Reaction products of D-Glucose, n-Butanol and C10-12 (even numbered) alcohols

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:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study tested with the source substance D-Glycopyranose, oligometric, C10-16-alkyl glycosides. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

110615-47-9

EC Number:

600-975-8

Cas Number:

110615-47-9

IUPAC Name:

110615-47-9

Details on test material:

- Name of test material (as cited in the study report): trade name given
- Physical state: white paste
- Analytical purity: 54.4% in water
- Lot/Batch number: 94061
- Storage conditions: room temperature in the dark

Test animals

Species:

mouse

Strain:

other: Crl:CD-1TM(ICR)BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Margate, UK
- Age at study initiation: approx. 5-8 weeks
- Weight at study initiation: 25-30 g
- Housing: in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding
- Diet: Rat and Mouse Expanded Diet No. 1 (Special Diets Services Limited, Witham, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle/solvent used: sterile water
- Amount of vehicle (gavage): 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared freshly by dissolving the test substance in sterile water yielding final concentrations of 25, 12.5 and 6.25 mg/mL (corresponding to 250, 125 and 62.5 mg/kg bw, respectively).

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single treatment

Post exposure period:

24 and 48 h (vehicle and high dose group)
24 h (low, mid and positive control group)

Doses / concentrations

Remarks:

Doses / Concentrations:
62.5, 125, 250 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

7 (vehicle and dose groups), 5 (positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 5 mg/mL in sterile water (corresponding to 50 mg/kg bw )

Examinations

Tissues and cell types examined:

Femur bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range finding study in 2 male and 2 female mice was performed to find the maximum tolerated dose level, a suitable route of administration and to assess gender-specific differences in toxicity.

TREATMENT AND SAMPLING TIMES:
24 h sampling time: 0, 62.5, 125 and 250 mg/kg bw (test substance); 50 mg/kg bw (positive control)
48 h sampling time: 0 and 250 mg/kg bw (test substance)

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (i.e. 24 or 48 h following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained with May-Grünwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 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. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a biologically relevant statistically significant, dose-related increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour time points, when compared to their corresponding control group.
If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent 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 were analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance. Statistical significance was indicated at *p < 0.05, **p < 0.01 and ***p < 0.001.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

≥ 500 mg/kg bw (range finding toxicity study): all animals died within the study period of 48 h; 250 mg/kg bw (main study): 2/7 animals died in the 48-h harvest time group

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000, 2000 mg/kg bw (intraperitoneal) and 2000 mg/kg bw (oral)
- Clinical signs of toxicity in test animals: in animals dosed with the test material via the intraperitoneal route premature deaths occurred at ≥ 500 mg/kg bw. Clinical signs were observed at ≥ 250 mg/kg bw (and increased in severity with increasing doses) and included: hunched posture, lethargy, pilo-erection, decreased respiratory rate, ptosis, ataxia, splayed gait, prostration, laboured respiration and pallor of the extremities.
- Sacrifice time: 48 h
- Dose selection: based on the results, the maximum tolerated dose of the test material was 250 mg/kg bw and thus selected as high dose for the main study.
- Other: no marked differences in the toxicity of the test substance were noted between male and female mice. Therefore, it was considered acceptable to use males only for the main study.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: there were two premature deaths seen in the 48-h 250 mg/kg bw test material dose group. Clinical signs were observed in animals dosed with the test material at 250 mg/kg bw in both the 24 and 48 h groups. These included hunched posture, lethargy, pilo-erection, decreased respiratory rate, ptosis and ataxia.
- Induction of micronuclei: there was a small, statistically significant increase in the frequency of micronucleated PCEs in the 24-h 125 mg/kg bw test material dose group when compared to the respective control group. However, the response was within the current historical range for vehicle controls, the 24-h vehicle control PCE+MN value was very low and no individual animal values for micronucleated PCEs were greater than it would be considered acceptable for vehicle control animals. Therefore, the response was considered to be of no biological relevance. 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.
- Ratio of PCE/NCE: there was a statistically significant decrease in the PCE/NCE ratio of the 24-h 250 mg/kg bw test material group when compared to the concurrent vehicle control group. The response was part of a dose-related reduction in PCE/NCE ratios and was taken to indicate that cytotoxicity and exposure of the bone marrow had been achieved.
- Appropriateness of dose levels and route: it was considered that the loss of animals due to premature death did not affect the integrity of the study, because at least five analysable animals were available in each group, as recommended in the OECD test guideline No. 474.

Any other information on results incl. tables

Table 1. Results of the in vivo micronucleus assay

Treatment group	Dose (Conc) [mg/kg] (mg/mL)	Sampling time [h]	Number of PCE with MN [per 2000 PCEs]		PCE/NCE ratio
			Mean value	SD	Mean value	SD
Vehicle control	0	48	1.0	1.3	1.87	0.29
	0	24	0.7	0.8	1.72	0.65
Positive control	(5)	24	54.8***	15.9	1.62	0.41
Test substance	250	48	2.2	1.6	1.25	0.80
	250	24	1.3	1.4	0.98*	0.28
	125	24	2.1**	0.9	1.42	0.26
	62.5	24	0.9	0.9	2.12	1.25

Positive control: cyclophosphamide; ***p < 0.001, Student's t-test (two tailed) and one way analysis of variance

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative