6-tert-butyl-2,4-xylenol

Endpoint summary

Description of key information

Key value determined in compliance wiith GLP and analytical verification performed, however no reference guidelines detailed in the report.

3month NOAEL 100 ppm in the rat.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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:

Not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: “The order for specifying the items for the study on new chemical substances, and the investigation on the designated chemical substances. Article 4: Test Facility,” Kankiken No. 233, Eisei No. 38, 63 Kikyoku No. 823 (November 18, 1988).

Deviations:

no

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals: Seventy males and 70 females of Crj:CD(SD) strain rats (SPF) were purchased from Japan Charles River Co., Ltd., (Atsugi-shi, Kanagawa) at 8-weeks of age on September 20, 1993, quarantined and acclimatized to the test environment for 7 days. 48 males and 48 females which showed no abnormalities in general condition during the acclimatization period were allocated to each group after 8 days of preliminary housing period at 10-weeks of age.
At the end of grouping, the males weighed 339-400 g and the females weighed 226-265 g.

Animal husbandry: The animals were housed in an animal room (W 5.7 x D 10.0 x H 2.5 m, 142.5m3) kept by barrier system under the target environmental conditions at 22-26°C of temperature, 45-65 % of relative humidity with 15 times per hour of ventilation frequency, and 12 hour lighting of 150-300 lux (lighting at 7: 00 am and turn off at 7:00 pm).
Animals were housed individually in a cage with aluminum front and stainless steel mesh-floor (W 15.8 x D 23.8 x H 16.0 cm, Space: 6017 cm3). Cages were set on a water-flush breeding instrument (W 691.0 x D 79.0 x H 195.0 cm) supplied by Tokyo Giken Service Co. Ltd. (Fuchu-shi, Tokyo). However, during the mating period, males were housed in cages with aluminum front and stainless steel mesh-floor (W 36.8 x D 25.0 x H 16.0 cm, Space: 14720 cm3). Dam after 18 days of gestation was housed in a cage with aluminum front and stainless steel mesh-floor (W 36.8 x D 25.0 x H 16.0 cm) with a lactation tray and nesting materials (Alpha-dry) until day 4 of lactation. The cages were exchanged once every other week, and food suppliers were exchanged once a week.
The food used was NMF solid food (treated with radiation sterilization) supplied by Oriental Yeast Co., Ltd. (Chuo-ku, Tokyo). The food was available ad libitum. The analysis of contaminant in the food used in this study was conducted in Japan Food Research Laboratories (Shibuya-ku, Tokyo) on the request by Oriental Yeast Co., Ltd.
The animals were allowed free access to drinking water of tap water. The analyses of tap water were performed 4 times in a year in Examination Center of Life Science of Shizuoka (Hamamatsu-shi, Shizuoka). The food, water and nesting material supplied did not contain any contaminants which were considered to have affected the results of the study.
There were no environmental deviations which might have affected the reliability of the study data during the duration of housing period.

Test groups: Animals were stratified by the body weights and allocated to each test group by randomization method on October 5, 1993.
Animals were identified by ear-punch and attaching animal identification card with animal identification number (animal ID-No.) to cages. Before grouping, animals were identified by tentative animal numbers.
The remained animals were euthanized by carbon dioxide.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Oral rout of administration was selected according to the indication described in OECD Guideline “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Study”.
Administration volume was 0.5 ml per 100g of body weight, and the administration volume was calculated based on the most recent individual body weight. The test substance was administered by gavage once a day (7 days/week) using a stomach tube. Control animals were treated with corn oil only in an identical manner.
Male animals were administered for 45 days consecutively from 14 days before mating, during 14 days of mating period and up to 17 days after the end of mating period. Female animals were administered from 14 days before mating, during mating period (until the day of copulation confirmation, 14 days at the longest), gestation period after copulation and up to day 3 of lactation after delivery (41-48 days). Infertile female animal was administered for 44 days until the day before necropsy (day 25 of gestation).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis of homogeneity and concentration of the dosing preparation was conducted on the sample taken randomly from batches of each group prepared at the start of administration. As shown in Reference data 5, mean values of the concentration in each preparation were within 99.3-104 %, and the coefficient of variation was lower than 0.8 % indicating that the preparations were properly adjuste

Duration of treatment / exposure:

Male animals were administered for 45 days consecutively from 14 days before mating, during 14 days of mating period and up to 17 days after the end of mating period. Female animals were administered from 14 days before mating, during mating period (until the day of copulation confirmation, 14 days at the longest), gestation period after copulation and up to day 3 of lactation after delivery (41-48 days). Infertile female animal was administered for 44 days until the day before necropsy (day 25 of gestation).

