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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Nov 2009 - 10 Mar 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
inconsistencies in the english translation; deviations to current guideline
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
(OECD 422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 Nov 2009 - 10 Mar 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
inconsistencies in the english translation; deviations to current guideline
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)
Version / remarks:
adopted on 22th March 1996
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29th July 1996
Deviations:
yes
Remarks:
substance administration only until nursing day 4, no blood samples taken from pups, no determination of serum T4 levels, no counting of nipples / aerolae in male pups
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
SD [Crl: CD (SD)]
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Tsukuba Breeding Centre (955 Kamibayashi, Ishioka-shi, Ibaraki-ken, Japan)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 weeks
- Weight at study initiation: Males: 390 g (mean) / 351 - 427 g (range); Females: 234 g (mean); 212 - 253 g (range)
- Fasting period before study: No
- Housing: In individual stainless steel mesh cages [260W x 380D x 180H (mm)] arranged on five-tier stainless steel racks; females that achieved copulation were housed in polycarbonate cages [265W x 426D x 200H (mm)] containing nesting material (white flake, Charles River Laboratories Japan) on day 0 of gestation, and their offspring were allowed to share the cage after delivery
- Diet: Solid feed lab MR stock, Nosan Corporation; ad libitum
- Water: Sterilised tap water filtered through a cartridge filter with a pore size of 1 µm and then irradiated with ultraviolet rays; ad libitum
- Acclimation period: 12 days (including quarantine and observation of the oestrus cycle)

DETAILS OF FOOD AND WATER QUALITY:
The results of the analyses of contaminants in the feed and nesting material were all confirmed to be within the permissible range at the laboratory set with reference to standards such as the “Limits on Contaminants in Feed and Solvents under the United States Environmental Protection Agency Toxic Substances Control Act" (1979), and drinking water was confirmed to conform to the water quality standards under the Water Works Law.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 - 23.0
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:

- VEHICLE
- Concentration in vehicle: 10 mg/kg bw/daygroup: 2 mg/mL, 50 mg/kg bw/day group: 10 mg/mL, and for 250 mg/kg bw/day group: 50 mg/mL
- Amount of vehicle: 5 mL administration solution/kg bw
- Lot/batch no: OG-17 (Kozakai Pharmaceutical Co. Ltd)

The administration solutions prepared were subdivided into amounts for 1 day and stored tightly stoppered and protected from light in a cool place (2 - 6°C) until use. Since the test substance was confirmed to be stable after storage for 7 days in cool place and protected from light and for 1 day at room temperature and protected from light, it was used within 8 days of preparation. The first and last administration solutions to be prepared were analysed and were confirmed to have been prepared at the designated concentrations.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To confirm that the test substance administration solutions had been prepared at the designated concentrations, the first and last prepared solutions of 2,5-bis(2,4,4-trimethylpentan2-yl)benzene-1,4-diol in olive oil prepared (for 10 mg/kg bw(day group: 2 mg/mL, for 50 mg/kg bw/day group: 10 mg/mL, and for 250 mg/kg bw/day group: 50 mg/mL) were each analysed three times. The results confirmed that the test substance administration solutions had been prepared at the designated concentrations.
Duration of treatment / exposure:
Duration of administration was from 14 days prior to the start of mating for both males and females and for 42 days in the case of males. In the case of females, administration continued during mating and gestation and until day 4 of nursing after delivery, with the duration ranging from 42 to 46 days. In the female recovery group, the duration of administration was 42 days as with the males.
Frequency of treatment:
Once daily
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Test substance groups: 12 animals/dose
Control group males: 12 animals (for the recovery group: 5 animals each were randomly selected from the control group and the maximum dose group on day 42 of administration)
Control group females: 12 animals; in addition: two satellite groups consisting of a control group and a recovery group (not mated) with five females in each group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a dose setting study, 0 (solvent only), 30, 100, 300 and 1000 mg/kg bw/day of the substance was repeatedly orally administered for 14 days to four male and four female rats per group, and observation of general condition, measurement of body weight and feed intake, urinalysis, haematological and blood biochemical tests, necropsy and organ weight measurement were performed. The results showed that in the 100 mg/kg bw/day and higher groups, low haematocrit was observed in males and females, low absolute weight of prostate was observed in males, and low haemoglobin levels and high absolute and relative weight of liver were observed in females. In addition, in the 300 mg/kg bw/day and higher groups, loose stools and high platelet count were observed in males and females, high activated partial thromboplastin time and ALT, low absolute and relative weight of prostate were observed in males, and increased feed intake and low red blood count were observed in females. In addition to these changes, in the 1000 mg/kg bw/day group, bIotted fur in the lower abdomen with urine, increased salivation and feed intake, dilation of the caecum and low chlorine were observed in males and females, low levels of neutral lipids, low relative weight of heart and high relative weight of liver were observed in males, and high γ-GTP and low urine pH were observed in females. On the basis of these results, 250 mg/kg bw/day, a concentration at which repeated dose toxicity was definitely expected to occur, was set as the maximum dose in the present study, and 10 mg/kg bw/day, a concentration at which no toxic effects were expected to be observed, was set as the minimum dose. A dose of 50 mg/kg bw/day falling between these two doses was also set, making three doses in total.

