Reaction mass of Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate and Octadec-9-en-1-yl ammonium mono- and di-butylphosphate

Genetic toxicity: in vivo

Administrative data

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

Study period:

The study were conducted between 10 December 2003 and 22 January 2004.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Octadec-9-en-1-yl ammonium mono and di-butylphosphate

Molecular formula:

C22H48NO4P - C26H56NO4P

IUPAC Name:

Octadec-9-en-1-yl ammonium mono and di-butylphosphate

Constituent 2

Reference substance name:

Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate

Molecular formula:

C30H64NO2PS2

IUPAC Name:

Octadec-9-en-1-yl ammonium di-n-hexyl phosphorodithioate

Test material form:

liquid: viscous

Details on test material:

Material is a yellow viscous liquid

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
Sufficient albino Crl:CD-1 ™(ICR)BR strain mice were supplied by Charles River (UK) Limited, Margate, Kent. At the start of the main test the male mice weighed 26 to 30g and were approximately five to eight weeks old. After a minimum acclimatisation period of seven days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
The animals were housed in groups of up to seven in solid-floor polypropylene cages with steel mesh bottoms suspended over absorbent paper. Free access to mains drinking water and food (Certified Rat and Mouse Diet 5LF2, BCM, IPS, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered 11ot to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

The vehicle was supplied by Analytical Supplies Limited, as follows:
Supplier's identification: Arachis oil
Supplier's lot number: T71
Safepharm serial number: V-2818
Date received: 28 May 2003
Description: Straw coloured slightly viscous liquid
Storage conditions: Room temperature

Details on exposure:

Range-finding Toxicity Test
A range-finding toxicity test was performed to determine a suitable dose level for the micronucleus test. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The range-finding toxicity study was also used to determine if the main study was to be performed using both sexes or males only. As a requirement of the protocol the oral route was not investigated.

All animals were dosed once only at the appropriate dose level using a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.

Animals were observed one hour after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.

Duration of treatment / exposure:

Single dose administered

Frequency of treatment:

Single dose administered

Post exposure period:

All animal.s were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

Doses / concentrations

Remarks:

Doses / Concentrations:
12, 18, 25, 50, 200, 500, 1000 and 2000 mg/kg
Basis:
other: administered intraperitoneally

No. of animals per sex per dose:

7 males per dose group

Control animals:

yes

Positive control(s):

The positive control material was supplied by Sigma-Aldrich, as follows:
Supplier's identification: Cyclophosphamide
Supplier's lot number: 91K1176
Safepharm serial number: R-2827
Date received: 10 June 2003
Storage conditions: 4°C in the dark
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire AGUETTANT batch no. F256207).
The concentration, homogeneity and stability of the positive control material and its preparation were not detennined by analysis.

Examinations

Tissues and cell types examined:

Micronucleated cells per 2000 polychromatic erythrocytes from bone marrow

Details of tissue and slide preparation:

Micronucleus Test
Groups, each of seven mice, were dosed once only via the intraperitoneal route with the test material at 4.5, 9 or 18 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test material at 18 mg/kg was killed after 48 hours. In addition, three further groups of mice were included in the study; two groups (seven mice) were dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.

Slide Preparation
Immediately following termination (i.e. 24 or 48 hours foliowing dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald /Giemsa, allowed to air-dry and coverslipped using mounting medium.

Slide Evaluation
Stained bone marrow smears were coded and examined blind using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCEblue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to nonnochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

Interpretation of Results
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.

If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.

A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

All data were statis tically analysed using appropriate statistical methods as recorrunended by the UK.EMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a ?(x + 1) transformation using Student's t-test (two tailed) and any significant results were confinned using the one way analysis of variance.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Range-finding Toxicity Test
In animals dosed with the test material via the intraperitoneal route premature deaths occurred at and above 25 mg/kg, and clinical signs were observed at and above 25 mg/kg as follows: hunched posture, ataxia, ptosis, lethargy, decreased respiratory rate, distended abdomen, emaciation, splayed gate, pilo-erection, pallor of the extremities, gasping respiration, hypothermia, prostration and laboured respiration.

The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated when administered via the intraperitoneal route, therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 18mg/kg, was selected for use in the main test, with 9 and 4.5 mg/kg as the lower dose levels.

Micronucleus Test
Mortality Data and Clinical Observations
There were no premature deaths seen in any of the dose groups. Only one clinical sign was observed in animals dosed with the test material at 18 mg/kg in the 48-hour group only, this was hunched posture.

Evaluation of Bone Marrow Slides
Statistically significant decreases in the PCE/NCE ratio were observed in the 24-hour 4.5 and 9 mg/kg test material dose groups when compared to their concurrent control group. With no statistically significant decreases in the PCE/NCE ratio being observed in either of the two 18 mg/kg dose groups the validity of the responses seen in the lower dose levels may be questioned. However, both of the 18 mg/kg dose groups had PCE/NCE ratio values lower than their concurrent vehicle controls and, therefore, the decreases in PCE/NCE ratios were taken to indicate that exposure to the bone marrow had been achieved.
There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

The positive control group showed a marked and statistically significant increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. However, it should be noted that the sample of cyclophosphamide used proved to be very toxic, as indicated by the NCE values and the significant reduction in the PCE/NCE ratio value. This also led to two animals (numbers 17 and 19) having very low individual score for micronucleated PCEs, however, the overall response was considered adequate and not to affect the integrity of the study. The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

In animals dosed with the test material via the intraperitoneal route premature deaths occurred at and above 25 mg/kg, and clinical signs were observed at and above 25 mg/kg as follows: hunched posture, ataxia, ptosis, lethargy, decreased respiratory rate, distended abdomen, emaciation, splayed gate, pilo-erection, pallor of the extremities, gasping respiration, hypothermia, prostration and laboured respiration.
The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated when administered via the intraperitoneal route, therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 18mg/kg, was selected for use in the main test, with 9 and 4.5 mg/kg as the lower dose levels.

Any other information on results incl. tables

Range-finding toxicity test

The mortality data are summarise below:

Dose level (mg/kg)	Sex	Number of animals treated	Route	Deaths on day			Total deaths
				0	1	2
2000	Male	1	ip	1e	-	-	2/2
	Female	1	ip	1	-	-
1000	Male	1	ip	1	-	-	2/2
	Female	1	ip	1	-	-
500	Male	1	ip	1c	-	-	2/2
	Female	1	ip	1e	-	-
200	Male	1	ip	0	1	-	2/2
	Female	1	ip	0	1	-
50	Male	1	ip	0	0	0	0/2
	Female	1	ip	0	0	0
25	Male	1	ip	0	1e	-	1/2
	Female	1	ip	0	0	0
18	Male	2	ip	0	0	0	0/2
12	Male	1	ip	0	0	0	0/2
	Female	1	ip	0	0	0