Frequency of treatment:

The test substance was administered by gavage once a day (7 days/week) using a stomach tube.

Remarks:

Doses / Concentrations:
0, 6, 30, 150 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

12 animals per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

Reason for selection of the species: The use of rat is indicated by OECD Guideline. The species was selected considering the stability in estrus cycle, reproduction and genetic.

Reasons for the selection of dose levels: Dose levels were selected by considering the results of “Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Preliminary Study in Rats” (study number 2265) conducted previously. In this preliminary study, dose levels of 0, 75, 150, 300 and 600 mg/kg were administered to males and females for 14 consecutive days, and all animals treated with 600 mg/kg died. Suppression in body weight gain was seen in male groups treated with 300 mg/kg or more and in females treated with 600 mg/kg. Decrease in food intake was seen in male groups treated with 150 mg/kg or more, and females treated with 300 mg/kg or more.
Among the organ weight at necropsy, both absolute and relative weights of liver increased in male groups treated with 75 mg/kg or more, and in female groups treated with 150 mg/kg or more.
At necropsy, enlargement of liver was seen in males treated with 150 or 300 mg/kg and females treated with 300 mg/kg.
From the above results and taking into account of the longer administration period in the main study, 150 mg/kg was selected as the highest dose level. The lower dose levels of 30 and 6 mg/kg were selected by dividing the highest dose level by common geometric ratio of 5.

Positive control:

Positive control not utilised in this study.

Observations and examinations performed and frequency:

Male animals
Clinical observations: All animals were observed with the frequency of once or more in a day during the study period.
Body weight: All animals were weighed on days 0 (start of administration), 7, 14, 21, 28, 35, 42 and 45 (day of necropsy). In addition, the body weight gain during the study period was calculated.
Measurement of food intake: Food given to all animals were weighed on days 0 (start of administration), 7, 14, 21, 28, 35, 42 and 44 (day before necropsy), and mean daily food intake was calculated by the amount on the measurement day and the amount on the next measurement day. In addition, cumulative food intake during the administration period was calculated. Food intake during mating period when males were housed together with females was not measured.
Hematology: All animals were fasted for about 16 hours from the evening of the last day of administration period (45 days) to the next morning, and then blood samples were obtained from the abdominal aorta under ether anesthesia. Blood samples obtained at the first bleeding were used for the examination. Blood samples were collected into sample cups containing anticoagulant (EDTA-3K) and examined on the items listed below using a total hematological examination system THMS H 6000 (Technicon Corporation, USA).
Hematocrit (HCT): Calculated from total volume of erythrocytes
Hemoglobin(HGB): Cyanmethemoglobin method
Erythrocyte count (RBC): Dark field plate method
Mean corpuscular volume (MCV): Calculated from RBC and HCT
Mean corpuscular hemoglobin (MCH): Calculated from HGB and RBC
Mean corpuscular hemoglobin concentration (MCHC): Calculated from HGB and HCT
Platelet count (PLT): Dark field plate method
Leukocyte count (WBC): Dark field plate method
Differential leukocyte count: Flow cytometry
Differential leukocyte counts were determined with the instrument described above. However, blood smear was prepared and stained with May-Grunwald-Giemsa stain and stored.
Reticulocyte (RC) counts were determined by the microscopic observation of blood smear stained with a glass capillary tube for reticulocyte staining (Terumo Corporation, Shibuya-ku, Tokyo).
Blood chemistry: At the same time of the sampling for the hematological examination, blood samples were obtained from the abdominal aorta and put into vessels, Clean Seal (Yatron Corporation, Chiyoda-ku, Tokyo). The samples were centrifuged at 3000 rpm for 7 minutes after being left for 30 minutes, and serum was obtained and used for the analysis. Parameters indicated below were assayed using Multi-item biochemistry auto-analyzer, CentrifiChem ENCORE II (Baker Corporation, USA) and EKTACHEM 700N (Kodak Corporation, USA).