- Post-exposure recovery period in satellite groups: 14 days after the end of administration.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were for mortality / morbidity and to examine their behaviour twice a day (in the morning and afternoon) from the start of administration until the date of necropsy. Gestation, delivery and nursing status were examined carefully.
- Cage side observations checked: Mortality, behaviour, gestation / delivery and nursing status

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the day prior to the start of administration and then once a week, animals were examined upon removal from the cage and outside the cage in an aluminium open field (370W x 560D x 40 H mm) for reactivity on removal from the cage, reactivity on handling, muscle tone (relaxed to rigid), skin (colour), fur, piloerection, eye discharge, palpebral closure, exophthalmos, lachrymation, smudge around mouth/nose (dirt), salivation, soiled fur in the lower abdomen with urine, soiled fur around anus with faeces, vocalisation, breathing, body position, convulsion, tremor, exploration (arousal), alertness, locomotor activity (activity), walk (wobbly), abnormal behaviour (self-biting, walking backwards etc.), stereotypy (excessive grooming, repeated turning movement etc.), failure of consciousness (confusion, catalepsy, coma), limb tone, urination and defecation.

BODY WEIGHT: Yes
- Time schedule for examinations: In males on administration day 1 (test substance administration start date, immediately prior to test substance administration), 7, 14, 21, 28, 35 and 42, the necropsy date and in the case of the recovery group, also on day 49 (recovery day 7) and 56 (recovery day 14) and the necropsy date. In females, body weight was measured on administration day 1, 7 and 14, gestation day 0, 7, 14 and 20, nursing day 0 and 4, and the necropsy date. In the female recovery groups set up as satellite groups, mating was not performed and body weight was therefore measured on the same days as in males.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes; the amount of feed consumed in each cage over the 24 h until the following day was measured on the body weight measurement days. However, the final feed intake measurement day was administration day 41 in males, nursing day 3 in females and recovery day 13 in the recovery groups.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, but as g food/rat/day
- 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 (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately prior to necropsy on the day following final test substance administration or the day following the end of the recovery period.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (the animals were no longer supplied with feed after 5 pm the previous day and were only provided with water)
- How many animals: Tests were performed on five animals randomly selected from each group. In the recovery groups, in the case of males, five animals were randomly selected from the control group and 250 mg/kg bw/day group, and in the case of females, all animals were used for the tests.
- Parameters checked: Red blood count, haemoglobin, haematocrit, mean erythrocyte volume, mean erythrocyte haemoglobin, mean erythrocyte haemoglobin concentration (calculated values), white blood count, reticulocyte count, white cell percentages and platelet count, prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately prior to necropsy on the day following final test substance administration or the day following the end of the recovery period.
- Animals fasted: Yes (the animals were no longer supplied with feed after 5 pm the previous day and were only provided with water)
- How many animals: Tests were performed on five animals randomly selected from each group. In the recovery groups, in the case of males, five animals were randomly selected from the control group and 250 mg/kg bw/day group, and in the case of females, all animals were used for the tests.
- Parameters checked: Total protein, albumin, A/G ratio (calculated value), blood glucose, total cholesterol, triglycerides, total bilirubin, urea nitrogen, creatinine, AST, ALT, ALP, γ -GTP, LDH, ChE, calcium and inorganic phosphorus, sodium, potassium and chlorine, glucokinase, glucose-6-phosphate dehydrogenase, cholesterol esterase, cholesterol oxidase, peroxidase, lipoprotein lipase, glycerol kinase, L-y-glycerophosphate oxidase, butyrylthiocholine, 5, 5'-dithiobis-2-nitrobenzoic acid, purine nucleoside phosphorylase, xanthine oxidase

