Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Dedicated phase of a GLP and OECD test guideline compliant repeated-dose toxicity study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Principles of method if other than guideline:
Test performed based on the methods described by Schmid (Schmid W. The micronucleus test. Mutation Research 31: 9-15, 1975) and modified by Salamone et al. (Salamone M, Heddle J, Stuart E and Katz M. Toward an improved micronucleus test. Studies on 3 model agents, mitomycin C, CPA and dimethylbenzanthracene. Mutation Research 74: 347-356, 1980).
Apart from detecting chromosome breakage events (clastogenesis), the micronucleus test is capable of detecting chemicals which induce whole chromosome loss (aneuploidy) in the absence of clastogenic activity. In the bone marrow of rats exposed to a chemical which induces cytogenetic damage, chromosomal fragments or entire chromosomes which are left behind at cell division were not incorporated into the nuclei of daughter cells. Most of these fragments condense and form one or more micronuclei in the cytoplasm. The visualization of micronuclei is facilitated in erythrocytes because their nucleus is extruded during erythropoiesis. Accordingly, the basis of this test is an evaluation of the increase in the number of Micronucleated Polychromatic Erythrocytes (MPE).
Substances which inhibit either proliferation or maturation of erythroblasts and those which are toxic for nucleated cells, decrease the proportion of immature erythrocytes (polychromatic, PE) when compared to mature erythrocytes (normochromatic, NE). Thus, the cytotoxicity of a substance can be evaluated by a decrease in the PE/NE ratio.
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vivo micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation:
. Main study: 9 (females) - 10 (males) weeks old
. Micronucleus phase: 15 weeks old
- Weight at study initiation:
. Main study: 216 g (females) - 392 g (males)
. Micronucleus phase: 277 g (females) - 498 g (males)
- Fasting period before study: No
- Housing: Individual (except during pairing) in polycarbonate 940 cm² cages with stainless stell lids and autoclaved dust
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days (main study) / 7 days (micronucleus phase) before dosing initiation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 10 May 2012 To: 20 July 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS (main study):
- The test item was administered as a solution in the vehicle, by mixing with the required quantity of vehicle.
- The dose formulations were prepared daily.

VEHICLE
- Concentration in vehicle: The concentration of the test item in samples of each control and test item dose formulation prepared for use in weeks 1, 3, 5 and 7 was determined.
- Administration volume: 5 mL/kg/day (main study) / 10 mL/kg (cyclophosphamide-treated group in micronucleus phase)
Duration of treatment / exposure:
The dose formulations were administered daily according to the following schedule (Day 1 corresponding to the first day of the treatment period):

MAIN STUDY:
. In the males:
- 2 weeks before pairing (from study day 1 to 14),
- during the pairing period (3 weeks, from study day 15 until study day 16 to 29),
- until sacrifice (at least 5 weeks in total, from study day 17 to 30 until study day 36).
. In the females:
- 2 weeks before pairing (from study days 1 to 14),
- during the pairing period (3 weeks, from study days 15 to 29),
- during gestation (from study days 16 to 30 until study days 36 to 50),
- during lactation until day 5 post-partum inclusive (from study days 37 to 51 until study days 42 to 56),
- until sacrifice for the non-pregnant females (at least 6 weeks in total, approximately, until study day 41 to day 45).

MICRONUCLEUS PHASE:
The positive control dose formulation was administered as a single dose on the day preceding the scheduled sacrifice (i.e. on completion of the treatment period for groups 1 to 4).
Frequency of treatment:
Once daily
Post exposure period:
None
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 (main study) / 5 (micronucleus phase)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Nature: cyclophosphamide (CPA, CAS No. 6055-19-2, Sigma, Saint-Quentin-Fallavier, France)
- Justification for choice of positive control: In the absence of any specific recommendation in the OECD guideline No. 474, the dose level was selected based on scientific literature, in order to insure a clear positive response.
- Route of administration: oral (gavage)
- Doses / concentrations: 30 mg/kg bw as a single dose 24 hours prior to euthanasia at the end of the treatment period

Examinations

Tissues and cell types examined:
Bone marrow cells (increase of the frequency of micronucleated cells) were examined in the first five animals in sultaine-treated groups and all CPA-treated animals.
Details of tissue and slide preparation:
- Preparation of the smears:
At the end of the treatment period, all animals (first five principal animals in sultaine-treated groups, all CPA-treated animals) were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination. The femurs were removed and bone marrows were flushed and suspended in fetal calf serum. The separation of anucleated erythrocytic cells from other myeloic cells was carried using a cellulose column. This elution step enables the production of slides containing only polychromatic and normochromatic erythrocytes without any nucleated cells or mast cell granules. After centrifugation of the eluate containing the cells, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air dried and stained with Giemsa.
The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring). Thereafter, CPA-treated animals were discarded without any further investigations.

