Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

repeated dose toxicity: oral, other
Combined repeated dose Toxicity study with Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
Limit test:

Test material

Test material form:
solid: granular
Specific details on test material used for the study:
- Source of test material: MANAC, Hiroshima, JAPAN; Lot No.: 70909
- Purity 99.8%
- Storage: cool dark place with appropriate ventilation

Test animals

Crj: CD(SD)
Details on test animals and environmental conditions:
- Source: Charles River Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 361-397 g in males, 233-258 g in females
- Fasting period before study: not stated; daily administration of dose
- Housing: individual cages with mesh floor, each cage 6000 cm3; after GD18, dams were housed in a rearing cage of 14,700 cm3 and additionally given a nursing tray and nesting materials (CareFRESH). Cages were changed every two weeks.
- Diet: ad libitum with NMF pellets provided by Oriental Yeast Co.
- Water: tap water ad libitum
- Acclimation period: 1-2 weeks (quarantined for one week, study began 2 weeks after arrival)

- Temperature (°C): 24 +/- 2
- Humidity (%): 55 +/-10
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on oral exposure:
The concentrations of dosing solutions were 20, 60, and 200 mg/mL; solutions were made and stored in a tightly closed container in a cool dark place, then used within 8 days.
The test compound was previously determined to be stable for 8 days at concentrations of 20 and 200 mg/mL under the given storage conditions
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The dosing solutions were sampled, and confirmed to be within a concentration range of 84.5-100% of the stipulated concentration.
Duration of treatment / exposure:
Total 48 days: Treatment started 14 days prior to mating, and continued daily through the premating period, mating, gestation, and until lactation day 3.
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
As acute oral LD 50 was previously reported as 2000 mg/kg bw., a preliminary test of 14 days was conducted with the doses of 0 (vehicle; corn oil), 100, 300 and 1000 mg/kg/day.
Based on the results of the preliminary test, the highest dose was set at 1000 mg/kg/day where clear adverse effects would appear.
Positive control:


Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes, Clinical observations were conducted for all animals of both sexes every day throughout the study period, and abnormalities and deaths noted.

Body weights and food consumption of males were measured prior to dosing, then on Days 1, 8, 15, 22, 29, 36, 43, and 48 (food) 49 (weight; necropsy date).
Body weights and food consumption of females were measured prior to dosing, then Days 1, 8, and 15. Non-pregnant females were measured Days 22, 29, 36, 43, and 49. Pregnant females were measured on gestation days 0, 7, 14, 21, and lactation days 0 and 4.
No food consumption was measured during mating and cohabitation.

- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked were examined: total protein, albumin, A/G, ALP, Creatinine, total bilirubin, potassium, chloride

Estrous cycles were monitored for 14 days before mating, which took place 1:1 with males every evening for up to 14 days; the morning a vaginal plug or sperm was detected in a vaginal smear, the female was defined as Day 0 of gestation.

Dams were allowed to deliver spontaneously. If delivery was complete by 10 am, the day was defined as Day 0 of lactation. If parturition continued after 10 am, the following day was designated Day 0 of lactation.
Sacrifice and pathology:
On lactation day 4, all dams and pups were sacrificed and necropsied. Pups that died before lactation day 4 were also subjected to necropsy.