URINALYSIS: Yes
- Time schedule for collection of urine: Males on day 36 of test sibstance administration and recovery day 8 in case of the recovery group
- Metabolism cages used for collection of urine: Yes (animals were placed for about 3 or 18 h in metabolism cages)
- Animals fasted: Yes (no feed or water was supplied during time in metabolism cages)
- Parameters checked: Observation of appearance, pH by the test paper method (Multisticks, Bayer Sankyo Co. Ltd.), occult blood, protein, glucose, ketone bodies, qualitative tests of bilirubin and urobilinogen, and sedimentation tests (microscopy after staining with URI-CELL solution, Cambridge Chemical Products). In addition, for urine obtained over 18 h, amount of urine, specific gravity (refractometer, Erma Inc.) sodium and potassium (automated electrolyte analyser, NAKL-132, DKK-Toa Corporation), were measured.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory function tests: Males on day 42 of administration (final administration date), in females on day 3 of nursing, and on day 42 of test substance administration (final administration date) and day 14 of recovery (recovery period end date) in males and females in the recovery groups; Grip strength and amount of spontaneous exercise: Males on day 41 of administration, in females on day 3 of nursing, and on the final administration date and day 13 of recovery in males and females in the recovery groups.
- Dose groups that were examined: Tests were performed on five animals randomly selected from each group. In the recovery groups, in the case of males, five animals were randomly selected from the control group and 250 mg/kg bw/day group, and in the case of females, all animals were used for the tests.
- Battery of functions tested: Sensory function tests, measurement of grip strength, measurement of amount of spontaneous exercise

IMMUNOLOGY: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals for planned sacrifice were bled out under ether anaesthesia on the day following administration day 42 in the case of males, nursing day 5 in the case of females and the day following recovery day 14 in the case of the recovery groups. The surface of the body, mucous membranes at the incision and internal organs were examined macroscopically. In addition, the liver, kidneys, adrenal glands, thymus, spleen, brain, heart, pituitary gland, thyroid gland, prostate, seminal vesicles, testes and epididymides of all males and females in each group were weighed (absolute weight), and the ratio to body weight (relative weight) was calculated based on body weight on the day of sacrifice. Weights for symmetrical organs were combined weights and the pituitary gland and thyroid gland were weighed after fixing. In females, number of corpora lutea on the ovaries and number of implantations in the uterus were examined and the implantation rate [(number of implantations/number of corpora lutea) x 100] was calculated.

