culture Conditions
The washed hepatocytes were centrifuged and transferred into Williams medium E (WME, Invitrogen, D-76344 Eggenstein) supplemented (I) with:
Hepes 2.38 mg/ml
Penicillin 100 units/ml
Streptomycin 0.10 mg/ml
L-GIutamine 0.29 mg/ml
Insulin 0.50 ug/ml
Fetal calf serum (FCS) 100 ul/ml

This complete medium was adjusted to pH 7.6.
At least three cultures were established from each animal. Aliquots of 2.5 ml with freshly isolated hepatocytes in complete culture medium (2.0 x 105 viable cells/ml) were added to 35 mm six-well dishes (Greiner, D-72603 Nürtingen) containing one 25 mm round plastic coverslip (Thermanox, Nunc, D-65203 Wiesbaden) per well coated with gelatine. After an attachment period of approximately 1.5 h in a 95 % air/ 5 % C02 humidified incubator at 370 C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells (9). Subsequently, 3HTdR (5 pCi/ml, specific activity 20 Ci/mmol; New England Nuclear, D-63033 Dreieich) in 2.0 ml culture medium
(WME, I % (v/v) FCS) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with I % (wv) FCS and 0.25 rnM unlabelled thymidine. Cultures were incubated overnight using the same medium (2). To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection (9). The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 wv) for 20 minutes each, rinsed with 96 % (VIV) ethanol, and air-dried.

8.4.7. Autoradiographic Processing
The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK N TB (Tecnomara, D-35463 Fernwald) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 40 C. The photographic emulsion was then developed with Ilford Phenisol (Ilford Imaging GmbH, 63265 Dreieich) at room temperature, fixed in Rapid Fixer (llford Imaging GmbH, 63265 Dreieich) and stained with hematoxylin/eosin.

8.4.8. Quantification of I-JDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the Sorcerer I-JDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of the most heavily labelled nuclear-sized cytoplasm area adjacent to the nucleus was counted (2). At least two slides per animal and 50 cells per slide were evaluated. Heavily radiolabelled cells undergoing replicative DNA synthesis were excluded from counting.
Three animals per group were evaluated as described above.

8.4.9. Data Recording
The data generated were recorded in the raw data. The results were presented in tabular form, including experimental groups with the test item, vehicle and positive controls.
The nuclear and cytoplasmic grain counts, the net grain counts (nuclear minus cytoplasmic grains) as well as the mean and percentage of cells in repair (cells with a net grain count larger than 5) are reported separately (5). Individual slide and animal data are provided. The mean counts with standard deviation are used to describe the distribution of 3HTdR incorporation in the nucleus, the cytoplasm and for the net grains, respectively.

Evaluation criteria:

Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains. Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test (4).
A test item producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system.

Results and discussion

Test results

Test results 1

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

ruffled fur, reduction in spontaneous activity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

ruffled fur, reductions in spontaneous activity, eye lid closure

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

8.6. Historical Controls (1999-2005)

59 finalised studies

Test group	Net grains		Treatment period	Number of animals
	Range	Mean
Vehicle control*	-14.3 to -3.2	-6.7±2.2	2 or 16 hrs	352
Positive controls (DMH)	3.1 to 39.1	13.4± 7.5	2 hrs	176
Positive controls (2-AAF)	2.9 to 35.9	15.1 ± 7.3	16 hrs	175

* : Vehicles: deionised water, sesame oil, corn oil, PEG 400, CMC (0.5-1.0%).

 

9. BIOMETRY

A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

 

10. RESULTS

10.1. Pre-Experiment

In the first pre-experiment 2 male and 2 females rats received orally a single dose of 200 mg/kg b.w. of Arquad 2C formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 ml/kg b.w..

10.1.1 The treated animals expressed toxic reactions as shown in the table:

toxic reactions	hours post-treatment male / female
	1h	2-4h	6h	24h
ruffled fur	0/0	2/0	1/0	0/0

 

In the second pre-experiment 2 male and 2 females rats received orally a single dose of 1000 mg/kg b.w. of Arquad 2C formulated in DMSO / PEG 400. The volume administered was 10 ml/kg b.w..

