Registration Dossier

Administrative data

Description of key information

The key study for this endpoint is a 90-day (14-week) study in both rats and mice by dietary administration, with NOAELs lower than 781 ppm (approximately 70 mg/kg) or equal to 1562 ppm (approximately 300 mg/kg), respectively, based on forestomach microscopic lesions, which are of low toxicological relevance to the human situation.
1000 ppm, i.e. approximately 40 mg/kg (males) or 50 mg/kg (females), based on statistically significant effects on body weight gain (both sexes) and liver weight (females).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - November 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 weeks old
- Weight at study initiation: 78 g (males) and 75 g (females)
- Fasting period before study: no
- Housing: 5 animals per cage (polycarbonate, changed twice per week)
- Diet: NTP-2000 mash feed, available ad libidum
- Water: tap water via automatic watering system, available ad libidum
- Acclimation period: 11 (males) or 12 (females) days

ENVIRONMENTAL CONDITIONS
- Temperature: ca. 22°C
- Humidity: 50% +/- 15%
- Air changes: 10/hour
- Photoperiod: 12 hours light/day

IN LIFE DATES:
26 (males) or 27 (females) August 1996 to 25 (males) or 26 (females) November 1996.
Route of administration:
oral: feed
Details on oral exposure:
DETAILS ON DIET PREPARATION
- Rate of preparation of diet: every 4 weeks
- Type of food: irradiated feed
- Storage of food : in plastic bags inside buckets at 5°C for up to 42 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analytical verification of homogeneity of doses/concentrations: Yes (performed in the 781 and 12500 ppm dose formulations)
- Analytical verification of stability of doses/concentrations: Yes (stability of the 781 ppm dose formulation was confirmed for 42 days for samples stored in plastic bags at temperatures up to 5°C. Samples subjected to animal room conditions showed declines of up to 37% in TBC concentrations with time.)
- dose formulations were analyzed at the beginning, midpoint, and end of the study; all dose formulations analyzed were within 10% of the target concentrations. Animal room samples of these dose formulations were also analyzed; the concentrations of 9 of 15 animal room samples were less than 90% of the target concentrations (these low concentrations are attributed to chemical degradation, oxidation, evaporation or binding with feed)
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
7 days per week
Remarks:
Doses / Concentrations:
0, 781, 1562, 3125, 6250 and 12500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 70, 135, 270, 525 and 1030 mg/kg bw
Basis:
other: males
Remarks:
Doses / Concentrations:
0, 70, 145, 265, 555 and 1010 mg/kg bw
Basis:
other: females
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
Dose selection rationale: based on the results in the 15-day study in rats: based on the body weight effects in the 25000 ppm groups and mortality in the 50000 ppm groups, the highest exposure concentration selected for the 14-week study was 12500 ppm.

Post-exposure period: none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule: initially, weekly and at the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 4 and 22 and at the end of the study
- Anaesthetic used for blood collection: Yes (mixture of carbon dioxide and oxygen)
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: haematocrit; haemoglobin concentration; erythrocyte, reticulocyte and platelet counts; erythrocyte and platelet morphology; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on days 4 and 22 and at the end of the study
- Anaesthetic used for blood collection: Yes (mixture of carbon dioxide and oxygen)
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase and bile salts

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
- Organ weights examined at necropsy (in all animals): heart, right kidney, liver, lungs, right testis, thymus.
- GROSS PATHOLOGY: yes
- HISTOPATHOLOGY (in all animals of the 0 and 12500 ppm groups): adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesentric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach, glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, uterus.
Statistics:
- The Fisher exact test was used to determine significance of the incidences of microscopic lesions;
- The parametric multiple comparison procedures of Dunnett and Williams were used to determine significance of organ and body weight data;
- Haematology, clinical chemistry, spermatid and epididymal spermatozoal data were analysed using the nonparametric multiple comparison methods of Shirley and Dunn;
- Jonckheere's test was used to assess the significance of the dose-related trends;
- Extreme values were identified by the outlier test of Dixon and Massey;
- Average severity values were analysed for significance with the Mann-Whitney U test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
There were no clinical findings of toxicity.

MORTALITY
All rats survived at the end of the study.

BODY WEIGHT AND WEIGHT GAIN
Final mean body weights and body weight gains of males exposed to 1562 ppm or greater and females exposed to 3125 ppm or greater were significantly lower than those of the controls (-4.5%, -7%, -12% and -23% for males, respectively and -4.5%, -8.5% and -9% for females, respectively).