HISTOPATHOLOGY: Yes
Histopathology was performed for all males and females in the control group and 250 mg/kg bw/day group. The following organs were examined: brain (including cerebrum, cerebellum and pons), pituitary gland, thyroid gland (including parathyroid glands), thymus, trachea and lungs (immersed in fixing solution after infusion), stomach, intestines (from the duodenum to the rectum, including Peyer patch), heart, liver, spleen, kidneys, adrenal glands, urinary bladder, testes, epididymides, prostate, seminal vesicles, vagina, uterus, spinal cord (cervical region, thoracic region, lumbar region), sciatic nerve, bone and bone marrow (femur), lymph nodes (submandibular lymph nodes, mesenteric lymph nodes), mammary glands.
The results showed that administration of the test substance had affected the liver, kidneys and thyroid gland in males and females, and the liver, kidneys and thyroid gland were therefore inspected in males and females in other groups, including the recovery groups. The pituitary gland and reproductive organs were also inspected in the pairs of males and females that did not achieve gestation. Macroscopically abnormal sites in any group were inspected in all animals. Inspections were performed by preparation of paraffin embedded sections in accordance with the usual method, H-E staining and microscopy. In addition, PAS staining was performed on the testes of animals for spermatogenetic cycle inspections, and PAS staining and alpha2u-globulin immunostaining was performed to identify deposits in the kidneys.
Other examinations:
In females, following the quarantine and acclimatisation period, Giemsa-stained vaginal smears were prepared from the administration start date until confirmation of copulation and examined by microscope to assess the stage of the oestrus cycle. In addition, the oestrus cycle was calculated based on the assessment of oestrus cycle from the administration start date until the mating start date. One oestrus cycle was treated as the time from the day on which signs of oestrus (III) were observed until the day prior to observation of signs of the next oestrus. If signs of oestrus were continuous, calculations were made from the day on which the first signs of oestrus were observed.
After completion of administration for 2 weeks prior to mating (afternoon of administration day 15), males and females in the same group were placed together continuously in the same cage at a 1:1 ratio until copulation was confirmed or up to a maximum of 2 weeks. During the mating period, copulation was checked at a set time (around 9:30) every morning and the copulation rate [(number of copulated animals/number of animals kept together) x 100] calculated. Copulation was confirmed by vaginal plug formation or by the presence of semen in the vagina, and the day of confirmation was used as gestation day 0. Delivery was confirmed by 10 am by checking that parent animals had built a nest and started lactation or by observation of the area around the vagina in parent animals and palpation of the abdominal region. The day on which delivery was confirmed was used as nursing day 0. Based on the results of observation of mating and delivery, the conception rate (%) [(number of conceiving females/number of copulated females) x 100], duration of gestation (number of days from gestation day 9 until the day of confirmation of completion of delivery) and the birth rate (%) [number of females delivering live offspring/number of gestating females) x 100] were calculated for each group.

