Oligomerisation products of ethylene oxide with reaction products of rape oil and ethanolamine

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 experimental phases of the study were performed between 20 January 2003 and 10 March 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 02/12/2002. Date of Signature: 13/02/2003

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

Description: Yellow slightly viscous liquid
Date received: 12 December 2002
Storage conditions: Room temperature, in the dark

Test animals

Species:

mouse

Strain:

other: Crl:CD-1(ICR)BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mice were supplied by Charles River (UK) Limited, Kent.
- Age at study initiation: Approximately five to eight weeks old.
- Weight at study initiation: 22 to 30g.
- Assigned to test groups randomly: yes
- Fasting period before study: Not reported.
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene cages with woodflake bedding.
- Diet (e.g. ad libitum): Free access to food (Certified Rat and Mouse Diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study.
- Acclimation period: Seven days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Set to acheive limits of 19 to 25°C
- Humidity (%): Set to acheive limits of 30 to 70%
- Air changes (per hr): Approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil used as vehicle control.

Details on exposure:

Groups, each of seven mice, were dosed once only via the intrapertoneal route (with a hypodermic needle attached to a graduated syringe) with the test material at 100, 200 or 400 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 400 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 cyclophoshamide, 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 groups were killed 24 hours following dosing.

Duration of treatment / exposure:

Dosed once.

Frequency of treatment:

Dosed once.

Post exposure period:

24 or 48 hours.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 200, 400 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): Known to produce micronuclei under the conditions of the test.
- Route of administration: Dosed orally.
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:

Erythrocytes in bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range-finding toxicity study was performed to determine a suitable dose level and route of administration for the micronucleus study.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Immediately following termination (24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centriguation and re-suspension.

DETAILS OF SLIDE PREPARATION:
The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue 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 were counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

OTHER:

Evaluation criteria:

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 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 statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committe on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √(x + 1) tranformation using Students t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs were observed in animals dosed at and above 200 mg/kg in both 24 and 48 hour groups.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In animals dosed with test material via the oral route no premature deaths or clinical signs were observed in animals dosed at the maximum recommneded dose of 2000 mg/kg.

In animals dosed with the test material via the intraperitoneal route, clinical signs were observed at and above 600 mg/kg as follows: hunched posture, pilo-erection, lethargy, ptosis, distended abdomen, hypothermia, pallor of the extremities, laboured respiration, ataxia, decreased respiratory rate, dehydration, emaciation and walking with splayed or tiptoe gate. The clinical signs observed in animals dosed with test material at and above 1000 mg/kg were considered so severe that animals were killed in extremis on grounds of animal welfare.

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 study. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration, therefore, this was selected for use in the main study. Because the clinical signs observed at 600 mg/kg were considered to be marginally too severe, the maximum tolerated dose (MTD) of the test material, selected for use in the main study was 400 mg/kg, with 200 and 100 mg/kg as the lower dose levels.


RESULTS OF DEFINITIVE STUDY
Mortality Data and Clinical Observations:
There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 200 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: hunched posture, ptosis and pallor of the extremities.

Evaluation of Bone Marrow Slides:
A summary of the results of the micronucleus study is given in Table 1 (see attached background material). Individual and group mean data are presented in Tables 2 to 8 (see attached background material).

No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 hour test material dose groups when compared to their concurrent control groups. However it was noted that a marked decrease in the PCE/NCE ratio was observed in the 48-hour 400 mg/kg and 200 mg/kg 24-hour test material groups. This, accompanied by the presence of clinical signs was taken to indicate that systemic absorption had occurred.

There was 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 increase in the incidence of micronucleated PCEs hence confirming the sensitivity of the system to the known mutagenic activity of cyclophospamide under the conditions of the test.

The test material was found not to produce significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Any other information on results incl. tables

Range-finding Toxicity Study:

The mortality data are summarised as follows:

Dose level (mg/kg)	Sex	Number of animals treated	Route	Deaths on day 0	Deaths on day 1	Deaths on day 2	Total deaths
2000	Male	1	ip	0	0	0	1/2
	Female	1		0	1e	-
1000	Male	1	ip	0	0	1e	2/2
	Female	1		0	0	1e
600	Male	1	ip	0	0	0	0/2
	Female	1		0	0	0
2000	Male	1	oral	0	0	0	0/2
	Female	1		0	0	0

 ip: Intraperitoneal

-: no data

e: animal killed in extremis

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test material was considered to be non-genotoxic under the conditions of the test.

Executive summary:

Introduction:

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complies with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Micronuncleus Test", Method B12 of the EC Commission Directive 2000/32/EC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods:

A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes; therefore the main study was performed using only male mice. The micronucleus study was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 400 mg/kg with 200 and 100 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NEC) erythrocyes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Results:

No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 hour test material dose groups when compared to their concurrent control groups. Though it was noted that a marked decrease in the PCE/NCE ratio was observed in the 48-hour 400 mg/kg and 200 mg/kg 24-hour test material dose groups. This, accompanied by the presence of clinical signs was taken to indicate that systemic absorption had occurred.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated PCEs hence confirming the sensitivity of the system to the known mutagenic activity of cyclophospamide under the conditions of the test.

Conclusion:

The test material was considered to be non-genotoxic under the conditions of the test.