10.1.2 The treated animals expressed toxic reactions as shown in the table:

toxic reactions	hours post-treatment male / female
	1h	2-4h	6h	24h
reduction of spontaneous activity	2/1	2/1	-/-	2/0
abdominal position	0/0	1/0	-/-	2/0
eyelid closure	2/0	2/0	-/-	0/0
ruffled fur	2/2	2/2	-/-	2/2
breathing difficulty	0/0	0/0	-/-	2/0
bloody mouth	0/0	0/0	-/-	2/0
diarrhoea	0/0	0/0	-/-	2/0

-/-: no observation made.

In the third pre-experiment 2 male and 2 females rats received orally a single dose of 1250 mg/kg b.w. of Arquad 2C formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 ml/kg b.w..

10.1.3 The treated animals expressed toxic reactions as shown in the table:

toxic reactions	hours post-treatment male / female
	1h	2-4h	6h	24h
reduction of spontaneous activity	2/2	2/2	2/2	1/1
eyelid closure	2/0	2/0	2/0	1/0
ruffled fur	2/2	2/2	2/2	1/1
breathing difficulty	0/0	1/1	1/1	0/0
death	0/0	0/0	0/0	1/1

 

On the basis of these data the dose of 1000 mg/kg b.w. was estimated to be suitable as the high dose

 

10.2. Toxic symptoms in the Main Experiment

In the main experiment with the 4 h treatment period 4 animals received orally a single dose of 500 and 1000 mg/kg b.w.. Arquad 2C formulated in 30% DMSO / 70% PEG 400.

The volume administered was 10 ml/kg b.w..

10.2.1 The treated animals expressed toxic reactions as shown in the table:

toxic reactions	hours post-treatment 500 / 1000 mg/kg b.w.
	1h	2h	4h
reduction of spontaneous activity	2/3	2/4	4/4
eyelid closure	2/4	2/4	4/4

 

In the main experiment with the 16 h treatment period 4 animals received orally a single dose of 500 and 1000 mg/kg b.w.. Arquad 2C formulated in 30% DMSO / 70% PEG 400. The volume administered was 10 ml/kg b.w..

10.2.2 The treated animals expressed toxic reactions as shown in the table:

toxic reactions	hours post-treatment 500 / 1000 mg/kg b.w.
	1h	2-4h	16h
reduction of spontaneous activity	4/4	-/-	4/3
ruffled fur	2/4	-/-	4/3
breathing difficulty	0/1	-/-	0/1
diarrhoea	0/0	-/-	4/3
death	0/0	-/-	0/1

-/-: no observation made.

 

10.3 Viability of Hepatocytes

	Treatment	Animal no.	Viability* [%]	Number of isolated cells [x10^6]
Vehicle control 30% DMSO / 70% PEG 400	4 h	1	78	452
		2	88	350
		3	73	297
		4	74	275
500 mg/kg b.w. Arquad 2C	4 h	5	83	314
		6	Perfusion failed
		7	86	306
		8	78	359
1000 mg/kg b.w. Arquad 2C	4 h	9	84	299
		10	80	177
		11	78	227
		12	78	234
Positive control DMH 80 mg/kg b.w.	4 h	13	76	396
		14	87	375
		15	88	303
		16	83	381
Vehicle control 30% DMSO / 70% PEG 400	16 h	17	93	498
		18	75	377
		19	Perfusion failed
		20	96	251
500 mg/kg b.w. Arquad 2C	16 h	21	76	186
		22	Perfusion failed
		23	82	453
		24	80	314
1000 mg/kg b.w. Arquad 2C	16 h	25	72	351
		26	77	254
		27	Animal died
		28	91
Positive control 2-AAF 100 mg/kg b.w.	16 h	29	Animal died#
		30	79	346
		31	79	249
		32	85	391

*Viability determined by means of trypan blue dye exclusion assay

#application failure

One spare animal was included for each dose group.