Regarding observations of the offsprings see section 7.8 Toxicity to reproduction.
Statistics:
Mean values and frequencies obtained were used to test for significant differences from the control group (ratio of not more than 5%):
(1) Parametric data: Bartlett variance test for comparisons of multiple groups. If variance was uniform: analysis of variance with a one-dimensional layout. If the results revealed a significant difference, comparative tests of each group against the control group by the Dunnett test method. If the variance was not uniform, test method using non-parametric data (body weight, body weight increase, feed intake, grip strength, amount of spontaneous exercise, qualitative data in urinalysis, haematological test data, blood biochemical test data, organ weights, number of corpora lutea, number of implantations, duration of achievement of copulation, duration of gestation, number of pups delivered, number of live pups, number of dead pups). An F test was used for comparisons between two groups. If the results revealed uniform variance, Student t test, and if the results revealed non-uniform variance, an Aspin-Welch t test.
(2) Non-parametric data: Kruskal-Wallis variance test for comparisons of multiple groups. If the results revealed a significant difference, the control group and each group were compared by the Dunnett test method (white blood cell percentages, qualitative data in urinalysis, spermatogenetic cycle tests, number of implantations, viability, delivery rate, oestrus cycle, live birth survival rate, sex ratio). Mann-Whitney U test for comparisons between two groups (graded data from qualitative data from urinalysis, white blood cell percentages and histopathological test findings).
(3) Categorical data
Fisher's exact test (incidence rate, copulation rate, conception rate and birth rate for animals with abnormalities in detailed clinical observations, sensory function tests, observation of general condition and pathological tests). Mann-Whitney U test for graded data from histopathological test findings.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the test substance administration period: Mild loose stools in 12/12 males and 9/12 females in the 250 mg/kg bw/day group and 5/5 females in the 250 mg/kg bw/day recovery group. Loose stools were observed transiently after test substance administration and the animals recovered prior to test substance administration the following day.
Mild soiled fur in the lower abdomen with urine was observed in 11/12 males and 7/12 females in the 250 mg/kg bw/day group and 5/5 females in the 250 mg/kg bw/day recovery group. In addition, salivation was observed in 11/12 males and 8/12 females in the 250 mg/kg bw/day group and 3/5 females in the 250 mg/kg bw/day recovery group. Salivation was observed in 1/12 females in the 50 mg/kg bw/day group. The salivation was transient and involved mild wetness around the mouth immediately after test substance administration. No changes in general condition were observed in the 10 mg/kg group. In addition, during the recovery period, no changes in general condition were observed in any group.
Please refer also to Table 3 / Table 4-1 / Table 4-2 in the attachement for further details.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female in the 250 mg/kg group died on gestation day 21. Another female of the same group was moribund and therefore sacrificed on gestation day 22 (administration day 39).
Dead animal: A tendency for suppression of body weight increase was observed during gestation as this female had not eaten when food intake was measured on gestation day 20 (test substance administration day 38); the animal was emaciated, and adopted the recumbent position and died the following day (gestation day 21).
Moribund animal: A similar tendency for suppression of body weight increase was observed during gestation in another female. Also this female had not eaten when food intake was measured on gestation day 20 (test substance administration day 38) and reddish lachrymation was observed from gestation day 21 (test substance administration day 38). The following day, the animal was observed in the prone position with low skin temperature, and moribund sacrifice was therefore performed.

