Phenol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 November 2021 to 2022 (draft report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Version / remarks:

2020 version

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

transgenic rodent mutagenicity assay

Test material

Test material information

Constituent 1

Reference substance name:

Phenol

EC Number:

203-632-7

EC Name:

Phenol

Cas Number:

108-95-2

Molecular formula:

C6H6O

IUPAC Name:

phenol

Test animals

Species:

mouse

Strain:

other: Muta™Mouse (CD2-lacZ80/HazfBR)

Details on species / strain selection:

Muta™Mouse was selected as the test system as it is the most commonly reported transgenic system in use and forms the majority of entries in the Transgenic Rodent Assays Information Database (TRAID; Lambert et al., 2005). There is also a large volume of background data in this transgenic strain in the laboratory.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK (Range-Finding Experiments and Main Experiment) or France (Main Experiment).
- Age at study initiation: approximately 7 to 9 weeks old on the first day of dosing in the range-finder experiment, and approximately 12 to 13 weeks old on the first day of dosing in the main experiment.
- Weight at study initiation: approximately 23-29 g (males) or 17-22 g (females) on the first day of dosing in the range-finder experiment, and approximately 24-32 g on the first day of dosing in the main experiment.
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in wire topped, solid bottomed cages; males singly and females with a maximum of three animals per cage.
- Diet: LF2 EU Rodent Diet (ad libitum).
- Water: Mains water (ad libitum).
- Acclimation period: at least 7 days before the start of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 40% to 70% (sometimes below 40% but it was transient and minor)
- Air changes: minimum of 15 air changes/hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 30 November 2021 To 09 March 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: it has been used in previous in vivo studies (soluble)
- Concentration of test material in vehicle: 1.25, 2.5 or 5.0 mg/mL (main experiment), 2.5-10 mg/mL in the range-finder experiment.
- Amount of vehicle: 20 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Phenol was weighed into a formulation container and an appropriate volume of vehicle (purified water) added to achieve the final volume. Formulations were stirred on a magnetic stirrer to homogenise.
Formulations were prepared daily in the range-finding experiments and once weekly in the main Experiment (when stability was confirmed).

Duration of treatment / exposure:

Main experiment: 28 days
Range-finder experiment: 14 days

Frequency of treatment:

once daily

Post exposure period:

Main experiment only: 28-day dose-free expression period (genotoxicity animals were sacrificed on Day 56)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

main experiment

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

main experiment

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

main experiment

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

range-finder experiment

Doses / concentrations 5

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

range-finder experiment

Doses / concentrations 6

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

range-finder experiment

Doses / concentrations 7

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

range-finder experiment

No. of animals per sex per dose:

Main experiment: seven males/group in the genotoxicity subgroup, three males/group for the satellite animals (for bioanalysis)
Ranger-finder experiment: 3 males and 3 females/group

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylnitrosourea
- Justification for choice of positive control(s): known mutagen
- Route of administration: no data
- Doses / concentrations: 50 mg/kg/day
Remark: no concurrent positive control animal treatments were specifically conducted in this study. Tissues from appropriate positive control treated animals (treated independently to this study) were used to provide DNA that were analysed alongside the DNA from animals in this study.

Examinations

Tissues and cell types examined:

Liver, glandular stomach and duodenum.
The developing germ cells from the seminiferous tubules was also taken and retained for possible future analysis.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on previous existing toxicological data and from the results of the range-finder experiment performed before the main experiment (see details in 'Additional information on results').

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
In the Main Experiment the liver, glandular stomach, dudoenum and the seminiferous tubules of the testes (developing germ cell) were taken from all Subgroup 1 animals (on day 56). Tissues were removed, rinsed (where appropriate) and flash frozen in liquid nitrogen prior to DNA extraction and determination of mutant frequency.
The seminiferous tubules of the testes were held in a freezer at nominally -80°C
In the Range-Finder Experiments, the liver, glandular stomach and duodenum were taken from all surviving animals for histopathology. Samples of liver glandular stomach and duodenum were removed, immediately preserved in neutral buffered formalin and stored at room temperature. The liver organ weights were recorded prior to storage.

DETAILS OF SLIDE PREPARATION:
Preserved liver, glandular stomach and duodenum samples form the survving Range-Finder animals along with the three male and three female untreated comparator animals were embedded in wax blocks and sectioned at 5 µm nominal. Slides were stained with haematoxylin and eosin and examined to evaluate any dose limiting microscopic changes which could impact endpoint assessment.