Total protein (T. protein): Burette method
Albumin (Albumin): B.C.G. method
A/G (A/G): Calculation
Glucose (Glucose): Glucose oxidase method
Urea nitrogen (BUN): Modified urease method
Creatinine (Creatinine): Jaffé method
Total bilirubin (T. Bilirubin): Diazo colorimetric analysis
Glutamic oxaloacetic transaminase (GOT): Modified Karmen method
Glutamic pyrubic transaminase (GPT): Modified Karmen method
γ-Glutamyltranspeptidase (Gamma-GTP): Modified Szasz method
Potassium (Potassium): Electrode method
Chloride (Chloride): Electrode method
Calcium (Calcium): Arsenazo III method
Inorganic phosphate (I. Phosphate): Ammonium molybdate method

Female animals
Clinical observations: All animals were observed with the frequency of once or more in a day during the study period.
Body weight: All animals were weighed on days 0 (start of administration), 7, 14 and 21. After copulation, animals were weighed on days 0, 7, 14 and 21 of gestation, and on days 0 and 4 of lactation after delivery. In addition, body weight gain during the administration period was calculated.
Measurement of food intake: Food given to all animals were weighed on days 0 (start of administration), 7 and 14, and on days 0 and 4 of lactation after delivery, and mean daily food intake was calculated by the amount on the measurement day and the amount on the next measurement day. In addition, cumulative food intake during the administration period was calculated.

Sacrifice and pathology:

Male animals
Necropsy and organ weight: Macroscopic observations of organs and tissues were performed on the animals euthanized by exsanguinations after blood sampling. Thymus, liver, kidney, testis and epididymides were weighed, and the ratio of organ weight to body weight (relative weight) was calculated. In addition, thymus, liver, kidney, brain, heart, spleen, adrenals, seminal vesicles, prostate, pituitary and the tissues with abnormal findings (lung, mass in the abdominal cavity) of all animals were fixed in 10% neutral buffered formaldehyde solution. The testis and the epididymides were fixed in Bouin solution.
Histopathology: Paraffin sections from the fixed organs and tissues indicated below were prepared by Histo Science Laboratory Co., Ltd., (Oume-shi, Tokyo) and routinely stained with hematoxylin-eosin. In addition, kidney was stained with PAS reaction. Microscopic observations were conducted in BioSafety Research Center.
Fertile male: Brain, thymus, heart, liver, kidney, spleen, adrenals, testis of all animals of the control group and 150 mg/kg group and the tissues with abnormal findings in all groups. Thymus, liver, kidney and adrenals of all animals of 6 and 30 mg/kg groups.
Infertile male: Brain, thymus, heart, liver, kidney, spleen, adrenals, testis, epididymides, seminal vesicles, prostate and pituitary.

Female animals
Necropsy and organ weight: The animals necropsied are shown below and the presence of abnormalities in the organs and tissues was observed.
Dead animal: Animals were necropsied immediately after found dead. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution.
Female with natural delivery: Animals were euthanized by exsanguination under ether anesthesia on day 4 of lactation, and main organs were observed macroscopically. And then, thymus, liver, kidney and ovaries were weighed, and the ratio of organ weight to body weight (relative weight) was calculated. In addition, thymus, liver, kidney, ovaries, brain, heart, spleen, adrenals, pituitary and the tissues with abnormal findings of all animals were fixed in 10% neutral buffered formaldehyde solution.
Female without natural delivery: On day 25 of gestation, animals were euthanized by exsanguinations under ether anesthesia, and macroscopic observations of main organs were performed. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution. Animal with no implantation sites was judged to be infertile.
Female whose pups all died: On the day or the next day when the death or cannibalism of pups were found, parental females were euthanized by exsanguinations under ether anesthesia, and macroscopic observations of main organs were performed. Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord were fixed in 10% neutral buffered formaldehyde solution. The numbers of corpora lutea of pregnancy and implantation sites were examined at the necropsy.
Histopathology: Paraffin sections from the fixed organs and tissues indicated below were prepared by Histo Science Laboratory Co., Ltd., (Oume-shi, Tokyo) and routinely stained with hematoxylin-eosin. In addition, kidney were stained with PAS reaction. Microscopic observations were conducted in BioSafety Research Center.
Dead animal: Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord.
Female with natural delivery: Brain, thymus, heart, liver, kidney, spleen, adrenals, ovaries of all animals of the control group and 150 mg/kg group, and the tissues with abnormal findings of all animals of all groups. Thymus, liver, kidney and adrenals of all animals of 6 mg/kg group and 30 mg/kg group.
Infertile female: Brain, thymus, heart, liver, kidney, spleen, adrenals, vagina, ovaries and pituitary.
Female whose pups all died: Skin, mammary glands, lymph nodes, salivary glands, sternum, femoral bone (with bone marrow), thymus, trachea, lung with bronchus, heart, thyroids with parathyroid glands, tongue, esophagus, stomach, duodenum, small intestine, large intestine, liver, pancreas, spleen, kidney, adrenals, urinary bladder, ovaries, uterus, vagina, eyes, Harderian glands, brain, pituitary and spinal cord.