Histopathological tests on dead animals revealed a high incidence of the changes due to administration of the test substance described under Histopathology, particularly changes in the kidneys, and death was inferred to have occurred as a result of this combined with stress from the end of gestation until the start of delivery.
Please refer also to Table 2 in the attachement for further details.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the test substance administration period: Significantly decreased body weight gain in males and significantly higher body weight gain during the period prior to mating in females in the 250 mg/kg bw/day group. Significantly higher body weight (compared to controls) on administration day 21 and 28 and body weightgain during the test substance administration period were observed in the female recovery group. No significant changes were observed in males or females in the 10 and 50 mg/kg bw/day groups.
During the recovery period: Significantly low body weight in males for the recovery group from the end of administration and throughout the recovery period, but no changes in body weight gain during the recovery period. However, the body weights of the animals selected for the recovery period were coincidentally lower than those for animals sacrificed at the end of the administration period. In the female recovery group, significantly low body weight gain was observed during the recovery period, and body weight, which had been rather higher than in the control group during the test substance administration period, now approached that in the control group, and there was no longer any difference in recovery days 7-14.
Please refer also to Figure 1 / Figure 2-2 / Table 11 / Table 12-1 / Table 12/2 in the attachement for further details.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the test substance administration period: In the 250 mg/kg bw/day group, significantly higher food intake in males on test substance administration day 14, 28, 35 and 41 and in females on test substance administration day 14 and gestation day 14, and lower food intake were observed in females on nursing day 3. In the female recovery group, significantly higher food intake was observed in the 250 mg/kg bw/day group on administration day 14. Although significantly low levels of food intake were observed in males in the 10 mg/kg bw/day group on administration day 35, these appeared to be incidental changes. No significant changes were observed in males or females in the 50 mg/kg bw/day groups.
During the recovery period: Significantly higher food intake was observed in males of the 250 mg/kg bw/day group on recovery day 0. No significant changes were observed in the female recovery group
Please refer also to Figure 3 / Figure 4-1 / Figure 4-2 / Table 13 / Table 14-1 / Table 14/2 in the attachement for further details.
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):
Tests on animals sacrificed at the end of the administration period: significantly low levels of haemoglobin and MCH in males in the 50 and 250 mg/kg bw/day group and significantly low levels for red blood count and haematocrit in males in the 250 mg/kg bw/day group. In females, significantly low levels of haemoglobin were observed in the 50 mg/kg bw/day group, and significantly low levels of MCH and a tendency for low levels of haemoglobin were observed in the 250 mg/kg w/day group. Furthermore, in males, for white blood cell percentages, significantly low levels of neutrophils and significantly high levels of lymphocytes were observed in the 50 mg/kg bw/day group and significantly low levels of neutrophils and a significantly high white blood count were observed in the 250 mg/kg bw/day group. In addition, significantly high platelet count and significantly low prothrombin time were observed in females in the 250 mg/kg bw/day group.
Tests on animals sacrificed at the end of the recovery period revealed a decrease in the amount of variation from the control group and a tendency for recovery, although a significant difference remained in terms of low levels of MCHC and high platelet count in females.
Individual values, however, were all within the reference range of the background data and no changes were observed in haematopoietic tissue or peripheral blood reticulocyte count, and no changes were observed that were suggestive of hyper-destruction of red blood cells in the reticuloendothelial system such as the spleen.
Please refer also to Table 16-1 / Table 17-1 / Table 17/2 in the attachement for further details.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly low levels of calcium in males and significantly high levels of γ-GTP, total protein, albumin and total cholesterol and significantly low levels of creatinine and chlorine in females of the 250 mg/kg bw group. Significantly high levels of calcium were observed in the 10 mg/kg bw/day group, but no dose correlation was observed. No significant changes in any test item were observed in males or females in the 50 mg/kg bw/day group.
Tests on animals sacrificed at the end of the recovery period revealed significantly high levels of total protein, albumin and total cholesterol and significantly low levels of creatinine and chlorine in females, but a decrease in the amount of variation from the control group and a tendency for recovery were observed in all cases in comparison with female animals sacrificed at the end of the test substance administration period. In addition, significantly low levels of alkaline phosphatase and significantly high levels of calcium and inorganic phosphorus were observed in females, but all changes were slight.
Please refer also to Table 18-1 / Table 19-1 / Table 19-2 in the attachement for further details.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Tests during the test substance administration period: significantly low levels of both specific gravity in the 10 mg/kg bw/day group and potassium in the 50 mg/kg bw/day group. Individual values, however, were all within the reference range of the background data from the research institute (specific gravity: mean 1.065 / range 1.042-1.087, potassium: mean 2.81 / range 0.63-4.99 mEq/18 hr) and, in addition, the changes had no dose correlation.They were therefore considered to be incidental. No significant changes were observed in the 250 mg/kg bw/day group. No significant changes were observed in animals during the recovery period.