FOOD CONSUMPTION AND COMPOUND INTAKE
Feed consumption by males and females in the 6250 and 12500 ppm groups at week 1 and the 12500 ppm groups at week 14 was lower than that by the controls.

HAEMATOLOGY
An erythrocytosis, indicated by increased hametocrit values, hemoglobin concentrations, and erythrocyte counts, was observed in 6250 and 12500 ppm rats on day 4 and in 12500 ppm rats on day 22.

CLINICAL CHEMISTRY
On days 4 and 22, a transient hepatic effect was demontrated by increases in serum alanine aminotransferase activities and bile salt concentrations in exposed rats. Increases in bile salt concentrations are, in general, used as a marker of hepatic cholestasis. There was evidence early in the current study of decreased feed consumption by rats in the higher exposure groups; thus, the increase in alanine aminotransferase activities and bile salt concentrations could suggest a transient, minimal liver effect that was not expressed in the other markers of liver injury or cholestasis.

ORGAN WEIGHTS
Organ weight differences in exposed reflected decreases in body weights.
Differences in reproductive organ weights were observed, as detailed below.

GROSS PATHOLOGY
There were no exposure-related gross lesions.

REPRODUCTIVE ORGANS
See details in corresponding section.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidence of hyperkeratosis of the forestomach epithelium were significantly increased in males and females in all exposed groups. The incidence of hyperplasia of the forestomach epithelium was significantly increased in males and females exposed to 3125 ppm or greater. The severities of the forestomach lesions were minimal to moderate in males and minimal to mild in females. All males exposed to 3250 or 12500 ppm had minimal cytoplasmic alteration of the liver.

Dose descriptor:
NOAEL
Effect level:
< 781 ppm
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified

Mean body weight:

Concentration (ppm)

Initial 

Final 

Change relative to controls (%) 

Male

 

 

 

 0

79

311

 

 781

79

319

+2

 1562

76

297*

-5

 3125

78

290**

-7

 6250

80

273**

-12

 12500

76

240**

-23

Female

 

 

 

 0

75

178

 

 781

76

183

+3

 1562

74

184

+3

 3125

76

170*

-4

 6250

75

163**

-9

 12500

72

162**

-9

* Significantly different (P ≤ 0.05) from the control group    ** P ≤0.01

Incidences of selected nonneoplastic lesions:

 

0 ppm

781ppm

1562 ppm

3125 ppm

6250 ppm

12500ppm

Male

 

 

 

 

 

 

Forestomacha

10

10

10

10

10

10

Epithelium, hyperplasiab

0

4* (1.0)c

8** (2.0)

9** (1.8)

10** (2.2)

10** (3.1)

Epithelium, hyperkeratosis

0

0

2 (1.0)

8** (1.8)

8** (2.0)

10** (2.6)

Liver

10

10

10

10

10

10

Cytoplasmic alteration

0

0

0

0

10** (1.0)

10** (1.0)

Female

 

 

 

 

 

 

Forestomach

10

10

10

10

10

10

Epithelium, hyperplasia

0

6** (1.0)

7** (1.4)

9** (1.6)

10** (2.0)

10** (2.2)

Epithelium, hyperkeratosis

0

0

0

5* (1.6)

8** (1.8)

10** (2.2)

* Significantly different (P ≤ 0.05) from the control group    ** P ≤0.01

anumber of animals with forestomach examined microscopically

bnumber of animals with lesion

caverage severity grade of lesions in affected animals; 1=minimal, 2=mild, 3=moderate, 4=marked

Conclusions:
Following 14 weeks of dietary administration to rats, the primary target organ of 4-tert-butylpyrocatechol toxicity was to the forestomach.
Forestomach toxicity was observed at all exposure concentrations, and the No-Observed-Adverse-Effect-Level (NOAEL) was not determined for this effect.
Executive summary:

In a 14 week dietary toxicity study (NTP study, 2002), 4 -tert-butylpyrocatechol was administered to 6 -week male and female Fischer 344 rats, 10/dose, in diet, at the dose levels of 0, 781, 1562, 3125, 6250 and 12500 ppm, resulting in doses of 0, 70, 135, 270, 525 and 1030 mg/kg bw to males and 0, 70, 145, 265, 555 and 1010 mg/kg bw to females.

There were no clinical findings of toxicity and no mortality. Mean body weights of males exposed to 1562 ppm or greater and females exposed to 3125 ppm or greater were significantly lower than those of the controls (from -4.5% to -23%). Feed consumption by males and females in the 6250 ppm (week 1) and 12500 ppm (weeks 1 and 14) was less than that by the controls.

