[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study to GLP without any deviations.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

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

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Justification for type of information:

See read across justification document

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

reduction of activity, ruffled fur all animals both doses and treatment times

Vehicle controls validity:

valid

Positive controls validity:

valid

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Proban STi

IUPAC Name:

Proban STi

Constituent 2

Reference substance name:

-

EC Number:

436-230-7

EC Name:

-

Cas Number:

359406-89-6

Molecular formula:

This substance is a UVCB formed from the multiple reactions. It is not possible to provide a molecular formula.

IUPAC Name:

Phosphonium, tetrakis(hydroxymethyl)-, chloride (1:1), reaction products with 1-tetradecanamine and urea

Details on test material:

- Name of test material (as cited in study report): Proban STi

Test animals

Species:

rat

Strain:

other: Wistar Hanlbm: WIST (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Animal Breeding Services, CH-4414 Fuellinsdorf and Harlan Winkelmann GmbH, D-33178 Borchen
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 177.2-205.6 g
- Fasting period before study: 6 hours or overnight
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: from 4 October 2004 to 14 December 2004

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: acceptable solubility and non-toxicity to the animals
- Concentration of test material in vehicle: 10 ml/kg bw

Duration of treatment / exposure:

2 and 16 hours

Frequency of treatment:

once

Post exposure period:

not applicable

Doses / concentrations

Remarks:

Doses / Concentrations:
175 and 350 mg/kg
Basis:
actual ingested
based on dry content (65.8%)

No. of animals per sex per dose:

4 (males only)

Control animals:

yes

Positive control(s):

2 hours preparation interval: DMH (N,N'-dimethylhydazinedihydrochloride)
- Justification for choice of positive control(s): validated for this assay
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw / 10 ml/kg bw

16 hours preparation interval: 2-acetylaminofluorene
2 hours preparation interval: DMH (N,N'-dimethylhydazinedihydrochloride)
- Justification for choice of positive control(s): validatd for this assay
- Route of administration: oral gavage
- Doses / concentrations: 100 mg/kg bw / 10 ml/kg bw

Examinations

Tissues and cell types examined:

primary hepatocytes

Evaluation criteria:

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 (based on historical data)
A group average between 0 and 5 net grains is considered a marginal response. A dose-related increase in nuclear and net nuclear 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 result with less than 5 net grains.

Statistics:

not required.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

reduction of activity, ruffled fur all animals both doses and treatment times

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100-500 mg/kg
- Solubility: soluble in water at all dose levels
- Clinical signs of toxicity in test animals:
500 mg/kg: Mortality of 1/2 males and 2/2 females. Reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur, apathy in all animals 6 h post dosing.
350 mg/kg: Mortality of 2/2 females. Reduction of spontaneous activity, abdominal position, ruffled fur, apathy in all animals 6 h post dosing.
250 mg/kg: Reduction of spontaneous activity, abdominal position, ruffled fur, apathy in all animals 6 h post dosing.
100 mg/kg: Reduction of spontaneous activity in all animals 6 h post dosing.

Any other information on results incl. tables

The change in the urine colour of the treated animals could be due to the systemic distribution of the test substance, thus demonstrating its bioavailability.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test substance at any of the treatment periods or dose groups. The inter-individual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of historical controls.

No dose level of the test substance revealed UDS induction in the hepatocytes of the treated animals 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 substance for 2 hours or 16 hours. Therefore the net grain values obtained after treatment with the test substance were consistently negative. No substantial shift to higher values was obtained in the percentage of cells in repair.

Appropriate reference mutagens [DMHat 40 mg/kg and 2-AAF at 100 mg/kg] were used as positive controls.In vivotreatment withDMHor 2-AAF revealed distinct increases in the number of nuclear and net grain counts.

Toxic symptoms in the Main Experiment

2 hour exposure

Toxic reactions	Hours post-treatment 175 / 350 mg/kg bw
	1 h	2 h
Reduction of spontaneous activity	4/4	4/4
Ruffled fur	4/4	4/4

 

16 hour exposure

Toxic reactions	Hours post-treatment 175 / 350 mg/kg bw
	1 h	16 h
Reduction of spontaneous activity	4/4	4/4
Ruffled fur	4/4	4/4
Urine colour	No observations	Orange

Viability and number of hepatocytes

Treatment	Period	Animal No.	Viability (%)	Number of isolated cells (x 10^6)
Deionised water	2 h	1	71	530
		2	79	300
		4	85	468
175 mg/kg bw Proban STi	2 h	5	73	226
		6	74	266
		7	71	309
350 mg/kg bw Proban STi	2 h	9	78	312
		10	72	281
		11	74	289
40 mg/kg bwDMH	2 h	13	70	375
		14	75	375
		15	73	314
Deionised water	16 h	17	75	225
		19	72	241
		20	71	249
175 mg/kg bw Proban STi	16 h	21	74	363
		22	81	267
		23	75	315
350 mg/kg bw Proban STi	16 h	25	72	317
		26	70	361
		27	81	271
100 mg/kg bw 2-AAF	16 h	29	76	235
		30	74	252
		31	75	296

 