Please refer also to Table 15-1 in the attachement for further details.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory function tests: No changes were observed for any test item in any test substance administration group in tests during the test substance administration period and recovery period.
Grip strength and amount of spontaneous exercise: No significant changes in grip strength were observed in measurements on the final test substance administration date. Regarding amount of spontaneous exercise, a significant decrease was observed in 60 min measurements in males in the 10 and 250 mg/kg bw/day groups. In the recovery group, a significant decrease was observed in 60 min measurements in tests on the final test substance administration date in males in the 250 mg/kg bw/day group. No significant changes were observed in 30 min measurements and in the amount of spontaneous exercise in the 50 mg/kg bw/day group. Since all values were within the reference range apart from those for one animal in the 250 mg/kg bw/day group, this was considered to be an incidental change.
Please refer also to Table 9-1 / Table 9-2 in the attachement for further details.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Among animals sacrificed at the end of the administration period: significantly high absolute and relative weight of the liver and the kidneys in males of the 50 and 250 mg/kg bw/day groups, significantly high absolute and relative weight of the liver in females of the 50 and 250 mg/kg bw/day groups; in addition, high relative weight of the kidneys and heart, absolute and relative weight of the adrenal and thyroid gland in females of the 250 mg/kg bw/day group, As there was no change in absolute heart weight, the variation in relative weight was slight, and there were no accompanying histopathological changes, this was determined to be an incidental change. No significant changes were observed in males or females of the 10 mg/kg bw/day groups.
Among animals sacrificed at the end of the recovery period: significantly high relative weight for the kidneys in males and females of the 250 mg/kg bw/day group, and a significantly high absolute and relative weight for the liver, a significantly high relative weight for the heart in females of the 250 mg/kg bw/day group. In addition, significantly low body weight immediately prior to dissection and significantly high relative weights for the thymus, pituitary gland and thyroid gland in males of the 250 mg/gk bw/day.
Please refer also to Table 22-1 / Table 22-2 / Table 23-1 / Table 23-2 in the attachement for further details.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Among animals sacrificed at the end of the administration period: enlargement of the liver in 2/12 males and 2/12 females in the 50 mg/kg bw/day group and 6/7 males and 10/10 females in the 250 mg/kg bw/day group. In addition, among females in the 250 mg/kg bw/day group, thymic atrophy in an animal that died and thymic atrophy and red urine in the urinary bladder in a moribund sacrifice. Apart from these changes and unrelated to administration of the test substance, yellowing of the liver among the two sets of 12 animals in one male and one female in the 10 mg/kg bw/day group, one male in the 50 mg/kg bw/day group, and one animal with renal enlargement, prostatomegaly, distension of the seminal vesicles and distension of the urinary bladder in the 10 mg/kg bw/day group.
Among animals sacrificed at the end of the recovery period: decrease in the size of the testes and epididymides (bilateral) in 1/5 males in the 250 mg/kg bw/day group.
Please refer also to Table 20 / Table 21 in the attachement for further details.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Among animals sacrificed at the end of the administration period: changes attributable to administration of the test substance in the liver, kidneys and thyroid gland.
- Liver: Centrilobular hepatocellular hypertrophy in 3/12 males and 5/12 females in the 50 mg/kg bw/day group and 5/7 males and 11/12 females in the 250 mg/kg bw/day group (significant difference in number of incidences compared to the control group in females in the 50 mg/kg bw/day group and males and females in the 250 mg/kg bw group). The degree of the change tended to be more severe in the 250 mg/kg group than in the 50 mg/kg group for both males and females. No hepatocellular hypertrophy was observed in the liver amon animals sacrificed at the end of the recovery period.
- Kidneys: PAS stain-negative hyaline droplets in the renal proximal tubular epithelium in all groups, including the control group, but the number increased in the 50 and 250 mg/kg bw/day groups (there was a tendency for enlargement, and a comparison of graded data with the control group revealed a significant difference in the 250 mg/kg bw/day group). These PAS stain-negative hyaline droplets were confirmed to be alpha2u-globulin immunostain-positive. In females: fatty degeneration of the renal proximal tubular epithelium (5/12 animals) and appearance of PAS stain-positive hyaline droplets in the renal proximal tubular epithelium (12/12 animals) in the 250 mg/kg bw/day group (significant difference from the control group). Hyaline droplets in males had recovered to the same level as the control group, the incidence rate of hyaline droplets and fatty degeneration of the renal proximal tubular epithelium in females had decreased to 1/5 and 2/5 animals, respectively, and a tendency for recovery was observed at the end of the recovery period.
- Thyroid gland: Diffuse follicular epithelial cell hyperplasia in 7/7 males and 12/12 females in the 250 mg/kg bw/day group (significant difference from the control group in both males and females). In the 250 mg/kg bw/day group, thyroid follicles had decreased in size, epithelial cells had become cuboidal or cylindrical, and colloids within the follicles tended to decrease. No follicular epithelial cell hyperplasia in the thyroid gland was observed among animals sacrificed at the end of the recovery period.

