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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 22,1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): 2,4,6-TRIMETHYLPHENOL
- Substance type: organic
- Physical state: White fine needle crystal
- Analytical purity: 99.8%

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
-Sources: Charles River Laboratories (Atsugi production plant)
-Age at study initiation : 9 weeks
-Weight at study initiation : It was confirmed that the body weight range of the animal is average body weight ±20%. Females 190-235 g, males: 293-338 g
-Animals were uniquely identified and kept in their cages for five days prior to the start of the study to allow for acclimatisation to the laboratory conditions
1 male and female animal was considered for mating period per 1 cage
-Feeding : Solid feed for the test animal, which is sterilized by radiation sterilization (CRF-1, Oriental Yeast Company Ltd.). Administration Oral (Compulsory oral administration). The rat was administered once a day by using a disposable cylinder in which a gastric tube was set. The administration solution was added while stirring with a stirrer.
-Water : drinking water which has undergone ultraviolet radiation after filtration through 5 μm filter
Temperature acceptable range 19-25 °C
Relative humidity 48 – 62.3%
Lighting hours: 12 hours/day (7.00 – 19.00)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.1w/v% Tween 80 + 0.5w/v% Sodium carboxymethylcellulose solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Males : Total of 42 days (from before 14 days of mating till the day after completion of mating)
Females : 14 days before mating, during mating, pregnancy and up to lactation day 4
Females (satellite), 42 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 60, 300 mg/kg bw/day
Basis:
other: Amount of solution administered: By considering 10 mL/kg, the fluid volume in each animal was calculated on the basis of body weight measured on the day of arrival
No. of animals per sex per dose:
Males, 12 (5 for recovery) ; females, 12; satellite females, 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The results of “Repeated administration toxicity test (Test number: B051509) for 14 days by oral administration using rats administered with 2, 4, 6-trimethylphenol” (dosage: 100, 300, 600 and 1000 mg/kg) conducted in the testing facility were set as the reference. Considering the results and the administration period of this test, a dosage of 300 mg/kg clearly forecasted the appearance of toxicity. Hereafter, the dosage was 60 mg/kg while decreasing approximately by a geometric ratio of 5 and the low dosage was set to 3 dosages of 10 mg/kg. Moreover, the control group for which only the medium (0.1w/v% Tween80 addition of 0.5w/v%CMC-Na) was administered was set.
Positive control:
not used

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes (see Appendix Study B051477)
- Time schedule and parameters checked:
Litter observations
PARAMETERS EXAMINED
The number of births (number of alive infants, number of stillborn infants), gender and presence of external abnormality, were examined on day 0 of lactation. Afterwards, general state and presence of death were observed every day. The weight was measured on 0 day and after 4 days of birth. External appearance that contains mouth cavity of the living infants was examined after 4 days of the birth.
GENERAL STATE: The administration period was observed twice a day (before administration and after administration). Other periods were observed once in the morning in 1 day.

BEHAVIORAL EXAMINATION: Observation of detailed symptoms (in home cage, at the time of handling and in open field) was carried out once before starting the administration, once a week during the administration period, and at any time after 13:00 till the sixth week (Pregnancy day and childbirth day of the female were not considered). The function test (Reactiveness to stimulation, Grip measurement) and the measurement of amount of motor activities were carried out after 13:00 of the sixth week. For 5 female animals of each group the measurement was carried out once when the birth date was near, and once during the breastfeeding period.

BODY WEIGHT: Males - on day 1, 8, 15, 22, 29, 36, 42, and 43. In addition, the weight of male recovery animals was measured on day 50 and 56. The weight of female satellite animals was measured with the same frequency as that of the male recovery animals. The weight of female test animal was measured on day 1, 8, and 15. The weight of pregnant female after mating was measured on day 0, 7, 14, and 20 and the weight of female during breast‐feeding after delivery was measured on day 0 and 4.

FOOD INTAKE : In the male test animal and the male recovery animal was measured on days 1~8, 8~15, 22~29, 29~36, 36~38, 43~50 and 52~56. The food intake in female satellite animals was measured on days 1~8, 8~15, 15~22, 22~29, 29~36, 36~42, 43~50 and 50~56. The food intake in the female test animal was measured with the same frequency as that of measurement of body weight. However, the food intake was not measured during the mating period when the animals were living together.