 

Table 10.4.1  Mean nucleus, cytoplasmic area, and net grains at 4 hr

Test group	Animal No.	Mean Nuclear Grain Count		Mean Cytoplasmic grain count§		Mean Net Grain Counts		Mean Nuclear Grains of Cells in Repair		% Cells  in Repair
		Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.
Vehicle control 30% DMSO / 70% PEG 400	1	8.14	3.73	14.82	5.55	-6.68	5.2	5	0	1
	2	13.43	5.95	18.71	7.67	-5.28	7	6.75	1.71	4
	4	6.83	3.85	12.4	4.35	-5.57	4	0	0	0
	Mean	9.47	3.49	15.31	3.18	-5.84	0.74	3.92	3.5	2
500 mg/kg b.w. Arquad 2C	5	5.41	2.63	9.86	2.59	-4.45	3.51	0	0	0
	7	6.01	2.85	10.24	2.92	4.23	3.09	0	0	0
	8	5.27	2.55	9.07	2.59	-3.8	3.1	6	0	1
	Mean	5.56	0.39	9.72	0.6	416	0.33	2	3.46	0
1000 mg/kg b.w. Arquad 2C	9	7.93	3.73	16.13	4.34	-8.2	4.42	5	0	1
	11	8.93	4.44	16.8	6.53	-7.87	6.73	8	0	1
	12	6.02	3.02	12.77	3.71	-6.75	4.03	0	0	0
	Mean	7.63	1.48	15.23	2.16	-7.61	0.76	433	4.04	1
Positive control DMH 80 mg/kg b.w.	13	39.6	12.08	14.5	4.98	25.1	10.58	25.75	10.05	97
	14	43.93	11.69	15.89	6.32	28.04	11.06	28.32	10.75	99
	15	31.94	10.34	11.38	3.73	20.56	9.03	20.76	8.85	99
	Mean	38.49	6.07	13.92	2.31	24.57	3.77	2494	3.85	98

SD = Standard deviation The Standard deviation shown for a each animal is tha deviation between the 100 analysed cells. The deviation shown for the mean of each group is the standard deviation between the results obtained for each test group consisting Of three animals.

 

Table 10.4.2  Mean nucleus, cytoplasmic area, and net grains at 16 hr

Test group	Animal No.	Mean Nuclear Grain Count		Mean Cytoplasmic grain count§		Mean Net Grain Counts		Mean Nuclear Grains of Cells in Repair		% Cells  in Repair
		Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.
Vehicle control 30% DMSO / 70% PEG 400	17	10.49	3.99	14.02	5.22	-3.53	4.75	6.5	1	4
	18	14.13	7.31	20.1	8.92	-5.97	8.24	8.88	3.68	8
	20	14.73	7.2	20.71	7.68	-5.98	5.95	8.33	2.08	3
	Mean	13.12	2.29	18.28	3.7	-5.16	1.41	7.9	1.24	5
500 mg/kg b.w. Arquad 2C	21	6.88	3.59	10.76	4.11	-3.88	4.89	6	1	3
	23	10.72	5.85	16	5.97	-5.28	5.9	11.67	5.77	3
	24	10.08	5.11	15	5.51	-4.92	5.27	6.5	2.38	4
	Mean	9.23	2.06	13.92	2.78	-4.69	0.73	8.06	3.14	3
1000 mg/kg b.w. Arquad 2C	25	12.55	6.53	17.41	6.69	-4.86	7.41	7.45	2.46	11
	26	12.41	6	15.5	6.79	-3.09	6.47	7.11	2.32	9
	28	11.63	7.47	15.13	8.26	-3.49	6.28	6.5	2.19	11
	Mean	12.2	0.49	16.01	1.22	-3.81	0.93	7.02	0.48	10
Positive control 2-AAF 100 mg/kg b.w.	30	20.57	6.17	12.45	488	8.12	5.66	1042	4.52	74
	31	37.69	15.57	25.94	9.56	11.75	11.32	16.23	8.07	77
	32	31.1	16.16	20.71	11.07	10.39	10.32	14.43	9.08	72
	Mean	29.79	8.64	19.7	6.8	10.09	1.83	13.69	2.98	74

SD = Standard deviation The Standard deviation shown for a each animal is tha deviation between the 100 analysed cells. The deviation shown for the mean of each group is the standard deviation between the results obtained for each test group consisting Of three animals.

