Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Bacterial Reverse mutation assay (OECD 471): Negative (with and without metabolic activation). Chromosome Aberration assay (OECD 473): Positive (with metabolic activation) Negative (without metabolic activation). Mammalian cell gene mutation assay (OECD 476): Negative with and without metabolic activation. Mammalian Erythrocyte Micronucleus Test (OECD 474): Negative.
Link to relevant study records
Reference
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:
July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern GLP study conducted in accordance with OECD Guideline 474 (OECD. 2014).
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: 6 weeks
- Weight at study initiation: Males 192.3-209.3g, Females 147.3-171.2g.
- Assigned to test groups randomly: yes, randomization function within Microsoft Excel
- Fasting period before study: No
- Housing: Animals of same sex housed up to five per Micro-barrier cage.
- Diet: Harlan 2018C Certified Global Rodent Diet, food provided ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ±3 F
- Humidity (%): 50± 20%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: stability and solubility has been demonstrated in corn oil
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 5mL/kg
Details on exposure:
Oral Gavage: All dose formulations were administered at a volume of 5 mL/kg by single oral gavage using appropriately sized disposable polypropylene syringes with gastric intubation tubes (needles)
Frequency of treatment:
Single dose
Post exposure period:
24 hour (control, low, mid and high dose groups)
48 hour (control and high dose groups)
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
actual ingested
Vehicle control (corn oil)
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes
Positive control(s):
- cyclophosphamide
- Justification for choice of positive control(s): OECD Guideline 474
- Doses / concentrations: 40 mg/kg
- Generated from BioReliance study AE19TL.125M012.BTL, bone marrow harvested at 24 hours after treatment
Tissues and cell types examined:
Tissue Examined: Femoral bone marrow
Cells Types Examined: Polychromatic Erythrocytes (PCEs), Normochromatic Erythrocytes (NCEs) and Micronucleated Polychromatic Erythrocytes (MnPCEs).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on acute toxicity and repeated dose toxicity of the registration substance.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Treatment oral gavage - day 0. Sampling time - 24 hours (vehicle control, 100, 300 and 1000 mg/kg) and 48 hours (vehicle control and 1000 mg/kg).

DETAILS OF SLIDE PREPARATION: Femoral bone marrow was collected at approximately 24 and 48 hours after dose administration. Animals were euthanized by carbon dioxide inhalation. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow aspirated into a syringe containing fetal bovine serum. The bone marrow was transferred to a centrifuge tube containing 2 mL fetal bovine serum, the cells were pelleted by centrifugation, and the supernatant was drawn off leaving a small amount of fetal bovine serumwith the pellet. Cells were resuspended and a small drop of the bone marrow suspension was spread onto a glass slide. At least four slides were prepared from each animal, air dried and fixed by dipping into methanol. One set of slides (including at least 5 sets of positive control slides) was stained with acridine orange for microscopic evaluation. The other set of two slides was kept as backup and will be archived. Each slide was identified by the harvest date, study number and animal number. Slides were coded using a random table by an individual not involved with the scoring process.

METHOD OF ANALYSIS: Bone marrow was evaluated by fluorescent microscopy. The staining procedure permitted the differentiation by color of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) (bright orange PCEs and ghost like, dark green NCEs, respectively).

The criteria for the identification of micronuclei are those of Schmid (1975). Micronuclei are brightly stained bodies that are generally round and that generally are between 1/20 and 1/5 the size of the PCE. The frequency of micronucleated cells was recorded with cells containing one or more micronuclei counted as one micronucleated PCE (MnPCE).

At least 4000 s/animal were scored for the presence of micronuclei (MnPCEs) whenever possible. In addition, at least 500 total erythrocytes (PCEs + NCEs) were scored per animal to determine bone marrow cytotoxicity. A reduction in the PCE proportions to less than 20% of the vehicle control value was considered excessively cytotoxic and the animal data were excluded from evaluation.

OTHER:
Evaluation criteria:
Evaluation of Test Results:

A test substance was considered to have induced a positive response if:

a) at least one of the test substance doses exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.

A test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the bone marrow was exposed to the test substance (unless intravenous administration was used).
If the response was neither clearly positive nor clearly negative, or in order to assist in establishing the biological relevance of a result, the data were evaluated by expert judgment and/or further investigations. Possible additional work may include scoring additional cells (where appropriate) or performing an additional experiment that could employ the use of modified experimental conditions. Such additional work was only carried out following consultation with, and at the request of, the Sponsor.

In some cases, even after further investigations, the data set precluded making a conclusion of positive or negative, at which time the response was concluded to be equivocal. In such cases, the Study Director used sound scientific judgment and reported and described all considerations.