HAEMATOLOGY:
- Time schedule for collection of blood: day 42 in male test animal, day 56 in male recovery animals and female satellite animals, day 4 of lactation for female test animals).
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: yes (more than 16 hours)
- How many animals: 10 animals (5 males -5 females) per group
- Parameters checked: Red blood cell count - Hemoglobin concentration - Hematocrit value - Mean corpuscular volume (MCV) - Mean corpuscular hemoglobin (MCH) - Mean corpuscular hemoglobin concentration (MCHC) - Reticulocyte count - Blood platelet count - Prothrombin time (PT) - Activated partial thromboplastin time (APTT) - White blood cell count - Differential leukocyte count.

BIOCHEMICAL EXAMINATION:
OF BLOOD - Parameters checked: ASAT (GOT) - ALAT (GPT) – γGT – ALP - Total bilirubin - Urea nitrogen – Creatinine – Glucose - Total cholesterol – Triglyceride - Total protein – Albumin - A/G ratio – Calcium – Inorganic phosphorous - Sodium (Na) - Potassium (K) - Chloride (Cl)
OF URINE Parameters checked: ( test on 5 male once on the 38th day) pH - Protein - Glucose - Ketone body - Bilirubin - Occult blood - Urobilinogen.
Sacrifice and pathology:
Postmortem examinations (Parental animals)
SACRIFICE:
Males, day 43 of treatment and day 15 of recovery.
Females, day 5 of lactation;
Females (satellite), day 15 of recovery

GROSS NECROPSY
- yes
Weight of below-mentioned organs were measured for 5 male test animals, for all the male recovery and the female satellite animals, and 5 female animal having early birth date: Brain, heart, liver, kidney, adrenal gland, thymus gland, spleen, testicles, epididymides

HISTOPATHOLOGICAL EXAMINATION
- Yes
Brain, pituitary gland, thymus gland, lymph node (lower jaw/mesentery), trachea, lung, stomach, intestinal tract (duodenum, jejunum, ileum, appendix, colon and rectum), thyroid gland, parathyroid gland (both sides), heart, liver, spleen, kidney (both sides), adrenal glands (both sides), urinary bladder, testicles (both sides), epididymides (both sides), seminal vesicles (including the coagulating gland), ventral prostate, ovaries (both sides), uterus, vagina, bone marrow (femur (one side)), sciatic nerve (one side), spinal cord, and abnormal parts that are visible by the naked eye.
For all abnormal parts visible with naked eyes, microscopic examination was carried out.
Postmortem examinations (Offspring)
SACRIFICE
- The F1 offspring was sacrificed at 4 days of age. A dissection was performed after the euthanasia and a search for abnormal finding was performed
Other examinations:
Estrous cyclicity (Parental animals)
In the morning from the day of starting the administration to the day of starting the hybridization, vaginal smear was collected; estrous cycle was examined and the incidence rate of average sexual cycle days and abnormal sexual cycle animals (animals whose sexual cycle is not 4~6 days) was calculated. Hybridization, observation of delivery and lactation and inspection after completion of breastfeeding were performed.
Statistics:
For the measurement data statistical significance Bartlett's test was used. When the variance was equal and when neither one-way analysis of the variance nor the variance was equal, the Kruskal-Wallis Test was conducted. When significant difference was observed between the groups the Dunnett methods or the Dunnett types was compared in multiple numbers. For some items, the Kruskal-Wallis Test was conducted. When significant difference was observed between the groups, the Dunnett types were compared in multiple numbers. Moreover, in histopathological examination, the enumeration data of 2 groups was compared with control group in the Wilcoxon rank sum test. Other enumeration data was tested by the Fisher's exact probability method. The data between 2 groups (control group and 300 mg/kg group) was analyzed conducting the Student's or the Welch's t-test. The enumeration data of 2 groups was compared with the control group by Wilcoxon rank sum test. The significance level of every test was assumed to be 5% using the safety test system MiTOX.
Multiple comparison tests:Weight, amount of food intake, hematological test, biochemical examination of blood, weight of organs, behavior examination measurement data (Strength of grip and motor activities), number of corpora lutea, number of implantations, number of born infants (number of alive infants, number of stillborn infants).
Multiple comparisons between Kruskal-Wallis type and Dunnett type: Urine test, Mating location and number of days, frequency of mating seasons missed till mating, average number of cycles, gestation period, implantation index, delivery rate, birth rate, incidence rate of external abnormality, viability index on day 4 of the newborn.
Wilcoxon rank sum test: Histopathological examination.
Fisher’s exact probability method: Incidence rate of abnormal estrous cycle, copulation rate, fertility index, birth rate, sex ratio.
F-test and Student’s t-test or Welch’s t-test: Variables data of recovered animal