METHOD OF ANALYSIS:
DNA from the liver, glandular stomach and duodenum was extracted using the Agilent RecovereaseTM DNA isolation and purification kits (Agilent Technologies UK Ltd., Stockport, UK). Prior to extraction a slice of tissue (or entire duodenum) was excised from each frozen sample of liver, glandular stomach or duodenum and homogenised in 5 mL chilled lysis buffer. The remaining tissues were retained at -80ºC (nominal).
Isolated genomic DNA from all tissues was stored at 2-8°C until required for packaging.

DNA Packaging into Bacteriophage Heads:
For each tissue/animal, approximately 5 µL of DNA solution were mixed with Agilent Transpack packaging reagents. Following the necessary incubation and mixing procedures, the resulting mixture contained DNA packaged into phage heads ready for transfection.

Transfection of Host Bacteria:
For the titration plates, packaged DNA (10 µL) was diluted with SM buffer (190 µL) and 10 µL of this dilution adsorbed to 500 µL suspension of E. coli C lac galE- Kanr (galE- Ampr) for approximately 20-30 minutes at room temperature. After adsorption, the phage/bacteria was suspended in 12 mL 1:3 LB:NaCl, 0.75% w/v agar (top agar) containing 10 mM magnesium sulphate and plated onto petri dishes (14 cm diameter) containing 12 mL 1:3 LB:NaCl, 1.5% w/v agar (bottom agar). Once the agar had gelled, the plates were inverted and placed in an incubator set to 37ºC overnight.
The remaining packaged DNA was divided equally into 3 tubes and incubated at room temperature with bacterial suspension (500 µL/tube) for approximately 20-30 minutes, suspended in top agar (as above) containing 10 mM magnesium sulphate and 0.3% w/v phenyl-galactose (P-gal), poured onto plates containing bottom agar (as above) and placed in an incubator set to 37°C overnight.

Scoring of Plates:
Following overnight incubation, plates were either scored immediately or stored at 2-8°C and scored as soon as possible.

Evaluation criteria:

All analysis was based on individual animal response.
The total number of mutant plaques and the total number of pfu for each packaging occasion were compiled to give the total number of pfu and mutant frequency (MF) for each animal.

A test article was considered positive, i.e. capable of inducing mutation, if:
1. A least one of the test doses exhibited a statistically significant increase in the MF, in any tissue, compared with the concurrent vehicle control
2. The increase exceeded the laboratory’s historical control data for that tissue.
A positive result may consist of either a dose-related increase in the MF, or a clear increase in the MF in a single dose group.
The test article was considered negative in this assay if none of the above criteria were met and tissue exposure was confirmed.
Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Evidence of a dose-related effect is considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between dose levels.
The biological relevance of any positive findings were considered in the context of the laboratory’s background control historical database.

Statistics:

Statistical analysis of MF per tissue was performed as follows:
Control and Test groups were analysed using one-way analysis of variance (ANOVA). Levene’s test for equality of variances across the groups was also performed and in all cases showed no evidence of heterogeneity (P>0.01). Comparisons between each treated group and control were performed using Dunnett’s test, performed with a one sided risk for increasing response. In addition, a linear contrast was used to test for an increasing dose response.
No statistical analysis was performed on the positive control tissues.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Remarks:

liver

Toxicity:

no effects

Remarks:

tested up to 100 mg/kg bw/day (maximum tolerated dose)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Sex:

male

Genotoxicity:

negative

Remarks:

glandular stomach

Toxicity:

no effects

Remarks:

tested up to 100 mg/kg bw/day (maximum tolerated dose)

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Sex:

male

Genotoxicity:

negative

Remarks:

duodenum

Toxicity:

no effects

Remarks:

tested up to 100 mg/kg bw/day (maximum tolerated dose)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs: no clinical or post dosing signs of toxicity were observed at 50, 100 and 30 mg/kg bw/day. At 200 mg/kg, moderate to severe post dosing signs of toxicity related to treatment with phenol commenced within 30 minutes to 1 hour of dosing on Day 1. One female was found dead at approximately 10 minutes post dose. In the remaining animals, signs of toxicity included moderate increased activity, ataxia and moderate whole body tremors. Due to the severity of the clinical observations, all remaining animals were subsequently removed from the study within 1 hour of dosing.
- Body weights: at 50, 100 and 30 mg/kg bw/day, all animals had gained bodyweight by the end of the 14-day dosing/observation period, with the exception of one female at 50 mg/kg bw/day.
- Food consumption: no notable effect observed.
- Organ weights: no notable effect of treatment on liver organ weight was observed.
- Histopathology: upon macroscopic and microscopic examination in surviving animals at 50, 100 or 30 mg/kg bw/day, no findings considered related to administration with phenol were recorded. Although phenol can be corrosive and an irritant, histopathology confirmed there were no confounding microscopic changes that could have confounded endpoint evaluation in stomach and duodenum.
From these results 100 mg/kg bw/day was considered to be a suitable projected maximum tolerated dose (MTD) for the 28-day Main Experiment and was therefore selected as the maximum dose.
As no substantial difference in toxicity was observed between male and female animals in the range-finder experiment, and as the developing germ cells of the seminiferous tubules were a potential organ for mutagenicity assessment, male animals only were used in the main experiment.

RESULTS OF DEFINITIVE STUDY
No clinical or post-dosing signs of toxicity were observed in the vehicle control group, or phenol dosed animals at 25, 50 or 100 mg/kg bw/day with the exception of squinting observed on Day 18.

Genotoxicity results:
* In the liver, no statistically significant increases in mutant frequency (MF) were observed at the low or high doses of 25 and 100 mg/kg bw/day. At the intermediate dose (50 mg/kg/day), there was a small, but statistically significant increase in MF (P≤0.05), which contributed to a statistically significant linear trend (P≤0.05). However, individual animal and group mean MF for all phenol treated groups fell within the laboratory’s historical vehicle control 95% reference range, and most animals fell around, or below the historical vehicle control mean value. It was therefore considered any increases in MF were within the normal variation for the assay and the statistical significance of no biological relevance.
* In the glandular stomach, no statistically significant increases in MF were observed and there was no evidence of a dose related response. Individual animal and group mean MF for all phenol-treated groups fell within the laboratory’s historical vehicle control range, with two exceptions which fell below this range (but within the range of the current test vehicle control group).
* In the duodenum, no statistically significant increases in MF were observed and there was no evidence of a dose related response. Individual animal and group mean MF for all Phenol-treated groups fell within the laboratory’s historical vehicle control 95% reference range.

Assay validity:
The vehicle control MF data for liver, glandular stomach and duodenum generally fell within the laboratory’s historical vehicle control ranges, with exceptions to this falling below, rather than above these ranges.
Concurrent positive control DNA, tissue matched and packaged at the same time as the study DNA, gave elevated MF compared to the concurrent vehicle control DNA and confirmed correct packaging procedures and efficient packaging reactions. The assay was therefore accepted as valid.

Formulation analysis:
Samples of vehicle control and formulations of phenol were taken once during Weeks 1 and 4 of dosing, showed that all formulations were homogeneous. Achieved concentrations of phenol formulations prepared at 1.25, 2.5 and 5 mg/mL generally fell within 100±10% of nominal. No test article was detected in the vehicle samples. Stability was demonstrated at 0.25 and 10 mg/mL for 8 days at 15-25°C, protected from light.

Bioanalysis:
Systemic exposure of phenol was confirmed in the blood at 50 and 100 mg/kg/day. At the low dose of 25 mg/kg/day, only one animal contained measurable levels of phenol above the quantification limit.

Any other information on results incl. tables

Summary of Liver Data:

Group	Treatment	Dose (mg/kg/day)	Group Mean MF (x10^6)	SD	P-value
1	Vehicle	0	41.54	13.459	-
2	Phenol	25	40.31	10.044	0.8156 (NS)
3	Phenol	50	56.93	12.575	0.0423 (P≤0.05)
4	Phenol	100	50.56	12.640	0.1992 (NS)
N/A	Positive Control	50	282.62	41.52	-

Dose response (Group 1, 2, 3, 4): P = 0.0232 (P≤0.05)

NS: not statistically significant (P>0.05)

N/A: Not Applicable

 

Summary of Glandular Stomach Data:

Group	Treatment	Dose (mg/kg/day)	Group Mean MF (x10^6)	SD	P-value
1	Vehicle	0	37.62	14.331	-
2	Phenol	25	43.71	14.729	0.5242 (NS)
3	Phenol	50	53.33	21.386	0.1435 (NS)
4	Phenol	100	45.57	20.868	0.4292 (NS)
N/A	Positive Control	50	899.09	141.89	-

Dose response (Group 1, 2, 3, 4): P = 0.1563 (NS)

NS: not statistically significant (P>0.05)

N/A: Not Applicable

 

Summary of Duodenum Data:

Group	Treatment	Dose (mg/kg/day)	Group Mean MF (x10^6)	SD	P-value
1	Vehicle	0	67.68	24.295	-
2	Phenol	25	64.24	25.218	0.8523
3	Phenol	50	46.96	10.560	0.9975
4	Phenol	100	62.28	13.767	0.8959
N/A	Positive Control	50	1473.38	292.28	-

Dose response (Group 1, 2, 3, 4): 0.8397 (NS)

NS: not statistically significant (P>0.05)

N/A: Not Applicable

 

Bioanalysis:

Dose Level (mg/kg/day)	Mean Plasma Conc. (mg/L) 5 minutes post-dose	Mean Plasma Conc. (mg/L) 30 minutes Â p ost-dose
25	3.38^a	BLQ
50	7.34	BLQ
100	37.2	12.5^b

BLQ: Below the limit of quantification (2.4 mg/L)

a: mean generated from a single animal as two were BLQ

b: mean generated from two animals as one was BLQ

 

For other result tables, see in the attached document.

Applicant's summary and conclusion

Conclusions:

In this study, phenol did not induce mutation in the lacZ transgene in the liver, glandular stomach or duodenum of male Muta™Mice when treated up to for 28 consecutive days at dose levels up to 100 mg/kg bw/day (the maximum tolerated dose) under the experimental conditions employed.

Executive summary:

Phenol was tested for its ability to induce gene mutation in the lacZ transgene (according to OECD TG 488 and GLP) in liver, glandular stomach and duodenum from Muta™Mice (CD2-lacZ80/HazfBR strain). The developing germ cells from the seminiferous tubules were also taken and retained for possible future analysis.
Groups of 7 males were administrated phenol by oral gavage during 28 consecutive days at the dose levels of 25, 50 and 100 mg/ kg bw/day (100 mg/kg was determined as the MTD in a range-finder assay) in purified water (20 mL/kg). Genotoxicicity animals were sacrificed on day 56 following a 28-day dose-free expression period. A concurrent vehicle control group was included. For the positive control, samples treated in a previous study were used, and tissue matched and packaged alongside the DNA from animals in this study were analysed. Formulation analysis and bioanalysis (satellite animals) were performed.

In-life observations consisted in clinical and post-dosing observation, and recording of body weights and food consumption.

On day 56, genotoxicity animals were euthanasied and necropsied.Tissue samples from liver, gladular stomach, duodenum and seminiferous tubules of the testes were removed, prepared and/or stored. Analysis of the mutant frequency was performed (except on seminiferous tubules).

 

No clinical or post-dosing signs of toxicity were observed in the vehicle control group, or phenol dosed animals at 25, 50 or 100 mg/kg/day with the exception of squinting observed on Day 18.
Systemic exposure of phenol was confirmed in the blood at 50 and 100 mg/kg/day. At the low dose of 25 mg/kg/day, only one animal contained measurable levels of phenol.
The vehicle and positive control mutant frequency (MF) data confirmed the validity of the assay.
In the liver, no statistically significant increases in MF were observed at the low or high doses of 25 and 100 mg/kg/day. At the intermediate dose (50 mg/kg/day), there was a small, but statistically significant increase in MF (P≤0.05), which contributed to a statistically significant linear trend (P≤0.05). However, individual animal and group mean MF for all phenol treated groups fell within the laboratory’s historical vehicle control 95% reference range, and most animals fell around the historical vehicle control mean value. It was therefore considered any increases in MF were within the normal variation for the assay and the statistical significance of no biological relevance.
In the glandular stomach, no statistically significant increases in MF were observed and there was no evidence of a dose related response. Individual animal and group mean MF for all phenol-treated groups fell within the laboratory’s historical vehicle control range, with two exceptions which fell below.
In the duodenum, no statistically significant increases in MF were observed and there was no evidence of a dose related response. Individual animal and group mean MF for all Phenol-treated groups fell within the laboratory’s historical vehicle control 95% reference range.

It is concluded that phenol did not induce mutation in the lacZ transgene in the liver, glandular stomach or duodenum of male Muta™Mice when treated up to for 28 consecutive days at dose levels up to 100 mg/kg/day (the maximum tolerated dose) under the experimental conditions employed.