Reaction product of 9-Octadecenoic acid, (Z)-, sulfonated, potassium salts, hydrogen peroxide and sulfuric acid

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:

2012-07-31 to 2012-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Age at study initiation: 9 - 12 weeks
- Weight at study initiation (mean ± SD): 304.5 ± 10.6 g
- Assigned to test groups randomly: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 45 - 100 %
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the vehicle was chosen due to its relative non-toxicity

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test material was dissolved in sterile water. All animals received a single standard volume (10 mL/kg bw) orally. The pH of the formulation was adjusted to 5 using NaOH.
At the beginning of treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight.

Duration of treatment / exposure:

Sinlge administration

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
300, 1000, 1500, 2000 mg/kg bw (prelminary toxicity study)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw (main study)
Basis:
nominal conc.

No. of animals per sex per dose:

2 males and 2 females (prelminary toxicity study)
7 males per dose group and sampling time (main study)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

Examinations

Tissues and cell types examined:

Femur bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice time, seven animals were sacrificed. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum. The nucleated cells were separated from the erythrocytes. The cell suspensions were then briefly passed through a column consisting of α cellulose and cellulose. The cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was discarded. The pellet was re-suspended in a small drop of foetal calf serum and spread on slides. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Evaluation criteria:

Evaluation of the slides was performed using NIKON microscopes with100X oil immersion objectives. Per animals at least 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

The test material was considered positive if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistical positive response for at least one of the test points. A test material producing neither a dose-related increase in the number of micronucleated polychomatic erythrocytes nor a statistically significant positive response at any of the test points was considered to give a negative result in this test system.

Statistics:

Statistical significance was determined by means of the nonparametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY STUDY
- No signs of toxicity were observed in animals dosed 300 mg/kg bw. Animals dosed at 1000 mg/kg bw and above exhibited articulation, breath sounds and diarrhoea from 24 hours post-treatment. Animals dosed at 2000 mg/kg bw were also observed to have ruffled fur and reduced spontaneous activity from 1 hour post-treatment onwards.
- On the basis of these data 2000 mg/kg bw was estimated to be suitable as the highest test material treatment dose.

MAIN STUDY
-Toxic symptoms in the main study were confined to ruffled fur in 7 males dosed at 2000 mg/kg bw 2-4 and 6 hours post-administration.
- After treatment with the test material the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test material did not exert cytotoxic effects in the bone marrow.
- In comparison to the corresponding vehicle controls there were no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test material with any dose level. The mean values of micronuclei observed after treatment with test material were below or identical to the value of the vehicle control group and within the historical vehicle control range.
- 20 mg/kg bw cyclophosphamide administered once orally was used as positive control which showed a significant increase in micronucleus frequency.

Any other information on results incl. tables

Table 2: Summary of Micronucleus Test Results

Test group	Dose (mg/kg bw)	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
Vehicle	0	24	0.121	1 - 4	981
Test material	500	24	0.107	0 - 7	887
	1000	24	0.121	1 - 5	975
	2000	24	0.114	0 - 4	877
Positive control	20	24	0.714	9 - 19	551
Test material	2000	48	0.093	1 - 3	942

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the test, the test material did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male rats.

Executive summary:

The genetic toxicity of the test material was investigated in vivo in accordance with the standardised guideline OECD 474. The test material was dissolved in water, which was also used as the vehicle control. The pH was adjusted to 5 with NaOH. The volume administered orally was 10 mL/kg bw. 24 and 48 hours after a single administration of test material the bone marrow cells were collected for micronuclei analysis. Seven males per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes were scored for micronuclei per animal.

After treatment with the test material the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control, thus indicating that the test material did not exert cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there were no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test material with any dose level. The mean values of micronuclei observed after treatment with test material were below or identical to the value of the vehicle control group and within the historical vehicle control range. The positive control showed a significant increase in micronucleus frequency.

In conclusion, it can be stated that under the conditions of the study, the test material did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat. Therefore, the test material is considered to be non-mutagenic in this micronucleus assay.