 

 

1. ASHBY, J., LEVEVRE, P.A., BURLINSON, B., PENMAN, M.G., 1985
An assessment of the in vivo rat hepatocyte DNA-repair assay
Mutation Res., 156, 1-18
2. BUTTERWORTH, B.E., ASHBY, J., BERMUDEZ, E., CASCIANO, DA, MIRSALIS, J.,
PROBST, G., WILLIAMS, G. M., 1987
A study plan and guide for the in vitro rat hepatocyte DNA-repair assay
Mutation Res., 189, 113-121
3. FAUTZ, R., HUSEIN, B., EFSTATHIOU, E., HECHENBERGER-FREUDL, C., 1993
Assessment of the relation between the initial viability and the attachment of freshly
isolated rat hepatocytes used for the in vivo / in vitro DNA-repair assay (I-JDS)
Mutation Res., 291, 21-27
4. KRAUTH, J.. 1971
Locally most powerful tied rank test in a Wilcoxon situation
Annals of Mathematical Statistics, 42, 1949-1956
5. LONATI-GALLIGANI, M., LOHMAN, PH M., BEHRENDS, F.. 1983
The validity of the autoradiographic method for detecting DNA-repair synthesis in rat
hepatocytes in primary culture
Mutation Res., 113, 145-160
6. MIRSALIS, J.C., BUTTERWORTH, B.E., 1980
Detection of unscheduled DNA-synthesis in hepatocytes from rats treated with
genotoxic agents: an in vivo / in vitro assay for potential mutagens and carcinogens
Carcinogenesis, 1, 621-625
7. MIRSALIS, J.C., TYSON, K.c., BUTTERWORTH, B.E., 1982
Detection of genotoxic carcinogens in the in vivo / in vitro hepatocyte DNA repair assay
Env. Mutagenesis, 4, 553-562
8. SEGLEN, p.o., 1976
Preparation of isolated rat liver cells
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9. WILLIAMS, G.M., 1977
Detection of chemical carcinogens by unscheduled DNA-synthesis in primary rat liver
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Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.
Therefore, Arquad 2C is considered to be non-genotoxic in this in vivo/ in vitro I-JDS test system.

Executive summary:

The test item Arquad 2C was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats with doses of 500 mg/kg b.w. and 1000 mg/kg b.w. (4 h and 16 h preparation interval).

The test item was formulated in 30% DMSO / 70% PEG 400, which was used as vehicle control. The volume administered orally was 10 ml/kg body weight. After a treatment period of 4 and 16 hours, respectively, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR (methyl-3H-thymidine) which is incorporated if UDS occurs (2).

The highest dose was estimated in a pre-experiment to be the maximum applicable dose, at which clinical signs of toxicity occurred. In the main experiment one animal died during the 16 h preparation interval.

The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test item at any of the treatment periods or dose groups. The interindividual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of our historical laboratory control (3).

No dose level of the test item revealed UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. Neither the nuclear grains nor the resulting net grains were distinctly enhanced due to the in vivo treatment of the animals with the test item for 4 hours or 16 hours, respectively. Therefore, the net grain values obtained after treatment with the test item were consistently negative. 

In addition, the obtained percentage of cells in repair were within the historical control range (up to 12%). Due to a great variation in the percentages of cells (26 and 4% for slides 28a and 28b, respectively) in repair observed in the two slides scored for animal no. 28, a third slide was additionally scored. Thus, for animal no. 28 a total of 150 cells were scored instead of 100. The data from the third slide (2% cells in repair) confirmed the value obtained from slide 28b (4% cells in repair). Thus, the observed increase in the percentage of cells in repair in slide 28a is not reproducible and, therefore, not biologically relevant.

Appropriate reference mutagens [DMH (10), 80 mg/kg b.w. and 2-AAF, 100 mg/kg b.w.] were used as positive controls. In vivo treatment with DMA or 2-AAF revealed distinct increases in the number of nuclear and net grain counts.