Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for Read Across is given in Section 13 of IUCLID.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1999

Reference 2

Reference Type:

secondary source

Title:

Unnamed

Year:

2004

Reference 3

Reference Type:

secondary source

Title:

Micronucleated Erythrocyte Frequency in Peripheral Blood of B6C3F1 Mice from Short-Term, Prechronic, and Chronic Studies of the NTP Carcinogenesis Bioassay Program

Author:

Witt, K.L., Knapton, A., Wehr, C.M., Hook, G.J., Mirsalis, J., Shelby, M.D., and MacGregor, J.T.

Year:

2000

Bibliographic source:

Environ. Mol. Mutagen. 36, 163-194.

Materials and methods

Principles of method if other than guideline:

The frequency of micronucleated erythrocytes in peripheral blood samples from 13-weeks dermal exposure of B6C3F1 mice to the substance was assessed. The protocol followed was the one described in MacGregor, J.T.et al.1990.

MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.D. (1990). The in vivo erythrocyte micronucleus test: Measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundam. Appl. Toxicol. 14, 513-522.

GLP compliance:

yes

Remarks:

Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)

Type of assay:

other: Mammalian Erythrocyte Micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-nitrilotriethanol

EC Number:

203-049-8

EC Name:

2,2',2''-nitrilotriethanol

Cas Number:

102-71-6

Molecular formula:

C6H15NO3

IUPAC Name:

2,2',2''-Nitrilotriethanol

Test animals

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

Mice provide a convenient model for these studies because, unlike rats, mice do not selectively remove micronucleated cells from the peripheral circulation. Therefore, the frequency of micronucleated cells in the circulating blood of mice would be expected to closely reflect the frequency in bone marrow, and unlike bone marrow sampling, repeated peripheral blood measurements can be made, without disturbing the physiology of the animals, by sampling a few microliters of blood at a time.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA).
- Age at study initiation: 6 weeks old.
- Housing: individually in Polycarbonate (Lab Products, Inc., Garfield, NJ) cages, equipped with Beta-Chips hardwood chips (Northeastern Products, Inc., Warrensburg, NY) (changed weekly). Cage filters: DuPont 2024 spun-bonded polyester filter (Snow Filtration Co Cincinnati, OH), changed every 2 weeks. Stainless steel racks were used and were changed every 2 weeks.
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc.,Gardners, PA), available ad libitum, changed weekly
- Water: tap water (City of Columbus municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI), available ad libitum.
- Acclimation period: 11-14 days.
- Other: five male and six female mice were randomly selected for parasite evaluation and gross observation for evidence of disease. At the beginning of quarantine and at the beginning and the end of the studies, serologic analyses were performed on five male and five female and mice using the protocols of the NTP Sentinel Animal Program.

ENVIRONMENTAL CONDITIONS
- Temperature: 20.0 - 23.9 °C.
- Humidity: 35 - 65 %.
- Air changes: 15 changes per hour.
- Photoperiod: 12 hrs dark / 12 hrs light.
- Other: fluorescent light was used.

Administration / exposure

Route of administration:

dermal

Vehicle:

- Vehicle/solvent used: acetone. Except for the highest dose, which was applied neat, all doses were administered in acetone.
- Justification for choice of solvent/vehicle: acetone is miscible with the test material and because acetone rapidly evaporates.
- Amount of vehicle: dose volumes were adjusted weekly, if necessary, according to the average body weights of the dosed groups.

Details on exposure:

TEST SITE
- Area of exposure: the area extending from the animal’s mid-back to the dorsal intrascapular region; the site of application was clipped weekly during the studies.

DOSING SOLUTIONS: periodic analyses of dose formulations of the substance were conducted by the study laboratory and the analytical chemistry laboratory with gas chromatography. The dose formulations were analyzed at the beginning, midpoint, and end of the studies; animal-room samples of the same dose formulations were also analyzed. All dose formulations and animal-room samples were within 10 % of the target concentrations.

Duration of treatment / exposure:

5 days per week, for 13 weeks

Frequency of treatment:

once per day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

4 000 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: doses for the 13-week study were based on the results of 14- and 16-day comparative studies in which the B6C3F1 mice received the substance by the dermal route in acetone. The results of the dermal studies included skin irritation of a severity that limited the highest dose for the 13-week studies to 4000 mg/kg for mice.

TREATMENT AND SAMPLING TIMES: peripheral blood samples were obtained from male and female B6C3F mice from the ventral tail vessel at the end of the 13-week exposure study.

DETAILS OF SLIDE PREPARATION: smears were immediately prepared and fixed in absolute methanol (for approximately 2 min) and were later stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y double fluorescent stain, using Sorensen’s M/ 15 phosphate buffer. Then they were coded.

METHOD OF ANALYSIS: slides were randomized and scored at 630 X magnification under oil immersion. They were scanned to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) and 10000 normochromatic erythrocytes (NCEs) in each animal per dose group. In addition, the percentage of polychromatic erythrocytes among 10000 erythrocytes was determined as a measure of bone marrow toxicity. Log transformation of the NCE data, testing for normality by the Shapiro-Wilk test, and testing for heterogeneity of variance by Cochran’s test were performed before statistical analyses.