Mean Nucleus, Cytoplasmic Area and Net Grains

Preparation interval: 2 hours

Test group	Animal No.	Mean Nuclear Grain Count		Mean cytoplasmic Grain Count		Mean Net Nuclear Grain counts		Mean Nuclear Grains of Cells in Repair		% cells in repair
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
Deionised water	1	8.4	3.8	14.4	6.0	-6.0	5.5	0.0	0.0	0
	2	11.9	7.3	17.1	7.5	-5.2	5.4	8.8	4.9	4
	4	13.9	6.8	19.9	9.1	-5.9	6.2	5.3	0.6	3
	Mean	11.4	2.8	17.1	2.7	-5.7	0.5	4.7	4.4	2
175 mg/kg bw Proban STi	5	15.9	9.9	27.1	12.7	-11.3	9.8	13.0	9.9	2
	6	11.8	5.9	20.5	8.5	-8.7	7.7	7.0	3.5	3
	7	24.1	14.2	39.5	20.8	-15.3	14.2	17.5	3.5	2
	Mean	17.3	6.3	29.0	9.6	-11.8	3.3	12.5	5.3	2
350 mg/kg bw Proban STi	9	18.3	8.4	28.6	12.4	-10.3	8.7	7.3	1.5	3
	10	11.4	7.2	21.6	10.6	-10.3	8.6	10.0	0.0	1
	11	16.2	6.5	24.7	8.9	-8.5	7.1	5.7	1.2	3
	Mean	15.3	3.6	25.0	3.5	-9.7	1.0	.7	2.2	2
40 mg/kg bwDMH	13	26.5	16.9	22.1	15.4	4.4	11.1	13.4	7.7	45
	14	22.8	10.2	20.0	8.9	2.8	11.2	12.2	6.8	46
	15	34.0	15.9	20.3	8.0	13.8	13.2	20.0	10.6	70
	Mean	27.8	5.7	20.8	1.1	7.0	5.9	15.2	4.2	54

SD=Standard Deviation. SD for each animal is deviation between the analysed cells. SD for means is the deviation between results for the animals in group.

 

Mean Nucleus, Cytoplasmic Area and Net Grains

Preparation interval: 16 hours

Test group	Animal No.	Mean Nuclear Grain Count		Mean cytoplasmic Grain Count		Mean Net Nuclear Grain counts		Mean Nuclear Grains of Cells in Repair		% cells in repair
		Mean	SD	Mean	SD	Mean	SD	Mean	SD
Deionised water	17	11.5	5.3	16.4	6.2	-4.9	5.3	6.7	2.9	3
	19	17.8	7.4	29.0	11.5	-11.3	9.6	9.5	2.9	4
	20	14.2	6.0	21.8	8.1	-7.6	7.3	6.0	0.0	2
	Mean	14.4	3.1	22.4	6.3	-8.0	3.2	7.4	1.9	3
175 mg/kg bw Proban STi	21	10.3	4.6	21.0	9.2	-10.7	8.2	0.0	0.0	0
	22	16.9	7.1	28.8	11.5	-11.9	9.9	7.0	0.0	1
	23	17.8	6.9	30.3	11.2	-12.5	8.8	5.0	0.0	1
	Mean	15.0	4.1	26.7	5.0	-11.7	0.9	4.0	3.6	1
350 mg/kg bw Proban STi	25	20.4	9.7	28.6	10.9	-8.1	8.8	11.4	5.6	7
	26	18.0	8.4	29.1	13.3	-11.2	10.9	6.4	1.1	5
	27	28.2	11.1	36.4	15.1	-8.2	11.3	9.8	6.2	12
	Mean	22.2	5.3	31.4	4.4	-9.2	1.7	9.2	2.6	8
100 mg/kg bw 2-AAF	29	26.2	10.3	22.5	6.4	3.7	9.5	11.9	7.2	43
	30	42.1	18.2	31.2	12.5	10.9	15.0	19.1	12.0	64
	31	27.3	9.5	24.0	8.0	3.3	10.3	12.5	6.4	45
	Mean	31.9	8.9	25.9	4.6	6.0	4.3	14.5	4.0	51

SD=Standard Deviation. SD for each animal is deviation between the analysed cells. SD for means is the deviation between results for the animals in group.

Historical Control Data (1995-2002)

Test group	Net Grains		Treatment period	Number of animals
	Range	Mean
Vehicle controls	1.82 to -13.51	-5.64 +/- 2.27	2, 3 or 16 hours	326
Positive controls (DMH)	0.270 to 47.46	15.69 +/- 9.57	2 or 3 hours	125
Positive controls (2-AAF)	2.60 to 80.18	20.44 +/- 10.98	16 hours	200

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the experimental conditions reported, the test substance did not induce DNA damage leading to increased repair synthesis in the hepatocytes of treated rats.

Executive summary:

Proban STi was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The test substance was formulated in deionised water, which was used as the vehicle control. The test substance was administered orally by gavage at 175 and 350 mg/kg bw (dose volume 10 ml/kg bw). After a treatment period of 2 and 16 hours respectively, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to³HTdR (methyl-³H-thymidine), which is incorporated if UDS occurs.

The highest dose of the test substance administered in the main experiment (350 mg/kg) was estimated in pre-experiments to be close to the maximum tolerated dose. The main experiment was performed using male rats only, since the males could be dosed higher than females. At a dose of 350 mg/kg of the test substance, the treated females died. However, due to the close proximity of the maximum tolerated dose for the females (250 mg/kg) to that of the males (350 mg/kg) and the limited number of animals assessed, the difference in the toxicity observed in males and females is not considered significant.

The change in the urine colour of the treated animals could be due to the systemic distribution of the test substance, thus demonstrating its bioavailability.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test substance at any of the treatment periods or dose groups. The inter-individual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of historical controls.

No dose level of the test substance revealed UDS induction in the hepatocytes of the treated animals 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 substance for 2 hours or 16 hours. Therefore the net grain values obtained after treatment with the test substance were consistently negative. No substantial shift to higher values was obtained in the percentage of cells in repair.

Appropriate reference mutagens [DMH at 40 mg/kg and 2 -AAF at 100 mg/kg] were used as positive controls. In vivo treatment with DMH or 2 -AAF revealed distinct increases in the number of nuclear and net grain counts.

It was concluded that under the experimental conditions reported, the test substance did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats.