Benzene, (1-methylethyl)-, oxidized, polyphenyl residues

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

other: read-across from most toxic ingredient

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is comparable to OECD Guideline 412 with acceptable restrictions (the high dose should result in toxic effects; histopathology of a limited number of organs [spleen, adrenals & heart not examined]; differential leucocyte count not determined in hematology; fasting glucose and ornithine decarboxylase not determined in clinical chemistry). According to REACH Regulation (EC) No 1907/2006 Annex VIII an exposure duration of 28 days is recommended.

Data source

Reference

Reference Type:

publication

Title:

Two-Week (Ten-Day) Inhalation Toxicity and Two-Week Recovery Study of Phenol Vapor in the Rat

Author:

Hoffman GM, Dunn BJ, Morris CR, Butala JH, Dimond SS, Gingell R, Waechter JM

Year:

2001

Bibliographic source:

Int J Toxicol 20: 45-52

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

(high dose not toxic)

GLP compliance:

yes

Limit test:

no

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 material form:

solid: crystalline

Details on test material:

- Physical state: a white solid
- Analytical purity: pure (100%)
- Purity test date: responsibility of the sponsor
- Lot/batch No.: lot number 112796; batch number 102596
- Expiration date of the lot/batch: none, considered stable for the duration of the study; storage at room temperature in glass container with N2-blanket
- Stability under test conditions: see analytics
- Source: Harrell Industries, Rock Hill, SC 29730 (USA)

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- inbred CDF (F-344/CrlBr)
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at study initiation: 8 weeks
- Weight at study initiation: 127 g (females; range 113-146 g) and 203 g (males; range 178-229)
- Fasting period before study: no
- Housing: individually
- Diet (ad libitum): certified and analysed diet, no contamination
- Water (ad libitum): city drinking water; monthly analysis, no contamination
- Acclimation period: 28 days; rats acclimated to plastic flow-past nose-only cones for 2 days prior to first exposure
- Randomization: randomly selected by body weight

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 23-75
- Air changes (per hr): 6.1-10.5
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

other: demineralized & distilled water; air in inhalation chamber

Remarks on MMAD:

MMAD / GSD: As expected, the measurements demonstrated similar MMAD for air control chamber and exposure chamber indicating vapour formation.

Details on inhalation exposure:

Rats acclimated to plastic flow-past nose-only cones for 2 days prior to exposure. During the exposure periods rats placed in the nose-only cones and rotated at each exposure to ensure uniform exposure; exposure chambers operated at 21 to 27°C with relative humidity of 32- 65%; airflow rate in the exposure chamber was 46, 46, 48, 42 l/min, respectively.

Phenol diluted in distilled water, mixtures of 0.05%, 0.5%, and 5.0% prepared for generation of the vapors for the 0.50, 5.0, and 25 ppm groups, respectively; phenol
solution pumped directly to a counter current volatilization chamber (glass helix heated and controlled by a variable auto transformer; flask of phenol solution was vented with a gas bag filled with nitrogen); houseline air (humidified) flowed up through the volatilization chamber and volatilized the phenol; phenol-laden air flowed through tubing of the volatilization chamber into the top of the flow-past nose-only exposure chamber.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples withdrawn hourly from each chamber (each day), samples extracted with methanol and High Performance Liquid Chromatograph (HPLC) with U'V-vis detector; particle size distribution measurements performed once during each exposure (TSI Model 3310A Aerodynamic Particle Sizer); phenol was totally vaporized, no aerosol was present.

Duration of treatment / exposure:

6 hours per day, 5 days per week for 2 weeks (10 exposures)

Frequency of treatment:

daily, 5 d/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.0, 0.50, 5.0, and 25 ppm (0, 1.9, 19, 96 mg/m³)
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.00, 0.52 ± 0.078, 4.9 ± 0.57 and 25 ± 2.2 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

20

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: based on reports of respiratory toxicity in rats following continuous exposure to 25 ppm (Dalin & Kristofferson, 1974, Ann. Zool. Fennici 11: 193-199)
- Rationale for animal assignment: randomization
- Rationale for selecting satellite groups: as recommended in OECD 412
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): no data

Ten animals per sex per dose terminated on the day after the 10th exposure; remaining 10 rats per sex per dose terminated after the 2-week recovery period.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (morning and afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly including neurobehavioral signs; also twice pretest

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- determined weekly (once pretest)

FOOD EFFICIENCY: no data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data, not mandatory

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination and after recovery period
- Anaesthetic used for blood collection: Yes, collected via the orbital plexus under C02/02 (60/40) anesthesia
- Animals fasted: yes, overnight
- How many animals: 10 per sex per dose per post exposure observation period
- Parameters examined: red blood cells; hemoglobin; hematocrit; prothrombin; activated partial thromboplastin time; platelet; white blood cells; mean corpuscular
volume; mean corpuscular hemoglobin; mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination and after recovery period
- Animals fasted: yes, overnight
- How many animals: 10 per sex per dose per post exposure observation period
- Parameters examined: aspartate aminotransferase; alanine aminotransferase; alkaline phosphatase; blood urea nitrogen; creatinine; glucose; total protein;
globulin; albumin/globulin ratio; total bilirubin; sodium; potassium; chlorine; calcium; phosphorus; gamma-glutamyltransferase

