Reaction products of amines, dicoco alkyl and glycollic 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:

30 May 2013 to 13 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study done according to OECD 474 guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

other: amber coloured liquid

Details on test material:

- Physical state: amber coloured liquid
- Storage condition of test material: room temperature in the dark

Test animals

Species:

mouse

Strain:

ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 6 to 10 weeks
- Weight at study initiation: 23 to 31g
- Assigned to test groups randomly: Yes
- Housing: solid-floor polypropylene cages with wood flake bedding
- Diet (e.g. ad libitum): Harlan Teklad 2014C Global Certified Rodent Diet
- Water (e.g. ad libitum): mains drinking water
- Acclimation period: minimum five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Concentration of test material in vehicle: 30, 60, 120 mg/kg
- Lot/batch no. (if required): V-5519

Details on exposure:

Intraperitoneal injection for main study. Oral gavage and intraperitoneal injection in the range-finder.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Single

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
300, 600, 1200 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 50 mg/kg / 5mg/ml

Examinations

Tissues and cell types examined:

Bone marrow polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Results of range-finding test provided the maximum tolerated dose level.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Treated at 0 hours and sampled at 24 and 48 hours.

DETAILS OF SLIDE PREPARATION: Bone marrow tissue smeared onto glass slides, fixed in methanol and stained with May-Grunwald and Giemsa.

METHOD OF ANALYSIS: Light microscopy, 2000 polychromatic cells per animal and the ratio of polychromatic to normochromatic cells.

Evaluation criteria:

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

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following square root transformation using Student's 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

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 to 2000 mg/kg
- Clinical signs of toxicity in test animals: None dosed orally at 2000 mg/kg. Hunched posture, ptosis, ataxia, splayed gate, distended abdomen at 1500 mg/kg intra peritoneal
- Evidence of cytotoxicity in tissue analyzed: No
- Rationale for exposure: No clinical signs via oral route, but evidence of absorbsion via intra peritoneal route

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No significant increases
- Ratio of PCE/NCE (for Micronucleus assay): No significant changes
- Appropriateness of dose levels and route: The maximum dose level caused clinical signs that were considered to be evidence of absorbsion and of exposure to the target tissue. The maximum dose level was considered to be the maximum tolerated dose level.
- Statistical evaluation: No statistically significant increases in micronucleus frequency.

Any other information on results incl. tables

Treatment	Number of PCE with Micronuclei per 2000 PCE		PCE/NCE Ratio
Group	Group Mean	SD	Group Mean	SD
Vehicle Control 24 hrs	2.0	1.7	0.83	0.31
Positive Control 24 hrs	41.4***	21.5	1.19	0.32
Test Item 1200 mg/kg 48 hrs	2.7	4.3	0.78	0.19
Test Item 1200 mg/kg 24 hrs	2.3	2.6	0.93	0.24
Test Item 600 mg/kg 24 hrs	1.3	1.4	0.96	0.28
Test Item 300 mg/kg 24 hrs	0.9	0.7	1.31	0.62

*** = P<0.001, SD = Standard deviation, PCE = polychromatic erythrocyte, NCE = normochromatic erythrocyte

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test item was considered to be non-mutagenic in vivo under the conditions of the test.

Executive summary:

Test Guidance

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Mammalian Erythrocyte Micronucleus Test", Method B12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

Method and materials

A range-finding test was performed to find suitable dose levels of the test item, route of administration, and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal (i.p.) route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 1200 mg/kg with 600 and 300 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 (NCE) erythrocytes were scored for the presence of micronuclei. Additional groups of mice were given a single ip dose of Arachis oil (7 male mice) or dosed orally with cyclophosphamide (5 male mice), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed after 24 hours.

Results

There were no premature deaths at any dose level. The clinical signs of hunched posture and ptosis were observed at 1200 mg/kg in both the 48 and 24 hour dose groups. No statistically significant decreases in the PCE/NCE ratio were observed in any dose groups. The observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditionsof the test.

Conclusions

The test item was considered to be non-mutagenic under the conditions of the test.