4-ethylphenol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-March-22 to 2022-May-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Gemany

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-ethylphenol

EC Number:

204-598-6

EC Name:

4-ethylphenol

Cas Number:

123-07-9

Molecular formula:

C8H10O

IUPAC Name:

4-ethylphenol

Details on test material:

- Name: 4-Ethylphenol
- CAS No.: 123-07-9
- Batch No.: STBK4225
- Physical State: solid
- Colour: white
- Purity: 99.4%
- Expiry Date: 31 August 2026
- Storage Conditions: 2-8°C, protected from light
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River, 97633 Sulzfeld, Germany
Number of animals: 5 males per dose group (7 animals for 1 MTD)
Initial age at start of acclimatisation: 6 - 8 weeks
Age at start of treatment: Minimum 7 weeks


ENVIRONMENTAL CONDITIONS
Housing: 5 animals of identical sex per cage
Cage type: IVC cage (Polysulphone), Type III H
Bedding: Altromin saw fiber bedding (Batch: 0131)
Feed: Free access to Altromin 1324 (Batch: 0939) maintenance diet for rats and mice
Air change: At least 10 x per hour
Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
Environment: Temperature 22 ± 3 °C
Relative humidity 55 ± 10%
Artificial light 6:00 - 18:00

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Name: Corn oil
Supplier: Sigma
Batch No.: MKCN9742

Details on exposure:

The test item was prepared in corn oil within 2 h before treatment. All animals received a single volume orally of 10 mL/kg bw.

Frequency of treatment:

All animals received a single volume orally of 10 mL/kg bw.

Post exposure period:

The sampling times were 48 h for all dose groups evaluated and additionally 72 h for the negative control and highest dose group.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Remarks:

1 MTD

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Remarks:

0.5 MTD

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day

Remarks:

0.25 MTD

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
CAS No.: 50-18-0
Supplier: Sigma
Catalogue No.: C0768
Batch No.: MKCL2547
Dissolved in: physiological saline
Dosing: 10 mg/kg bw
Route and frequency of
administration: ip, single
Volume administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:

peripheral blood erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
A pre-study on acute toxicity was performed with both gender of the same strain and under identical conditions as in the mutagenicity study. The maximum dose administered was 2000 mg/kg bw according to the OECD 474 guideline. The maximum volume which was administered at one time was 10 mL/kg bw. The animals received the test item once. The sampling times were 48 h for all dose groups evaluated and additionally 72 h for the negative control and highest dose group. Since no relevant differences in systemic toxicity were observed for male and female animals in the pre-experiment, the main experiment was only performed with male animals.

METHOD OF ANALYSIS
Evaluation of all samples, including those of positive (CPA) and negative controls, were performed using a flow cytometer (FACSLyric, BD Biosciences). Anti-CD71 antibodies were labeled with fluorescein isothiocyanate (FITC), anti-CD61 antibodies were labelled with phycoerythrin (PE). Particles were differentiated using forward scatter (FSC) and side scatter (SSC) parameters of the flow cytometer. Fluorescence intensities were recorded on PMT position E, D and B for FITC, PE and PI, respectively. 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect a possibly occurring cytotoxic effect of the test item, the ratio between immature and mature erythrocytes was determined. The result was expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes).

Evaluation criteria:

Providing all acceptability criteria are fulfilled, a test item is considered clearly positive if:
- at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
- any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
If only the highest dose is examined at a particular sampling time, a test item is considered clearly positive if there is a statistically significant increase compared with the concurrent negative control and the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and
- bone marrow exposure to the test item occurred.

Statistics:

Pairwise comparison of the proportion of PCE among total erythrocytes as well as the proportion of micronucleated polychromatic (immature) erythrocytes (PCE) among total PCE between the control groups and each of the treatment groups was performed by means of the non-parametric Mann-Whitney test at a statistical significance level of 5% (p < 0.05, two-tailed).
The X² test for trend at a statistical significance level of 5% (p < 0.05, two-tailed) was used to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 44 h sampling time.
Statistical methods were performed using the software GraphPad Prism version 6.0.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Animals treated with the highest dose of 500 mg/kg bw showed toxic effects.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY

Clinical signs of toxicity in test animals:
In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. One male and one female rat received a single dose of 2000 mg/kg bw orally and showed strong toxicity such as reduction of spontaneous activity, hunched posture, piloerection, ataxia and half eyelid closure. Both animals were euthanised 1.5 h after application for animal welfare reasons. The dose was reduced to 500 mg/kg bw. Three male and three female rats received a single dose of 500 mg/kg bw orally and showed toxicity such as reduction of spontaneous activity, prone position, hunched posture, ataxia, piloerection and eyelid /half eyelid closure.