URINALYSIS: No, not obligatory

NEUROBEHAVIOURAL EXAMINATION: No, because no neurobehavioral signs that were seen at detailed clinical examinations (weekly)

Sacrifice and pathology:

Mixed sacrifice order. Rats exsanguinated via the abdominal aorta under C02 anesthesia; necropsy performed; adrenals, brain, gonads, heart, kidneys, liver, lungs, prostate, spleen, and uterus were weighed and mean and relative organ weights calculated.
Histopathology: kidneys, larynx, liver, lungs, nasal turbinates, trachea, and gross lesions embedded, sectioned, stained with hematoxylin and eosin, and examined microscopically for control and high dose groups.

Other examinations:

no

Statistics:

Group means and standard deviations (SD) calculated and then analyzed for homogeneity of variance between the control group and each of the treatment groups using Bartlett's test; parametric procedures (ANOVA plus Dunnett's test) used if variance was equal; if not, nonparametric procedures used (Kruskal- Wallis test plus summed rank test (Dunn)).
Statistical test for trend in the exposure levels: in the parametric case (i.e., equal variance), standard regression techniques with a test for trend and lack-of-fit and in the nonparametric case, Jonckheere's test for monotonic trend was used.
Bartlett's test conducted at the 1% two-sided risk level. All other statistical tests were conducted at the 5% and 1% two-sided risk levels.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
One male died unscheduled at 0.50 ppm (not treatment related; turning itself within the nose-only restraint tube)
Scattered observations of chromodacryorrhea and nasal discharge noted during exposure period but not clearly treatment-related (no toxicological relevance, mostly abated during recovery period); no further effects detected.

FOOD CONSUMPTION
25 ppm: statistically significant decrease in females in the 2nd week; in the 1st week of the recovery period significant increase in food consumption in males. Not considered to be of toxicological relevance (decrease not seen in recovery group).

HAEMATOLOGY
slight but statistically significant increase in prothrombin time of females at 0.50 ppm at the recovery sacrifice, no increase at higher dose groups (9.5 +- 0.2, 10.1*+-0.5, 9.8 +- 0.6, 9.9 +- 0.4 seconds at 0, 0.5, 5, 25 ppm, respectively; no data on historical controls). No toxicological relevance. No further effects.
CLINICAL CHEMISTRY
25 ppm: slight but statistically significant increase in albumin concentration in females at the recovery sacrifice (4.3 +- 0.1, 4.4 +- 0.2, 4.4 +- 0.1, 4.5* +- 0.1 at 0, 0.5, 5, 25 ppm, respectively; no data on historical controls; no effects in females after 2 weeks). No toxicological relevance. No further effects.

ORGAN WEIGHTS
relative liver weight in 5.0 ppm-exposed females decreased at the terminal sacrifice as well as relative spleen weight and spleen-to-brain weight ratio in these same females at the terminal sacrifice; no such effects at the high dose level. No toxicological relevance (no effects in histopathology).

HISTOPATHOLOGY
Microscopic findings either similar for the treatment and control groups, or appeared sporadically.

Effect levels

Dose descriptor:

NOAEC

Effect level:

25 ppm

Sex:

male/female

Basis for effect level:

other: overall effects (higher concentrations not tested)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

25 ppm estimated to be equivalent to an oral dose of approximately 28 mg/kg bw/day (authors calculation).

Applicant's summary and conclusion

Conclusions:

No toxic effects were found in rats in any portal of entry or any systemic toxicity at exposure levels up to 25 ppm (96 mg/m³).

Executive summary:

The study is comparable to OECD Guideline 412 with acceptable restrictions (the high dose should result in toxic effects; histopathology of a limited number of organs; minor restrictions in hematology and clinical chemistry). According to REACH Regulation (EC) No 1907/2006 Annex VIII an exposure duration of 28 days is recommended.

In a 14 day-inhalation study male and female F344 rats (n=20 per dose per sex) were exposed to phenol vapour by nose-only exposures for 10 exposures (5 days/week, 6 hours/day) at target concentrations of 0, 0.5, 5.0 and 25 ppm (0, 1.9, 19, 96 mg/m³). 10 rats/sex/group were sacrificed after 10 exposures, and after a 14-day recovery time for the remaining animals. No signs of toxicity in clinical observations (including neurological signs), body weights, food consumption, clinical chemistry, haematology, organ weights, macroscopic pathology or microscopic pathology were seen during the exposures or at either sacrifice interval.

Conclusion: Under the conditions of this study no local or systemic toxic effects were detected in rats at exposure concentrations up to 25 ppm (96 mg/m³).