Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 21, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

S 611411

IUPAC Name:

S 611411

Details on test material:

- Name of test material (as cited in study report): C-SAT 050084, DHW 80 (50%)
- Physical state: Liquid to solid, yellow to orange
- Composition of test material, percentage of components: approximately 50 % active substance, 49.3 % water
- Impurities (identity and concentrations): Ammonium-9,10-dihydroxystearat: ~ 35-40%, Polycondensationproducts: ~10-15%, NH4OH: <= 2%
- Lot/batch No.: CD53120004
- Expiration date of the lot/batch: 2006-11-08
- Stability under test conditions: not indicated
- Storage condition of test material: at room temperature
- Other: well soluble in water, test item was heated up to 50°C in order to obtain a homogenous solution

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: males mean value 32g (SD ± 2.2g), females mean value 29.7g (SD ± 1.89g)
- Assigned to test groups randomly: yes
- Housing: individual, on granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen) in Makrolon Type I cages with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Diet: pelleted standard diet (Harlan Winkelmann, D-33178 Borchen), ad libitum
- Water: tap water (Gemeindewerke, D-64380 Roßdorf), ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): artificial light 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: item is well soluble in water
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): 18.11.05

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
at beginning of treatment animals were weighed and individual volume to be administered was adjusted to animal body weight

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

single exposure

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw after 24 hours
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000 mg/kg bw after 48 hours
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide - Sigma-Aldrich Vertriebs GmbH, 82041 Deisenhofen
- Catalogue no.: C 0768 (purity: >98%)
- dissolved in: deionised water
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg bw (10 mL/kg bw administered)

Examinations

Tissues and cell types examined:

tissue: bone marrow
cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
a preliminary study on acute dose selection was performed with two animals per sex under identical conditions as the mutagenicity study; animals were treated orally and examined for acute toxicity symptoms at 1, 2-4, 6, 24, 30 and 48 h after administration

DETAILS OF SLIDE PREPARATION:
slides were air dried and stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain), cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg)
METHOD OF ANALYSIS:
ten animals (5 males/5 females) were evaluated per test group, at least one slide was made from each bone marrow sample, evaluation was performed using NIKON microscopes with 100x oil immersion objectives, at least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei, cytotoxicity was determined by ratio of polychromatic and normochromatic erythrocytes

Evaluation criteria:

- test item is classified as mutagenic, if it induces a dose-related or a clear increase in number of micronucleated polychromatic erythrocytes in a single dose group
- primary point of consideration is the biological relevance of the results, if test item fails to produce biological relevant increase in number of micronucleated PCEs it is considered non-mutagenic in this system

Statistics:

nonparametrix Mann-Whitney test was used as an aid in evaluating results

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

animals of all dose groups showed: reduction of spontaneous activity and ruffeled fur; animals of high dose group: difficulty breathing and one male/female animal died

Vehicle controls validity:

valid

Negative controls validity:

other: vehicle control is negative control

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: reduction of spontaneous activity and ruffled fur in all dose groups; eyelid closure in 100 and 500 mg/kg bw group
- Harvest times: up to 48 hours after exposure

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): in comparison to vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of detected micronuclei at any preparation interval or dose level
- Ratio of PCE/NCE (for Micronucleus assay): no differences observed between treated groups and vehicle control groups
- Statistical evaluation: non-parametric Mann-Whitney test
(for details of toxicity and micronucleustest refer to tables1-4)

Any other information on results incl. tables

table1a-c: toxic symptoms in main experiment

Toxic reactions (a) 500 mg/kg bw	Hours post-treatment male/female
	1 h	2-4 h	6 h	24 h
Reduction of spontaneous activity	2/3	4/5	0/0	0/0
Ruffeled fur	4/3	4/5	0/0	0/0
Toxic reactions (b) 1000 mg/kg bw	Hours post-treatment male/female
	1 h	2-4 h	6 h	24 h
Reduction of spontaneous activity	0/0	4/5	0/0	0/0
Ruffeled fur	4/3	4/5	0/0	0/0
Toxic reactions (c) 2000 mg/kg bw	Hours post-treatment male/female
	1 h	2-4 h	6 h	24 h
Reduction of spontaneous activity	5/4	12/12	0/0	0/0
Eyelid closure	0/0	0/2	0/0	0/0
Ruffeled fur	7/7	12/12	0/0	0/0
Difficulty in breathing	0/0	0/0	0/0	0/0
Death	0/0	0/0	0/0	1/1

table2: historical controls micronucleus test

	Vehicle Controls			Positive Controls (CPA)
	Males	Females	Total	Males	Females	Total
Mean* ± SD	0.078±0.04	0.058±0.033	0.069±0.028	1.867±0.57	1.368±0.497	1.632±0.468
Range**	0.01±0.23	0.0±0.19	0.01±0.15	0.70±3.46	0.49±3.55	0.77±3.48
No. of experiments	229	217	230	228	217	229

* mean value (percent micronucleated cells)

** range of the mean group values (percent micronucleated cells)

table3: summary of micronucleus test result

test group	dose mg/kg bw	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
vehicle	0	24	0.075	0 - 7	1188
test item	500	24	0.120	1 - 4	1103
test item	1000	24	0.090	0 - 5	1162
test item	2000	24	0.100	0 - 5	1103
positive control	40	24	2.770	32 – 91	1042
test item	2000	48	0.100	0 - 5	1194

table4: biometry, statistical significance (p<0.05) evaluated by non-parametric Mann-whitney test

vehicle control versus test group	Significance	P
500 mg/kg bw; 24 h	no	0.0684
1000 mg/kg bw; 24 h	no	0.3598
2000 mg/kg bw; 24 h	no	0.2185
40 mg/kg bw CPA; 24 h	yes	<0.0001
2000 mg/kg bw; 48 h	no	0.2809

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in bone marrow cells of the mouse.