Evaluation criteria:

In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.
Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

Statistics:

- The frequency of micronucleated cells among NCEs was analyzed by analysis of variance using the SAS GLM procedure.
- The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the control group.
- In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

treatment-related decreases in sorbitol dehydrogenase activities in all dosed groups (males, females); significant increase in absolute kidney and liver weights of males and females of the highest groups.

Vehicle controls validity:

not specified

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

No significant increases in the frequencies of micronucleated NCEs or PCEs were observed at any dose tested.
The percentages of polychromatic erythrocytes in the dosed groups were similar to those in the vehicle control groups, indicating an absence of bone marrow toxicity.

Any other information on results incl. tables

The Frequency of Micronuclei in Peripheral Blood Erythrocytes of mice following treatment with the test material by dermal Application for 13 Weeks is presented in the table below. The following data are a combination of results from the current study and from data presented in NTP report, 2004.

10000 NCEs (normochromatic erythrocytes) and 2000 PCEs (polychromatic erythrocytes) were scored per animal.

Table: Frequency of micronuclei in peripheral blood erythrocytes.

	Dose (mg/kg)	Number of mice with erythrocytes scored	Micronucleated NCEs/ 1000 NCEs^a	p value^b	Micronucleated PCEs /1000 cells^a	PCEs (%)
Male	vehicle control	10	1.75 ± 0.20		2.13 ± 0.40	2.2
	1000	10	1.88 ± 0.17	0.3138	1.04 ± 0.36	2.2
	2000	10	1.36 ± 0.10	0.9350	2.41 ± 0.40	2.4
	4000	10	1.90 ± 0.30	0.3047	2.00 ± 0.48	2.1
			P=0.420^c
Female	vehicle control	10	1.16 ± 0.12		2.06 ± 0.35	2.1
	1000	10	1.20 ± 0.08	0.3890	1.60 ± 0.34	2.2
	2000	10	1.08 ± 0.12	0.7393	2.38 ± 0.43	2.2
	4000	10	0.99 ± 0.11	0.8813	2.12 ± 0.39	2.0
			P=0 .925

a = Data are presented as mean ± standard error.

b = Pairwise comparison with the vehicle controls, significant at P ≤0.008.

c = Significance of micronucleated NCEs/1000 NCEs tested by the one-tailed trend test, significant at P ≤ 0.025.

Toxicity of animals

- The final mean body weight and weight gain of males in the 250 mg/kg group were less than those of the vehicle controls.

- Clinical findings were observed only in mice in the 4000 mg/kg groups and included scaliness, irritation, and discoloration at the site of test material application for males and females and skin erosion at this site in one male.

- Serum sorbitol dehydrogenase activity was significantly lower in comparison with the vehicle control; treatment-related decreases in sorbitol dehydrogenase activities occurred in all dosed groups of males and females.

- Differences in other hematology and clinical chemistry parameters were minimal or transient and were not considered to be biologically relevant.

- The absolute kidney and liver weights of males and females in the 4000 mg/kg groups were significantly greater than those of the vehicle controls; relative kidney weights of males administered 1000 mg/kg or greater and of all dosed groups of females were also greater than those of the vehicle controls. The absolute and relative spleen weights of females in the 4000 mg/kg group were significantly greater than those of the vehicle controls.  Males administered 4000 mg/kg had a greater relative heart weight than the vehicle controls.

- At necropsy, the skin of males and females administered 4000 mg/kg was crusted (7/10 males 4/10 females) and white (1/10 males, 8/10 females), scaly (1/10 males), or both (5/10 males, 2/10 females) at the site of application.  One female in the 2000 mg/kg group also had crusted skin.  Minimal epidermal thickening (acanthosis), up to twice the normal thickness, occurred in nearly all dosed animals and in one vehicle control female; the severity of acanthosis was greater in the 4000 mg/kg groups than in the lower dose groups. Chronic active inflammation occurred in the 4000 mg/kg groups and in one female in the 2000 mg/kg group, with some animals having erosion, inflammation, or both. In these animals, the underlying dermis was often thickened by chronic active inflammation, including fibrosis.

Applicant's summary and conclusion

Conclusions:

No significant increases in the frequencies of micronucleated NCEs or PCEs at any dose tested were observed.

Executive summary:

The substance was evaluated for its potential to induce formation of micronucleated polychromatic erythrocytes in the peripheral blood of mice. For this reason 10 male and 10 female B6C3F₁ mice were dermally exposed to three concentrations of the substance (at 1000, 2000 and 4000 mg/kg) per day, for 5 days, for 13 weeks. After the exposure period, peripheral blood samples were taken from the ventral tail vessel of the mice. The smears were fixed in absolute methanol, they were stained, fixed and coded. 10000 NCEs (normochromatic erythrocytes) and 2000 PCEs (polychromatic erythrocytes) were scored for micronuclei per animal per dose group.

No significant increases in the frequencies of micronucleated NCEs or PCEs were observed at any dose tested.

The substance is considered as not mutagenic under the test conditions.