Other examinations:

Observations and examinations on the reproduction of parental animals and neonates was also performed as part of the reproduction/developmental toxicity study.

Statistics:

Data of this study were recorded using a computer system, and the results of the study were statistically analyzed using the following methods.
The mean value per dam was used as one sample for the results of neonates during lactation period. Levels of significance were two steps of * : P < 0.05 and ** : P < 0.01.

Multiple comparison analysis was applied for body weight, food intake, number of corpora lutea of pregnancy, implantation site, number of birth, number of still birth, sex ratio, mean value of estrus cycle duration, gestation period, implantation index, delivery index, live birth index, incidence of external anomaly, viability index on day 4 of neonates, organ weight, ratio of organ weight to body weight (relative organ weight), hematology and blood chemistry.
First, the homogeneity was assessed by Bartlett test. If homogeneity was obtained, one way analysis of variance was applied. If variance was significant and the numbers of samples were equal among groups, Dunnett multiple comparison was applied. In the case of unequal numbers of samples, the significances of difference between the control group and each treated group were assessed by Scheffé multiple comparison.
When the data showed non-homogeneity by Bartlett test, the data were analyzed by Kruskal-Wallis rank sum test. Dunnett rank test was applied if significance was obtained and numbers of sample were equal, and in the case of unequal numbers of samples, Scheffé rank test was applied for the analysis of significant difference between the control group and each treated group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Mortality:

mortality observed, treatment-related

Description (incidence):

Detailed in results

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Higher mean values of daily food intake were seen in 150 mg/kg group from day 28 to 35 of administration compared to the control group. However, cumulative food intake showed no differences between these groups.

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

Description (incidence and severity):

Deatiled in results

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Detailed in results

Histopathological findings: neoplastic:

not examined

Details on results:

Effect on male animals
Mortality and clinical observation: There was no death during the administration period.
In clinical observations, alopecia (fore limb) in a rat of 6 mg/kg group, eye discharge in 2 rats of 30 mg/kg group, urogenital hemorrhage in a rat of 150 mg/kg group, and teeth abnormality (broken incisive tooth of upper jaw) in 1, 2 and 1 rats were observed in the control, 30 mg/kg and 150 mg/kg groups, respectively.
Body weight: No differences between the control group and test substance administered groups were seen.
Food intake: Higher mean values of daily food intake were seen in 150 mg/kg group from day 28 to 35 of administration compared to the control group. However, cumulative food intake showed no differences between these groups. No differences from that in the control group were seen in 6 mg/kg and 30 mg/kg groups.
Hematology: Compared to the control group, hematocrit value, hemoglobin concentration and erythrocyte count were lower, and reticulocyte count was higher in 150 mg/kg group. Reticulocyte count was also higher in 6 mg/kg group. In addition, percentage of lymphocytes was low and the ratio of neutrophils was high in 150 mg/kg group, but these changes were slight. Erythrocyte count in 6 mg/kg group and leukocyte count in 30 mg/kg group showed higher values. However, the changes were not dose dependent.
Blood chemistry: Chloride level was higher and potassium level was lower in all of test substance administered groups compared to the control group. In 30 and 150 mg/kg groups, levels of total protein, albumin and γ-GTP were higher and GOT level was lower than compared to the control group. In addition potassium level showed low level in 150 mg/kg group. High inorganic phosphate level was seen in 30 mg/kg group, but this was not dose dependent change.
Organ weight: Compared to the control group, relative weight of liver was higher in 30 mg/kg group, and relative and absolute weights of liver and kidney were higher in 150 mg/kg group. No differences from the control group were seen in organ weight of 6 mg/kg group.
Macroscopic findings
Fertile male: Macroscopic finding often observed in the groups treated with test substance was enlargement of liver which was seen in 1 and 9 rats of 30 mg/kg group and 150 mg/kg group, respectively. Other changes considered to be spontaneous occurrence were colored patch/zone (brown) in lung, deformed liver (abnormal lobulation), mass in the abdominal cavity (fat necrosis), cyst and scar in kidney, atrophy of testis and epididymis were sporadically seen in 30 mg/kg group and 150 mg/kg, and yellowish liver was seen 2 control animals.
Infertile male: One infertile male was noted in the control group and no abnormalities were observed in this animal.
Histopathology
Fertile male: The finding with apparently higher incidence in 150 mg/kg group compared to the control group was hypertrophy of centrilobular hepatocytes (Photo. 2) which were seen in 9 animals out of 12 animals. In addition, the incidence of vacuolation of adrenal glands was slightly higher in the test substance administered groups compared to the control group. The other findings showed no differences in the incidence between the control group and test substance administered groups. Thymus, liver, kidney and adrenals were examined in 6 mg/kg group and 30 mg/kg group and no abnormalities were observed in liver or kidney.
Infertile male: One infertile male was noted the control group. Pigmentation in the spleen, fatty change of hepatocytes and basophilic change of tubules, and vacuolation of adrenal glands were observed in the infertile animal.

Effect on female animals
Mortality and clinical observation: Two females died on day 23 of gestation in 150 mg/kg group. Decrease in locomotor activity was seen on the day before death in one animal (animal No. 2302). The other animal (animal No. 2305) died during delivery. No deaths were seen throughout the administration period in the other administration groups.
In the clinical observation, eye discharge was seen in one female of 30 mg/kg group, alopecia (fore limb, hind limb, neck) was seen in one control animal and 2 animals in 6 mg/kg group.
Body weight: Lower body weight gain compared to the control group was seen in 150 mg/kg group from day 0 to day 21 of gestation. No differences were seen in 6 mg/kg group and 30 mg/kg group compared to the control group.
Food intake: No differences between the control group and test substance administered group were seen throughout the administration period.
Organ weight: Compared to the control group, relative organ weights of liver and kidney were higher in 150 mg/kg group, and absolute weights of these organs showed tendency of high values. No differences were seen in 6 and 30 mg/kg groups compared to the control group.
Macroscopic findings
Dead animal: Two animals died in 150 mg/kg group. Enlargement of liver was seen in 2 animals. Reddish lung, yellowish small intestine, white patch/zone in liver and enlargement of kidney and adrenals were observed in one animal.
Female sacrificed on day 4 of lactation: The findings often observed in the groups treated with test substance were as follows. Atrophy of thymus was observed in one rat in 6 mg/kg group, 2 rats in 30 mg/kg group and one rat in 150 mg/kg group. White patch/zone in liver and enlargement of adrenal glands were observed in 2 animals each in 150 mg/kg group. Pale kidney was seen in one female each in 6 and 150 mg/kg groups. Sporadic findings observed in test substance administered groups were red patch/zone in thymus, colored patch/zone (yellowish) in small intestine, anomaly nodule in liver (hyperplasia), pale or red liver, dilatation of pelvis and enlargement of kidney and thinning of hair.
Infertile female: Infertile female was seen in one rat in the control group. No abnormalities were observed in the animal.
Female whose pups all died: Females whose pups all died until day 4 of lactation were one in 30 mg/kg group and 3 in 150 mg/kg group. Enlargement of liver was seen 3 females (all animals) and red patch/zone was seen in 2 females in 150 mg/kg group. Sporadic changes observed were scar in heart, colored patch/zone in thymus (brown), black patch/zone and a nodule in lung, white patch/zone in stomach, white small intestine, white liver, black kidney, enlarged and pale kidney and enlargement of adrenal glands.
Histopathology
Dead animal: Two animals died in 150 mg/kg group. Pigment deposition in spleen, thickening of tunica media of blood vessels in lung, parakeratosis in tongue and esophagus, hyaline droplet degeneration, centrilobular necrosis, single cell necrosis and centrilobular hypertrophy of hepatocytes in liver, hyaline droplet degeneration, granular proteinaceous casts in kidney and hyperplasia in the mammary gland were seen in both 2 animals.
Female sacrificed on day 4 of lactation: The finding with apparently higher incidence in 150 mg/kg group than in the control group was hypertrophy of centrilobular hepatocytes. In addition, the incidence of atrophy of thymus was slightly higher in the test substance administered groups. Findings observed in several animals in 150 mg/kg group but not in the control group were necrosis of centrilobular hepatocytes and granuloma in liver, deposition of PAS positive granules at the papilla of kidney, and granular proteinaceous casts and necrosis of the proximal convoluted tubules. Other observed findings showed no differences in the incidence between the control group and test substance administered groups.
In 6 mg/kg group and 150 mg/kg group, thymus, liver, kidney and adrenal glands were examined. There were no abnormalities in liver and kidney. However, the incidence of thymus atrophy was similar to that in 150 mg/kg group.
Infertile female: Infertile female was seen one in the control group. In this animal, cell infiltration in heart, pigment deposition in spleen and granulation foci in liver were observed.
Female whose pups all died: Females whose pups all died were seen one in 30 mg/kg group and 3 in 150 mg/kg group. In all of these animals, atrophy of thymus, pigment deposition in spleen, parakeratosis in tongue and fatty change of liver were observed. In addition, hemorrhage in lung, parakeratosis in esophagus, mitosis of hepatocytes, centrilobular necrosis of hepatocytes, single cell necrosis of hepatocytes, hypertrophy of hepatocytes, proteinaceous casts, basophilic change of tubules, mitosis, dilatation of tubules, necrosis and vacuolation of proximal convoluted tubules in kidney were observed in 2 or 3 animals out of 3 animals treated with 150 mg/kg.

Key result

Dose descriptor:

NOEL

Effect level:

6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Critical effects observed:

not specified

This report was translated from the original Japanese report. The translated copy is appended below for reference.

Conclusions:

Based on the results, no observed effect level (NOEL) was estimated to be 6 mg/kg in male and 30 mg/kg in female.

Executive summary:

In order to evaluate the toxicological nature of existing chemical substances, the repeated dose toxicity as well as the effect on reproduction and development were studied by the administration of 6-tert-butyl-2, 4-xylenol at the dose levels of 0 (vehicle control), 6, 30 and 150 mg/kg/day to rats for the period from 14 days before mating, during mating period, gestation period and to day 3 of lactation.

The general toxicological effect as well as the effect on reproduction and development were studied following the OECD Guidelines for “Testing of Chemicals; Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Screening Test, Extended Steering Group Document No. 3” (March 22, 1990).

The conduct of the study was designed to fulfill the standard described in “The order for specifying the items for the study on new chemical substances, and the investigation on the designated chemical substances. Article 4: Test Facility,” Kankiken No. 233, Eisei No. 38, 63 Kikyoku No. 823 (November 18, 1988).

No effect of the test substance administration were seen in the general condition of animals, but 2 female rats treated with 150 mg/kg died in the last stage of gestation (one rat died during delivery). In the females of 150 mg/kg group, suppression in body weight gain was observed during gestation period, however, no effect of the administration of test substance were seen in the body weight of male and the food intake of male and female rats.

In the hematological examination, low values in hematocrit, hemoglobin content and erythrocyte count were seen together with the high values in reticulocyte count and slight tendency of anemia in the male animal of 150 mg/kg group. In blood chemistry, low level of GOT and high level of γ-GTP were observed in 30 and 150 mg/kg groups.

Organ weights of liver and kidney were increased or showed a tendency to increase in male groups treated with 30 mg/kg or more and in female group treated with 150 mg/kg. In macroscopic observation, enlargement of liver was seen in male groups treated with 30 or 150 mg/kg, and enlargement of liver and kidney was observed in female group treated with 150 mg/kg. In the histopathology examination, the following findings were considered to be due to the test substance administration. Hypertrophy of centrilobular hepatocytes was observed in the male group treated with 150 mg/kg. Hypertrophy of centrilobular hepatocytes, degeneration of hepatocytes, necrosis of centrilobular hepatocytes and single cell necrosis in liver were seen in 150 mg/kg females at necrosis on day 4 of lactation. Parakeratosis in tongue and esophagus, hypertrophy of centrilobular hepatocytes as well as various degeneration, single cell necrosis and increase in mitotic figures in liver were observed in dead animals and the animals whose pups all died. In addition, degeneration of proximal convoluted tubules, proteinaceous casts and deposition of PAS positive granules at the papilla in kidney were observed in the female of this group.

 

From the results, repeated administration of 6-tert-butyl-2, 4-xylenol induced low level of GOT and high level of γ-GTP in the males of 30 and 150 mg/kg group, death and suppression of body weight gain, increase in organ weight and enlargement in liver and kidney in the females of 150 mg/kg group, anemic tendency, increase in organ weight of liver and kidney, and enlargement of liver in the male of 150 mg/kg group. In addition, by histopathology examination, abnormal findings were seen in liver of males and females, and in kidney of females in 150 mg/kg group. Thus, the target organs of the test substance were considered to be liver and kidney. Based on the results, no observed effect level (NOEL) was estimated to be 6 mg/kg in male and 30 mg/kg in female.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

6 mg/kg bw/day

Study duration:

subacute

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

Two studies are available for inspection for the repeated dose endpoint as follows:

 

OECD Guideline 422 – 28 days – Substance itself.

 

NOEL (male): 6 mg/kg bw/day

NOEL (female): 30 mg/kg bw/day

 

90-Day study – Read across to 85-60-9

 

NOAEL (male/female): 10 mg/kg/day

 

Effects were noted in the liver and kidney which are considered to be indicative of toxicity. These are as follows:

 

On the basis of the CLP Definition for classification:

 

3.9.2.7.Effects considered to support classification for specific target organ toxicity following repeated exposure

 

3.9.2.7.3. Evidence from appropriate studies in experimental animals can furnish much more detail, in the form of clinical observations, haematology, clinical chemistry, and macroscopic and microscopic pathological examination, and this can often reveal hazards that may not be life-threatening but could indicate functional impairment. Consequently all available evidence, and relevance to human health, shall be taken into consideration in the classification process, including but not limited to the following toxic effects in humans and/or animals:

 

 (e) multi-focal or diffuse necrosis, fibrosis or granuloma formation in vital organs with regenerative capacity;

 

Substance: OECD Guideline 422

 

Hypertrophy of centrilobular hepatocytes, degeneration of hepatocytes,necrosis of centrilobular hepatocytes and single cell necrosisin liver were seen in 150 mg/kg females at necrosis on day 4 of lactation. Parakeratosis in tongue and esophagus, hypertrophy of centrilobular hepatocytes as well as various degeneration,single cell necrosisand increase in mitotic figures in liver were observed in dead animals and the animals whose pups all died. In addition, degeneration of proximal convoluted tubules,proteinaceous casts and deposition of PAS positive granules at the papilla in kidneywere observed in the female of this group.

 

SIGNIFICANT

 

Read Across - 90-Day study

 

Microscopic Pathology: Microscopic examination of a palpable mass from female F3002 revealed a single mammary gland adenoma. Microscopically, the occurrence of uterine dilatation was slightly higher than that observed at necropsy. Other microscopic changes which were apparently related to chemical administration occurred in the livers and mesenteric lymph nodes of both sexes. Centrilobular hepatocellular vacuolization occurred at a higher incidence in males at the two highest dose levels and in females at the highest dose level. Instances of hepatocellular hypertrophy, karyomegaly, multinucleated hepatocytes and increased mitoses were probably a result of chemical exposure. Macrophages with distended cytoplasm were present in sinusoids and portal areas of livers from both sexes at the highest dose level.

Likewise, accumulations of macrophages which apparently contained foreign material were prevalent in mesenteric lymph nodes of both sexes from the two highest dose levels. Other changes observed microscopically were considered to be of spontaneous origin and were un associated with chemical exposure.