An erythrocytosis, indicated by increased hematocrit values, hemoglobin concentrations, and erythrocyte counts, was observed in 6250 and 12500 ppm groups on day 4 and in 12500 ppm groups on day 22. At these time points, a transient hepatic effect was demonstrated by increases in alanine aminotransferase activities and bile salt concentrations in exposed rats.

Organ weight differences in exposed animals reflected decreases in body weights.

There were no exposure-related gross lesions. The incidence of hyperkeratosis of the forestomach epithelium were significantly increased in males and females in all exposed groups. The incidences of hyperplasia of the forestomach epithelium were significantly increased in males and females exposed to 3125 ppm or greater.

The NOEL was < 781 ppm (equivalent to 70 mg/kg bw/day).

This subchronic toxicity study in rats is acceptable and was conducted comparably to the guideline requirements for a subchronic oral study (OECD 408) in rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
70 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The subchronic repeat-dose toxicity of 4-tert-butylpyrocatechol was assessed in both rats and mice over a 3-month (14-week) dosing period by dietary administration, at dose levels ranging from 781 to12500 ppm, equivalent to approximate dose ranges of 70 to 1000 mg/kg for rats and 140 to 2600 mg/kg for mice. The two rodent species were used during the same study (US NTP study report No. 70, 2002), considered as a key study of validity 1 according to Klimisch cotation criteria. Examinations and measurements included changes in appearance and behaviour, body weight, food consumption, hematology, clinical chemistry, organ weights at necropsy, gross pathology and histopathology. Specific investigations on reproductive or genetic toxicity were also included, as detailed in the corresponding sections of this dossier.

 

Repeated dietary administration of the test substance had an effect on body weight (which was decreased versus controls from 1562 ppm in rats and 6250 ppm in mice), together with decreased food consumption from 6250 ppm in rats only. Changes in hematology and clinical chemistry were observed in rats only. They occurred transiently at the beginning of the dosing period and consisted of erythrocytosis (characterized by increased hematocrit, hemoglobin concentration and erythrocyte count) from 6250 ppm, and a minimal liver effect (characterized by increased alanine aminotransferase activity and bile salt concentration) at all dose levels (together with minimal cytoplasmic alteration in the liver of male rats given 6250 or 12500 ppm). Post-mortem findings were similar in both species. A significantly increased incidence of forestomach hyperkatosis was observed at all dose levels in rats and at12500 ppm in female mice. A significantly increased incidence of forestomach epithelial hyperplasia was also noted from 3125 ppm in rats and from 6250 ppm in mice. Therefore, the primary target organ for subchronic toxicity was forestomach in both rodent species and, based on forestomach histopathology, the No-Effect-Level (NOEL) after 14 weeks of dosing was lower than 781 ppm (equivalent to approximately 70 mg/kg bw) or equal to 1562 ppm (equivalent to approximately 300 mg/kg bw) in rats and mice, respectively.

However, a functional human counterpart for the rodent forestomach does not exist. Histologically related organs in humans are oesophagus and stomach, but tissue dosimetry for these tissues is different from the rodent situation, where forestomach is a holding compartment, which allows the tissue considerably longer exposure to the chemical than oesophagus where transit time is rapid in case of oral administration (1). Furthermore, the oral route of administration is irrelevant to the worker situation.

Therefore, no relevant target organ or sign of severe toxicity was identified from rodent subchronic studies, and no classification is warranted for repeated dose toxicity.

(1) Proctor DM, Gatto NM, Hong SJ & Allamneni KP. Mode-of-action framework for evaluating the relevance of rodent forestomach tumors in cancer risk assessment. Toxicol. Sci. 98(2): 313 -26, 2007.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study was GLP-compliant and comparable to an OECD-guideline study.

Justification for classification or non-classification

The guidance values for classification of a substance as harmful on repeated exposure by the oral route according to the criteria of Annex VI Directive 67/548/EEC (R48/22) relate to severe effects (clear functional disturbance or morphological change with toxicological significance) reported at 50 mg/kg bw/day or below in a 90-day study in rats. The equivalent guidance values for classification according to EU/GHS criteria (STOT Category 2) relate to effects reported at 10-100 mg/kg bw/day in a 90-day study in rats.

In the present case, no classification is warranted as to repeated dose toxicity, because:

  • the oral route is of limited relevance to the human situation,
  • the target organ identified in rodents (forestomach) is of limited relevance to human health,
  • and no major organ function impairment was evidenced.

4-tert-butylpyrocatechol is not classified for repeat-dose toxicity according to the criteria of Annex VI Directive 67/548/EEC or UN/EU GHS.