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
M: Salivation (300); F: Salivation (300), Abnormal gait (300)
Mortality:
mortality observed, treatment-related
Description (incidence):
M: Salivation (300); F: Salivation (300), Abnormal gait (300)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
M: Edema in the forestomach (300), Globule leukocyte in the glandular stomach ↑ (300); F: Erosion in the glandular stomach (300); M/F: Foveola hyperplasia in the glandular stomach (300), Squamous hyperplasia in the forestomach (60, 300)
Details on results:
Hyperplasia of stomach squamous epithelium was noticed in both the male and the female groups of 60 mg/kg and above. Fertility was not affected.
No change in the test substance was noticed in case of the estrous cycle, copulation rate, conception rate, delivering rate, gestation period, corpus luteum, implantation, implantation rate, birth rate, delivery and breastfeeding behavior in the parent animals. For details on stomach epithelial hyperplasia it is referred to the chapter of repeated dose toxicity.

Repeated dose toxicity
2,4,6-Trimethylphenol was studied for oral toxicity in rats in accordance with the
OECD guideline on combined repeated dose and reproductive/developmental toxicity screening test at dosages of 0, 10, 60, and 300 mg/kg/day. No deaths occurred in any of the treated groups. In the 300 mg/kg group, salivation in both sexes and abnormal gait in females were noted as post-dose symptoms. Histopathological examination revealed squamous hyperplasia in the forestomach in the 60 and 300 mg/kg groups of both sexes, and edema in the forestomach in one male were noted. In addition, foveola hyperplasia and erosion in the glandular stomach in the 300 mg/kg group were noted. Almost all the changes disappeared after a 14-day recovery period.

Effect levels

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Dose descriptor:
NOAEL
Remarks:
Target organ: stomach.
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Squamous hyperplasia in the forestomach.
Dose descriptor:
NOEL
Remarks:
Target organ: stomach.
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Squamous hyperplasia in the forestomach.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Regarding the repeated dose, NOEL and NAOEL related to both male and female animals were considered as 10 mg/kg/day because hyperplasia of stomach squamous epithelium was noticed in both the male and the female groups of 60 mg/kg and above.

Applicant's summary and conclusion

Conclusions:
It was concluded from the above results that NOEL and NOAEL for the repeated dose toxicity of 2,4,6-Trimethylphenol were 10 mg/kg/day for both sexes, and that the NOEL and NOAEL for the reproductive/developmental toxicity were 300 mg/kg/day for parental animals and offspring.
Executive summary:

Repeated dose toxicity

2,4,6-Trimethylphenol was studied for oral toxicity in rats in accordance with the OECD guideline on combined repeated dose and reproductive/developmental toxicity screening test at dosages of 0, 10, 60, and 300 mg/kg/day.

No deaths occurred in any of the treated groups. In the 300 mg/kg group, salivation in both sexes and abnormal gait in females were noted as post-dose symptoms. Histopathological examination revealed squamous hyperplasia in the forestomach in the 60

and 300 mg/kg groups of both sexes, and edema in the forestomach in one male were noted.

In addition, foveola hyperplasia and erosion in the glandular stomach in the 300 mg/kg group were noted. Almost all the changes disappeared after a 14-day recovery period.

Reproductive and developmental toxicity

The compound had no effects on reproductive parameters such as the estrous cycle, mating index, fertility index, numbers of corpora lutea or implantations, implantation index, delivery index, gestation index, gestation length, parturition, or maternal behavior. On the

examination of neonates, there were no significant differences in the number of offspring or live offspring, sex ratio, live birth index, or viability index on day 4. No abnormal findings ascribable to the compound were found in the external features, clinical signs, body weights or necropsy of the offspring.

Conclusion

It was concluded from the above results that NOEL and NOAEL for the repeated dose toxicity of 2,4,6-Trimethylphenol were 10 mg/kg/day for both sexes, and that the NOEL and NOAEL for the reproductive/developmental toxicity were 300 mg/kg/day for parental animals and offspring.