- Microscopic examination of slides:
In a first instance, the micronucleus analysis was performed on the slides of the males only. Based on the toxicological results obtained in this study (as differences were not observed between males and females), the slides analysis was not performed for the females.
For each sampled male, the number of Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio were established by scoring a total of 1000 erythrocytes (PE + NE). Scoring was performed "blind".
Evaluation criteria:
All individual data of MPE/2000PE and the PE/NE ratio were presented in tabular form. Mean and standard deviations of MPE/1000PE and PE/NE ratios were given for each experimental group. Because MPE/1000PE is considered as a percentage, this parameter was statistically analyzed after Arcsine transformation. The model was validated by a comparison between the vehicle group and the positive control. Effect of the test item was determined by comparisons of each group to the control one.
For a result to be considered positive, there must be: either a dose-related increase in the frequency of MPE when compared to the vehicle control group, or an increase in the frequency of MPE in a single dose group, of at least 2-fold the frequency of the vehicle control group.
Biological relevance of the results was considered first. Statistical analysis was used as an aid in evaluating the test results.
Statistics:
Normality and homogeneity of variances were tested using a Kolmogorov-Smirnov test and a Bartlett test. If normality and homogeneity of variances are demonstrated, the statistical comparisons are performed using a Student t-test (two groups) or a one-way analysis of variance (three groups) followed by a Dunnett test (if necessary). If normality or homogeneity of variances is not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (three groups) will be performed followed by a Dunn test (if necessary). All these analyses were performed using the software SAS Enterprise Guide Version 2.05.89 (SAS Release 8.02 TS Level 02M0, SAS Institute Inc), with a level of significance of 0.05 for all tests.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The positive control cyclophosphamide induced a significant increase (p < 0.001) in the frequency of MPE when compared with the vehicle group, indicating the sensitivity of the test system under our experimental conditions. The test conditions were therefore considered to be valid.
The mean values of the PE/NE ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals.
The mean frequencies of MPE in the three test item treated groups were not found significantly different from that in the vehicle group. These results met the criteria of a negative response.
(See Table below)

Any other information on results incl. tables

Table: Results of the cytogenetic test: data summary

Group

Dose(1)

MPE/1000PE

PE/NE ratio

(mg/kg/day)

mean

(sd)

mean

(sd)

Males

Vehicle

0

0.8

(0.4)

0.42

(0.19)

Test item

30

1.3

(0.8)

0.54

(0.09)

100

1.5

(0.9)

0.38

(0.11)

300

0.8

(0.3)

0.48

(0.13)

Cyclophosphamide

30

15.1***

(3.3)

0.22*

(0.06)

(1): expressed as active material

Five animals per group

MPE: Micronucleated Polychromatic Erythrocytes

PE: Polychromatic Erythrocytes

NE: Normochromatic Erythrocytes

sd: standard deviation

*** p<0.001         * p<0.05

The mean values of the polychromatic/normochromatic erythrocytes (PE/NE) ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals. However, based on the effects observed in kidneys (distant from the site of administration) in the repeated-dose toxicity phase of the study, the target tissue (bone marrow) was considered to have been exposed to the test substance and/or its metabolites or degradation products.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions of this study, Cocamidopropyl hydroxysultaine, did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period.
Executive summary:

As part of an OECD 422 compliant study, following daily oral administration (by gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until day 5 postpartum (p.p.)inclusive, an evaluation of the potential of the test item to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells (increase in the frequency of micronucleated cells) was performed.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Cocamidopropyl hydroxysultaine, as a 36.2% aqueous solution, daily, by oral administration (gavage), over the administration period, at doselevels of 30, 100 or 300 mg/kg/day.An additional group of 10 males and 10 females received the vehicle control, drinking water, under the same experimental conditions. The dosing volume was 5 mL/kg/day. Another group of five males and five females received Cyclophosphamide (CPA) as a single dose of 30 mg/kg on the day preceding schedule sacrifice, and acted as a positive control group for micronuclei induction. At necropsy, the femur of the first five principal animals in groups 1 to 4 and all group 5 animals were sampled for bone marrow micronucleus analysis.

There were no pathological findings in animals given 30 mg/kg/day of CPA at microscopic examination.

The positive control cyclophosphamide induced a significant increase (p < 0.001) in the frequency of micronucleated polychromatic erythrocytes (MPE) when compared with the vehicle group, indicating the sensitivity of the test system under our experimental conditions. The test conditions were therefore considered to be valid.

The mean values of the polychromatic/normochromatic erythrocytes (PE/NE) ratios in animals treated with the test item at 30, 100 or 300 mg/kg/day were not statistically significantly different from that of the vehicle control animals. However, based on the effects observed in kidneys (distant from the site of administration) in the repeated-dose toxicity phase of the study, the target tissue (bone marrow) was considered to have been exposed to the test substance and/or its metabolites or degradation products.

The mean frequencies of MPE in the three test item treated groups were not found significantly different from that in the vehicle group. These results met the criteria of a negative response.

In conclusion, under the experimental conditions of the study, Cocamidopropyl hydroxysultaine did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells up to 300 mg/kg administered daily for the whole dosing period (i.e., approximately 5 to 6 weeks).