Methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate

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:

from 1 August 2011 to 23 february 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

Reference substance name:

methyl (2R)-4-(dimethylcarbamoyl)-2-methylbutanoate; methyl (2S)-4-(dimethylcarbamoyl)-2-methylbutanoate

EC Number:

700-204-6

Cas Number:

1174627-68-9

Molecular formula:

C9H17NO3

IUPAC Name:

methyl (2R)-4-(dimethylcarbamoyl)-2-methylbutanoate; methyl (2S)-4-(dimethylcarbamoyl)-2-methylbutanoate

Details on test material:

Name in study report: Rhodiasolv Polarclean

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: approximately 6-10 weeks
- Weight at study initiation: 22-30g
- Fasting period before study: not applicable
- Housing: pre-mating: groups of up to 7
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: from 16August 2011 to 3 November 2011

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Distilled water

Duration of treatment / exposure:

Single exposure

Frequency of treatment:

Once

Post exposure period:

24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

7 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, intraperitoneal route, 50 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow

Evaluation criteria:

The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
A positive mutagenic response would be 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 the vehicle control group.
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 a (x 1) 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

Remarks:

Hunched posture, ptosis and ataxia at and above 1000 mg/kg in 24 and 48-h groups

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The group mean data from the micronucleus study are summarised in Table 3. The individual data from each group are summarised in Tables 4-9.

 

Table 3: Micronucleus Test - Summary of Group Mean Data

Treatment group		Number of PCE with Micronuclei per 2000 PCE		PCE/NCE ratio
		Group mean	SD	Group mean	SD
1	Vehicle control (Distilled water) 10 ml/kg 24-hour sampling time	1.1	1.5	0.98	0.30
2	Positive control (Cyclophosphamide) 50 mg/kg 24-hour sampling time	38.4***	20.8	0.97	0.23
3	Rhodiasolv Polarclean 2000 mg/kg 48-hour sampling time	0.7	0.8	0.79	0.24
4	Rhodiasolv Polarclean 2000 mg/kg 24-hour sampling time	0.7	0.8	0.66	0.24
5	Rhodiasolv Polarclean 1000 mg/kg 24-hour sampling time	0.7	0.8	0.94	0.27
6	Rhodiasolv Polarclean 500 mg/kg 24-hour sampling time	0.4	0.5	1.03	0.33

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standared deviation

*** = P < 0.001

Table 4: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Vehicle Control (10 ml/kg): 24-Hour Sampling Time

Treatment group	Animal number	BW (g) when dosed	PCE			NCE		PCE/NCE ratio
			Number scored	PCE+MN	%PCE+MN	Number scored	NCE+MN
1. Vehicle control 10 ml/kg 24-h sampling time	1	26	2000	1	0.05	435	1	1.30
	2	22	2000	2	0.10	691	0	0.45
	3	24	2000	1	0.05	465	0	1.15
	4	24	2000	4	0.20	488	0	1.05
	5	24	2000	1	0.05	446	0	1.24
	6	25	2000	0	0.00	554	0	0.81
	7	28	2000	0	0.00	541	0	0.85
	Group mean	24.7	2000	1.3	0.06	517	0.1	0.98
	SD	1.9	0	1.4	0.07	89	0.4	0.30

BW = bodyweight

 

Table 5: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Cyclophosphamide (50 mg/kg): 24-Hour Sampling Time

Treatment group	Animal number	BW (g) when dosed	PCE			NCE		PCE/NCE ratio
			Number scored	PCE+MN	%PCE+MN	Number scored	NCE+MN
1. Vehicle control 10 ml/kg 24-h sampling time	8	27	2000	45	2.25	449	1	1.23
	9	22	2000	49	2.45	528	2	0.89
	10	26	2000	18	0.90	467	0	1.14
	11	26	2000	64	3.20	614	0	0.63
	12	29	2000	16	0.80	508	0	0.97
	Group mean	26.0	2000	38.4	1.92	513	0.6	0.97
	SD	2.5	0	20.8	1.04	65	0.9	0.23

 

Table 6: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Test item (2000 mg/kg): 48-Hour Sampling Time