Statistics:
Statistical analysis was performed on the micronucleus frequency (MnPCE%) and PCE% using the animal as the unit. The mean and standard deviation of MnPCE% and PCE% were presented for each treatment group.

The use of parametric or non-parametric statistical methods in the evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test substance groups at the respective sampling time were compared using Levene’s test (significant level of p  0.05). Since the variation between groups was found not to be significant, a parametric one-way ANOVA was performed followed by a Dunnett’s post hoc analysis to compare each dose group to the concurrent vehicle control.

A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p 0.01).

A pair-wise comparison (Student’s T-test) was used to compare the positive control group to the concurrent vehicle control group.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mortality - No mortality occurred at any dose level during the course of the definitive assay (see table 2 attached).

Body Weight - Body weight cahnges were comparable with vehicle control (see table 2 attached).

Clinical Signs - Signs of lethargy and piloerection were observed in both males and females at 100 mg/kg. All other animals appeared normal throughout the observation period (see table 1 attached).

The incidence of MnPCEs per 20,000 PCEs scored (4000 PCEs/animal) and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time in Table 3 (attached). Individual animal data are presented in Table 4 and 5 (attached).

Historical Controls - see attachment
Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the assay described in this report, 2-(2-naphthyloxy)ethanol was concluded to be negative for the induction of micronucleated polychromatic erythrocytes in both male and female rats.
Executive summary:

The test substance, 2-(2-naphthyloxy)ethanol, was evaluated for its clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in rat bone marrow. Corn oil was selected as the vehicle. Test and/or vehicle control substance formulations were administered at a dose volume of 5 mL/kg by single oral gavage.

A dose range finding assay (DRF) was not performed. Based upon toxicity information provided by the Sponsor (Charles River Study Nos. 20016973, 20016974), the high dose for the definitive assay was 1000 mg/kg. The definitive assay dose levels tested were 100, 300 and 1000 mg/kg.

No statistically significant increase in the incidence of MnPCEs was observed in male rats at either 24 or 48 hours after treatment relative to the vehicle controls. 

A statistically significant increase in the incidence of MnPCEs was observed in female rats 24 hours after treatment at 1000 mg/kg relative to the vehicle control. Conversely, the group mean and individual animal results in females at 1000 mg/kg were within the historical vehicle control range. 

A statistically significant decrease in %PCE was observed in female rats 24 and 48 hours after treatment at 1000 mg/kg relative to the vehicle controls; however, this decrease was not considered biologically significant, because it was only 9% lower than the vehicle control. In order to call it significant the decrease should in the range of ~80%.

There was no dose dependent response in the incidents of micronuclei in the test article treated groups.

The positive control induced a statistically significant increase in the incidence of MnPCEs, indicating that the test was performing normally. 

The number of MnPCEs in the vehicle control groups were within the historical control range. 

Under the conditions of this study, the administration of 2-(2-naphthyloxy)ethanol at doses up to and including a dose of 1000 mg/kg was concluded to be negative in both male and female rats in the Micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Three in vitro mutagenicity studies have been conducted to fulfil the testing requirements outlined in REACH Annex's VII and VIII. Each of the three tests has been conducted in accordance with OECD guidelines and GLP standards. Of the three assays both the in vitro gene mutation study in bacteria and the in vitro gene mutation study in mammalian cells gave negative results for mutagenicity in the presence and absence of metabolic activation. The in vitro cytogenicity study in mammalian cells gave a positive result in the presence of metabolic activation for structural aberrations but not numerical aberrations, and a negative result in the absence of metabolic activation for both structural and numerical aberrations. This result alone is not sufficient to conclude on the mutagenic hazard of EC-202-228-8, therefore in accordance with REACH Annex IX a further in vivo mutagenicity study (OECD 474) was conducted to determine the mutagenic potential of the registration substance. Under the conditions of this study, the administration of 2-(2-naphthyloxy)ethanol at doses up to and including a dose of 1000 mg/kg was concluded to be negative in both male and female rats in the Micronucleus assay.


Justification for selection of genetic toxicity endpoint
GLP study conducted in accordance to OECD Guideline 474. Klimisch score 1.

Justification for classification or non-classification

The results from the three key studies for genotoxicity show some variation, with negative results seen in the in vitro gene mutation assay in bacteria and in the in vitro gene mutation assay in mammalian cells, and a positive result seen in the in vitro cytogenicity study in mammalian cells. An in vivo mutagenicity study (OECD 474) was conducted to determine the mutagenic potential of the registration substance. Under the conditions of this study, the administration of of 2-(2-naphthyloxy)ethanol at doses up to and including a dose of 1000 mg/kg was concluded to be negative in both male and female rats in the Micronucleus assay. Based on the available data, the registration substance is concluded to be non-genotoxic based on the criteria laid out in Regulation (EC) No. 1272/2008.