 

NOT SIGNIFICANT

 

(f) morphological changes that are potentially reversible but provide clear evidence of marked organ dysfunction (e.g., severe fatty change in the liver);

 

Substance: OECD Guideline 422

 

In the histopathology examination, the following findings were considered to be due to the test substance administration. Hypertrophy of centrilobular hepatocytes was observed in the male group treated with 150 mg/kg. Parakeratosis in tongue and esophagus (Parakeratosisis a mode of keratinization characterized by the retention of nuclei in the stratum corneum), hypertrophy of centrilobular hepatocytes as well as various degeneration,single cell necrosisand increase in mitotic figures in liver were observed in dead animals and the animals whose pups all died. In addition, degeneration of proximal convoluted tubules.

 

SIGNIFICANT

 

Read Across - 90-Day study

 

Gross Pathology: There was a slight (although statistically significant) decrease in the mean terminal body weights of high dose group females. The only notable changes in organ weights were slight increases in absolute liver weights for males at the highest dose level, and for females at the two highest dose levels. Liver to body weight ratios were slightly increased for both sexes at the highest dose level. Other increases in organ to body weight ratios were attributed to low terminal body weights, rather than a direct treatment effect on the organs.

 

A slight increase in the occurrence of uterine dilatation at the highest dose level was observed at necropsy. Other changes observed at necropsy were considered to be of spontaneous origin and were not associated with chemical exposure.

 

NOT SIGNIFICANT

 

Conclusion:

 

Substance should be classified as

 

Substance should be classified as

 

STOT RE-2

H373: May cause damage to organs (liver, kidney) through prolonged or repeated exposure (oral)

Justification for classification:

 

(e) multi-focal or diffuse necrosis, fibrosis or granuloma formation in vital organs with regenerative capacity;

 

Substance: OECD Guideline 422

 

Hypertrophy of centrilobular hepatocytes, degeneration of hepatocytes,necrosis of centrilobular hepatocytes and single cell necrosisin liver were seen in 150 mg/kg females at necrosis on day 4 of lactation. Parakeratosis in tongue and esophagus, hypertrophy of centrilobular hepatocytes as well as various degeneration,single cell necrosis and increase in mitotic figures in liverwere observed in dead animals and the animals whose pups all died. In addition, degeneration of proximal convoluted tubules, proteinaceous casts and deposition of PAS positive granules at the papilla in kidney were observed in the female of this group.

 

3.9.2. Classification criteria for substances (from CLP)

 

Table 3.9.1 – Categories for specific target organ toxicity – repeated exposure provides guidance for classification. On the basis of this tabulated data:

 

Cat 1 is not applicable as effects are not considered to be severe.

 

Car 2 is applicable, on the basis of the liver / kidney at 150 mg/kg/day results.

 

CLP Section 3.9.2.9.5 states that:

 

The guidance values refer to effects seen in a standard 90-day toxicity studyconducted in rats. They can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration, using dose/exposure time extrapolation similar to Haber's rule for inhalation, which states essentially that the effective dose is directly proportional to the exposure concentration and the duration of exposure.The assessment shall be done on a case-by-case basis; for a 28-day study the guidance values below is increased by a factor of three.

 

Table 3.9.3 – Guidance values to assist in Category 2 classification states as follows:

 

Oral (rat) mg/kg body weight/day 10 < C≤100

 

Value for classification is therefore multiplied by 3 on the basis of CLP Section 3.9.2.9.5

 

Therefore Cat 2 = 30 < C ≤ 300 for a 28-day study

 

Effects seen at C=150; therefore Cat 2 is appropriate.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Study performed in compliance with GLP and analytical verification performed, however no reference guidelines detailed in the report.

Justification for classification or non-classification

The above studies were ranked reliability 1 or 2 according to the Klimisch et al system. This ranking was deemed appropriate because the OECD 422 study conducted on the substance itself was conducted to GLP in compliance with agreed protocols. The 90-day report utilising read across was not conducted to GLP or to a recognised method; however it documents dose levels and responses in detail, so is deemed appropriate for use in the support of a formal registration. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

Justification for classification or non classification

The above results triggered classification under the CLP Regulation (EC No 1272/2008) as follows:

STOT Rep. Exp. 2

H373: May cause damage to organs <liver, kidney> through prolonged or repeated exposure <oral>