RESULTS OF DEFINITIVE STUDY

- Toxicity in the main experiment
500 mg/kg bw was tested as the maximum tolerated dose (1 MTD) in the main experiment. The volume administered orally was 10 mL/kg bw. Animals treated with the highest dose (1 MTD) showed toxic effects with reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture and half eyelid closure up to 4 h after application. Male rats treated with 250 mg/kg bw (0.5 MTD) and 125 mg/kg bw (0.25 MTD) showed no clinical signs of systemic toxicity after treatment with the test item


- Induction of micronuclei (for Micronucleus Assay):
For all dose groups, including positive and negative controls, 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.
The negative controls (48 h and 72 h) evaluated were within the historical control limits of the negative control (0.03 – 0.14% for males). The mean values of micronuclei observed for the negative control were 0.09% after 48 h and 0.10% after 72 h .
The mean value of micronuclei observed after treatment with 0.25 MTD was 0.07%. The mean value observed was within the range of the concurrent negative control as well as within the historical control limits of the negative control.
The mean value noted for the 0.5 MTD dose group was 0.06%. The mean value observed was within the range of the concurrent negative control as well as within the historical control limits of the negative control.
The dose group treated with 1 MTD (48 h sampling) showed a mean value of 0.07%. The mean value observed was within the range of the concurrent negative control as well as within the historical control limits of the negative control. The mean value observed for the 1 MTD (72 h sampling) was 0.06%. The mean value was statistically significantly decreased compared to the concurrent negative control and within the historical control limits of the negative control. No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.


- Ratio of PCE
The relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes) was determined for each animal. The relative PCE is the supportive endpoint to assess cytotoxicity, which helps to demonstrate a target cell exposure with the test item.
The negative control (48 h) was within the historical control limits of the negative control for the 48 h sampling time (0.64% - 2.49% for males). The mean value noted for the 48 h negative control was 2.20%. For the 72 h negative control a mean value of 2.56% was observed (Table 4 and Table 9). The value lay slightly above the upper control limit which can be attributed to blood loss 24 h earlier for the first sampling.
The animal group treated with 0.25 MTD showed a mean value of the relative PCE of 2.39% (Table 6). The mean value observed was within the range of the concurrent negative control and within the historical control limits of the negative control.
The dose group, which was treated with 0.5 MTD, showed a mean value of the relative PCE of 1.95% (Table 7). The mean value observed was within the range of the concurrent negative control and within the historical control limits of the negative control.
The animals who received 1 MTD (48 h sampling) showed a mean value of the relative PCE of 1.67% (Table 8). The mean value observed was within the range of the concurrent negative control and within the historical control limits of the negative control.


- Statistical evaluation
The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated. Additionally, the X² Test for trend was performed to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 48 h sampling time. No statistically significant increase in the frequency of micronucleated cells was observed.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study and under the experimental conditions reported, the test item 4-Ethylphenol did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the rat.
Therefore, the test item 4-Ethylphenol is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

In a Wistar rats peripheral blood micronucleus assay, five male  animals per dose group were treated orally with the test item at doses of 500, 250 and 125 mg/kg bw.  Peripheral blood cells were harvested at 48 h (all dose and control groups) and 72 h (negative control and 1 MTD group) post-treatment.  The vehicle was Corn Oil. The animals received the test item once orally. There were signs of toxicity during the study.  The animals treated with doses of 0.2 and 0.5 MTD showed no signs of systemic toxicity. The animals treated with a dose of 1 MTD showed signs of systemic toxicity such as reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture and half eyelid closure up to 4 h after application.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood cells after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.