GROSS PATHOLOGY: Yes, liver, kidney, thymus, brain, adrenals testes


Clinical laboratory tests were performed in all males of each group at necropsy. The males were fasted for 16 hours, then anaesthetized with ether, and blood collected from the abdominal aorta for haematology testing.
Multiple comparison assay for body weight, for food consumption, for numbers of corpora lutea, implantation sites, pups born, and stillborns, for sex ratio, for mean estrous cycle, for gestation period, for indices of implantation, delivery, viability on lactation day 4, and live births, for incidence of external anomaly, for organ weight and ratio of organ and body weights, and for haematological and blood chemistry values. Bartlett's test for variance was initially performed , followed by a one-way analysis of variance if variance was similar within groups. If variance was unequal within groups, Kruskal-Wallis rank test was done, followed by Dunnett's multiple comparative assay (non-parametric). The Chi-squared test was used for indices of delivery, copulation, and fertility. Fisher's direct probability test was used for incidence of pathological results, and the Mann-Whitney U test used to examine the rank sum. Results were noted as significant at p<0.05 or p<0.01.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in both males and females within 30 minutes of dosing at 300 and 1000 mg/kg. Numbers of animals with salivation were increasing by increased dose. This salivation, therefore, was considered as an adverse effect caused by the administration of this chemical. At 100 mg/kg/day no abnormality was observed.
Other signs were sporadic across treatment groups.
no mortality observed
Description (incidence):
No mortality was observed for all groups up to the highest doses, 1000 mg/kg/day for the test period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males dosed at 1000 mg/kg had statistically lower body weights compared to controls beginning on Day 8 of the study. Male body weight gain at 1000 mg/kg was also significantly low. Females did not show any difference in body weight or body weight gain until after Day 7 of gestation at the 1000 mg/kg dose level. Body weight and body weight gain were both low in the 1000 mg/kg group to Day 4 of lactation (date of sacrifice).
At 300 and 100 mg/kg/day: No significant changes were observed.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
In males, the 1000 mg/kg group had significantly lower food consumption compared to controls from Day 1 to 8 of the study. In females, the 1000 mg/kg group also had significantly lower values from Day 1 to 8 in the premating period, but also from Day 0 to 4 of lactation. No differences were noted during the gestation period.
In the 300 and 100 mg/kg/day dose groups no significant changes were observed.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
There was not considered to be a significant difference between treatment groups and contols, although the blood coagulation test showed a slight but statistically significant prolongation time compared to controls.
At 100 to 1000 mg/kg/day: No adverse effects were observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically elevated creatinine levels were seen in the 300 and 1000 mg/kg dose groups compared to controls. In 1000 mg/kg groups only, elevated protein was seen in total protein, albumin, A/G, ALP, and chlorine, as well as a higher trend in BUN, and significantly lower values of total bilirubin and potassium.
At 300 mg/kg/day or more: Significant increase in creatinine was observed. This increase suggested the toxicity of this chemical to liver and kidneys.
At 100 mg/kg/day: No significant adverse effects were observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day: Significant absolute and relative weight increases of liver and significant relative weight increase of kidneys were observed. Significant absolute weight decrease of thymus was observed. In addition, relative weight increases of brain, adrenals and testes were observed. However these were not considered as adverse effects because no histopathological abnormalities were found.
300 and 100 mg/kg/day: No significant changes were observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg male group, atrophy of thymus (4 males) and enlarged liver (3 males), white patch on the kidney (2 males) and hypertrophy of the adrenals (2 males) were noted. A red patch on the thymus, white patch on the liver, subcutaneous mass, abdominal cavity mass, epidydimus nodule, and thin hair were noted sporadically in all male dose groups except 300 mg/kg.
In females, a red patch was noted on the thymus, brown patch in the lung, liver abnormalities (enlargement, scars, white patch), ovarian cysts, dilated uterine lumen, were observed sporadically across dose groups and controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Pups in one litter treated with 1000 mg/kg all died after birth; the dam of this litter showed pigment deposits in the spleen, cellular infiltration in the lung, fatty changes in the liver, and microgranuloma in the uterus.
In males treated with 1000 mg/kg, changes seen were atrophy of the thymus (3 animals), hypertrophy of hepatocytes (12 animals), hyaline cast in kidney (8), dilatation of kidney tubles (7), renal papillary necrosis (5), lumphocyte infiltration (6), resulting in an increases incidence of observations compared to control. Fatty changes in liver and basophilic tubules were noted as mild in other treatment groups, but the degree of severity was greater in the 1000 mg/kg group. Elevated incidence of eosinophilic bodies were seen in the kidney of the 100 mg/kg group, but there was no dose-response or increase in severity. Pigment deposits in spleen, hemorrhage in thymus, microgranuloma in liver, and vacuolar degenertaion in adrenal zona fasciculata were observed frequently across groups including control. Othe sporadic findings were seen but not specified.
In females, the observed changes were either sporadic or without difference in incidence among groups.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
HISTORICAL CONTROL DATA (if applicable): not stated

In animals which failed to copulate, pigment deposits and lymphocyte infiltration were seen in the kidneys of both sexes. The male additionally had lymphocyte infiltration in liver, basophilic tubules in the kidney, and angiectasis and vacuolar degeneration of the zona fasciculata in the adrenals. The female additionally had capsulitis in the spleen, pneumonia, microgranulation in the liver, pyelitis in kidney, dilatation of the lumen and cellular infiltration of the uterus.

Copulation and fertility: copulation was achieved in all animals of the treatment groups, but not complete in the control group (copulation index 91.7% in controls). All copulated females conceived. All females indicated a 4-5 day estrus cycle, and no difference was noted between groups.
Delivery and lactation: The 1000 mg/kg group showed significantly lower gestation period compared to controls, although still within the normal range of 22 days. The number of live pups was lower in the 1000 mg/kg group, as was the viability index. No abnormalities were noted in delivery conditions, number of corpora lutea, number of implantation sites, number of newborns, or stillborns, indicating a lack on inter-group differences in the gestation index, implantation index, delivery index, or live birth index.
Morphological findings of newborns: In the 1000 mg/kg group, body weight from lactation day 0-4 were low. Various developmental defects were seen across treatment groups, but were not dose-related or seen at high incidence.

Effect levels

Key result
Dose descriptor:
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food efficiency
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

Applicant's summary and conclusion

The NOAEL for 2,4,6-tribromophenol in this adequate repeated-dose study is 100 mg/kg/day in both sexes. The effect seen at the next higher level, 300 mg/kg/day, was salivation within 30 minutes of dosing, and some changes in liver and kidney, including a high level of creatinine in the blood.