Fatty acids, C12-18 (even numbered)-methyl esters, sulfonated, sodium salts

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 1994 to 25 February 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

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)

Version / remarks:

Commission Directive 92/69/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: damage to chromosomes and/or aneuploidy

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C12-18, α-sulfo, 1-Me esters, sodium salts

EC Number:

288-224-7

EC Name:

Fatty acids, C12-18, α-sulfo, 1-Me esters, sodium salts

Cas Number:

85681-86-3

Molecular formula:

C13H25NaO5S - C19H37NaO5S

IUPAC Name:

Fatty acids, C12-18, α-sulfo, 1-Me esters, sodium salts

Constituent 2

Reference substance name:

Fatty acids, C12-18, α-sulfo, disodium salts

EC Number:

286-085-7

EC Name:

Fatty acids, C12-18, α-sulfo, disodium salts

Cas Number:

85186-99-8

Molecular formula:

C12H22Na2O5S - C18H34Na2O5S

IUPAC Name:

Fatty acids, C12-18,α-sulfo, disodium salts

Test animals

Species:

mouse

Strain:

CD-1

Remarks:

albino

Sex:

female

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- Sufficient male and female albino CDI strain mice were supplied by Charles River (UK) Ltd, Manston, Kent, UK.
- At the start of the main study animals weighed 22-29 g (males) or 20-24 g (females) and were approximately five to eight weeks old.
- After a minimum acclimatisation period of five days, animals were selected at random and given a unique number within the study by ear punching and a number written on a colour coded cage card.
- Animals were housed in groups of up to five by sex in solid-floor polypropylene cages with woodflakes bedding.
- Free access to mains drinking water and food (Rat and Mouse Expanded Diet No 1, Special Diets Services, Limited, Witham, Essex, UK) was allowed throughout the study.
- The animal room was maintained at a temperature of 19 to 21 °C with relative humidity of 44 to 46 %.
- Rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Sterile distilled water supplied by Steripak and stored at room temperature.

Details on exposure:

RANGE-FINDING TOXICITY STUDY
- A range-finding study was performed to determine a suitable dose level and route of administration for the micronucleus study.
- The dose level selected should ideally be the maximum tolerated dose or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 2000 mg/kg.
- Intraperitoneal dose levels were 50, 100, 200, 500, 1000 and 2000 mg/kg (one male and one female per dose level with administration via a hypodermic needle attached to a graduated syringe).
- Gavage dose levels were 500, 1000 and 2000 mg/kg (one male and one female per dose level with administration via a metal cannula).
- 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 per day for two days.
- Any deaths and evidence of overt toxicity were recorded at each observation.
- No necropsies were performed.

MICRONUCLEUS STUDY
- Groups of ten mice (five males and five females) were dosed once only via the intraperitoneal route with 50, 100 or 200 mg/kg test material.
- Dose groups, kill time and associated information are provided in the table below.
- Animals were killed by cervical dislocation.
- All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily and immediately prior to sacrifice.

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

Single dose

Post exposure period:

Not applicable

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 other: mg/kg

Remarks:

Single dose; intraperitoneal route

Doses / concentrations 2

Dose / conc.:

100 other: mg/kg

Remarks:

Single dose; intraperitoneal route

Doses / concentrations 3

Dose / conc.:

200 other: mg/kg

Remarks:

Single dose; intraperitoneal route

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide was supplied by the Sigma Chemical Company and stored at 0-5 °C.
- The positive control material was freshly prepared when required as a solution of appropriate concentration in distilled water.
- The concentration, homogeneity and stability of the positive control material and its preparation were not determined by analysis.

Examinations

Tissues and cell types examined:

One femur was dissected from each animal immediately following sacrifice.

Details of tissue and slide preparation:

SLIDE PREPARATION
- Femurs were aspirated with foetal calf serum and bone marrow smears were prepared following centrifugation and resuspension.
- Smears were air-dried, fixed in absolute methanol and stained in May-Grünwald/Giesma.

Evaluation criteria:

- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification.
- The incidence of micronucleated cells per 1000 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.
- 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 with appropriate group mean values for males, females and all animals.

Statistics:

- A comparison was made between the number of micronucleated polychromatic erythrocytes occuring in each of the test material groups and the number occuring in the corresponding vehicle control group.
- A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the corresponding vehicle control group.
- If these criteria are not demonstrated, the test material is considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio is shown to be statistically significant from the concurrent vehicle control group.
- All data were statistically analysed using appropriate methods as recommended by the UKEMS sub-committee on guidelines for mutagenicity testing, Report, Part III (1989).
- Data was analysed following a square route of (x+1) transformation using Student's t-test (two tailed) and any significant results were confirmed using one-way analysis of variance.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING STUDY
- Mortality data from the range-finding study are presented in the attached table.
- No premature animal deaths took place after dosing with test material via the oral route.
- Clinical signs oversed in animals dosed with test item via the intraperitoneal route were lethargy, decreased respiratory rate, laboured respiration, ptosis and hunched posture.
- Premature animal deaths occurred in animals dosed at and above 500 mg/kg via the intraperitoneal route. Clinical observations seen only at 500 mg/kg were ataxia, pallor of the extremities and walking with splayed gait.
- Based on these results, the main study was conducted using the intraperitoneal route with 200 mg/kg as the maximum tolerated dose (MTD) and 100 mg/kg and 50 mg/kg as the other dose levels.

MICRONUCLEUS STUDY
- One premature animal death was seen at 48 hours in the 200 mg/kg test item group.
- Clinical signs observed in animals dosed with test material were hunched posture, lethargy, decreased respiratory rate, laboured respiration, pilo-erection, ptosis and tiptoe gait.
- Lethargy, decreased respiratory rate and laboured respiration were observed one hour after dosing at 50 mg/kg and 100 mg/kg.
- With the exception of one animal in the 100 mg/kg group no clinical signs were observed at 24 hours after dosing.
- In the 200 mg/kg group, clinical signs were seen at one hour, 24 hours and 48 hours after dosing.

EVALUATION OF BONE MARROW SLIDES
- Results are summarised in Table 1 (attached).
- Individual and group mean data are presented in Tables 2 to 8 (attached).
- There was no statistically significant increase in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
- There was no significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups.
- The presence of clinical observations demonstrates that systemic absorption of test material had taken place.

EVALUATION OF BONE MARROW SLIDES
- 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.

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

A study was performed in accordance with OECD 474 and EU Method B.12 to assess the potential of the test item to produce damage to chromosomes and/or aneuploidy when administered via the intraperitoneal route to mice. Following a preliminary range-finding study to confirm the toxicity of the test material and route of administration, the micronucleus study was conducted using the intrperitoneal route in groups of ten mice (five male and five female) at 50 mg/kg, 100 mg/kg and the maximum tolerated dose of 200 mg/kg. Animals were killed 24 or 48 hours later, the bone marrow was extracted and smear preparations were made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of distilled water to serve as vehicle controls or dosed orally with cyclophosphamide to serve as positive controls.

There was no evidence of an increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control group. No significnat change in the PCE/NCE ratio was observed after dosing with test item although the presence of clinical observations indicated that systemic absorption had occurred. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes. The test material was considered to be non-genotoxic under the conditions of the test.