Treatment group	Animal number	BW (g) when dosed	PCE			NCE		PCE/NCE ratio
			Number scored	PCE+MN	%PCE+MN	Number scored	NCE+MN
1. Vehicle control 10 ml/kg 24-h sampling time	13	27	2000	1	0.05	596	0	0.68
	14	28	2000	0	0.00	551	0	0.81
	15	29	2000	1	0.05	703	0	0.42
	16	27	2000	0	0.00	514	0	0.95
	17	24	2000	2	0.10	463	0	1.16
	18	28	2000	1	0.05	627	0	0.59
	19	27	2000	0	0.00	526	0	0.90
	Group mean	27.1	2000	0.7	0.04	569	0.0	0.79
	SD	1.6	0	0.8	0.04	80	0.0	0.24

 

Table 7: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Test item (2000 mg/kg): 24-Hour Sampling Time

Treatment group	Animal number	BW (g) when dosed	PCE			NCE		PCE/NCE ratio
			Number scored	PCE+MN	%PCE+MN	Number scored	NCE+MN
1. Vehicle control 10 ml/kg 24-h sampling time	20	30	2000	0	0.00	653	0	0.53
	21	24	2000	1	0.05	688	2	0.45
	22	25	2000	1	0.05	497	1	1.01
	23	26	2000	0	0.00	680	0	0.47
	24	24	2000	1	0.05	649	1	0.54
	25	30	2000	0	0.00	613	0	0.63
	26	25	2000	2	0.10	505	0	0.98
	Group mean	26.3	2000	0.7	0.04	612	0.6	0.66
	SD	2.6	0	0.8	0.04	80	0.8	0.24

 

Table 8: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Test item (1000 mg/kg): 24-Hour Sampling Time

Treatment group	Animal number	BW (g) when dosed	PCE			NCE		PCE/NCE ratio
			Number scored	PCE+MN	%PCE+MN	Number scored	NCE+MN
1. Vehicle control 10 ml/kg 24-h sampling time	27	23	2000	1	0.05	628	1	0.59
	28	25	2000	0	0.00	553	0	0.81
	29	26	2000	0	0.00	415	0	1.41
	30	26	2000	2	0.10	533	2	0.88
	31	29	2000	1	0.05	524	0	0.91
	32	24	2000	0	0.00	554	0	0.81
	33	26	2000	1	0.05	466	0	1.15
	Group mean	25.5	2000	0.7	0.04	525	0.4	0.94
	SD	1.9	0	0.8	0.04	68	0.8	0.27

 

Table 9: Micronucleus Test - Individual Data and Group Means and Standard Deviations: Test item (500 mg/kg): 24-Hour Sampling Time

Treatment group	Animal number	BW (g) when dosed	PCE			NCE		PCE/NCE ratio
			Number scored	PCE+MN	%PCE+MN	Number scored	NCE+MN
1. Vehicle control 10 ml/kg 24-h sampling time	34	25	2000	0	0.00	602	0	0.66
	35	22	2000	0	0.00	546	0	0.83
	36	26	2000	0	0.00	524	0	0.91
	37	26	2000	0	0.00	408	0	1.45
	38	26	2000	1	0.05	521	0	0.92
	39	28	2000	1	0.05	391	0	1.56
	40	29	2000	1	0.05	522	0	0.92
	Group mean	26.0	2000	0.4	0.02	502	0.0	1.03
	SD	2.2	0	0.5	0.03	76	0.0	0.33

 

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The potential of Rhodiasolv Polarclean to produce damage to chromosomes or aneuploidy was investigated in GLP-compliant study according to OECD Guideline 474 (1997).

The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 2000 mg/kg and with 1000 and 500 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 intraperitoneal dose of distilled water (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.

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs consisting of hunched posture, ptosis and ataxia were observed in animals dosed with the test item at and above 1000 mg/kg in both the 24 and 48-hour dose groups Animals dosed at 2000 mg/kg in both the 24 and 48-hour dose groups exhibited decreased respiratory rate, laboured respiration and loss of righting reflex up to 10 minutes after dosing. The observation of the clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. No statistically significant decreases in the PCE/NCE ratio were observed in any of the test item dose groups when compared to the vehicle control group. There were no statistically significant increases in the frequency of micronucleated PCE in any test item dose groups. 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 conditions of the test.

Rhodiasolv Polarclean was considered to be non-genotoxic under the conditions of the test.