Regarding the death of one and moribund sacrifice of one female animal in the 250 mg/kg bw/day group: In addition to the observation of the changes in the animals sacrificed at the end of the administration period, severe changes in the kidneys in both animals, formation of hyaline casts and erythrocyte casts in the moribund animal, and thymic cortical atrophy in both animals.

In addition to these changes, fatty degeneration of hepatocytes in the perilobular zone (males: 0, 10, 50, 250 mg/kg bw/day groups, females:10 mg/kg bw/day group) was observed in animals such as those with yellowing of the liver revealed by necropsy to be unrelated to administration of the test substance, and dilation of the renal tubules in the kidneys, interstitial oedema in the prostate, inflammation of the seminal vesicles and dilation of the lumen, calculus in the urinary bladder and inflammation of the transitional epithelium were observed in the animal in the 10 mg/kg bw/day group with renal enlargement, prostatomegaly, distension of the seminal vesicles and distension of the urinary bladder. In addition, microgranuloma (males: 0, 10, 50 mg/kg bw/day groups, females: 0, 50, 250 mg/kg bw/day groups, recovery group males: 250 mg/kg bw/day group, females: 0, 250 mg/kg bw/day groups), focal necrosis (males: 0, 50, 250 mg/kg bw/day groups, females: 50 mg∕kg bw/day group, recovery group males: 0 mg/kg bw/day group) and granuloma that appeared to be a sign of repairs (males: 10 mg/kg bw/day group, females:10, 50, 250 mg/kg bw/day groups) in the liver; so-called eosinophilic bodies with a connection to PAS stain-negative hyaline droplets in the renal proximal tubular epithelium (males: 10 mg/kg bw/day group), basophilic renal tubules (males: 0, 10, 50, 250 mg/kg bw/day groups, females: 10, 50, 250 mg/kg bw/day groups, recovery group males: 0, 250 mg/kg bw/day groups, females: 0 mg/kg bw/day group), cortical lymphocyte infiltration (males: 10, 50 mg/kg bw/day groups, recovery group males: 250 mg/kg bw/day group), cortical fibrosis (males: 0 mg/kg bw/day group, females: 10 mg/kg bw/day group), mineralisation of the medullary tubules (females: 10 mg/kg bw/day group), hyaline casts (males: 0, 10, 50, 250 mg/kg bw/day groups, recovery group females: 0 mg∕kg bw/day group, females: 250 mg/kg bw/day group), solitary cyst (males: 0, 10, 250 mg/kg bw/day groups) and regenerating renal tubules (males:10 mg/kg bw/day group) in the kidneys; foam cell accumulation (males: 0, 250 mg/kg bw/day groups, females: 0 mg/kg bw/day group) and arterial wall mineralisation (males: 0, 250 mg/kg bw/day groups, females: 0, 250 mg/kg bw/day groups) in the lungs; myocardial degeneration and fibrosis (males: 0, 250 mg/kg bw/day groups, females: 0, 250 mg/kg bw/day groups) in the heart; dilation off the gastric fundal gland (males: 0, 250 mg/kg bw/day groups, females: 0, 250 mg/kg bw/day groups) in the glandular stomach; vacuolation of cytoplasm in cortical cells (males: 250 mg/kg bw/day group) and cortical focal fatty metamorphosis (females: 0 mg/kg bw/day group) in the adrenal glands; interstitial lymphocyte infiltration and sperm granuloma (males: 250 mg/kg bw/day group) in the epididymides; interstitial lymphocyte infiltration (males: 250 mg/kg bw/day group) in the prostate; and follicular cysts (females: 250 mg/kg bw/day group) in the ovaries were observed at a low incidence rate, but these changes are all spontaneously occurring lesions in rats, and no increase in incidence rates due to administration of the test substance was observed.

Extramedullary haematopoiesis and brown pigmentation in the spleen were both observed at high incidence rates in males and females, but no difference in the incidence rate or severity of changes was observed between the 250 mg/kg bw/day group and the control group. The changes were therefore deemed to have no relationship to administration of the test substance in any case.

Regarding the centrilobular hepatocellular hypertrophy, this was not associated with regressive changes in hepatocytes, blood biochemical tests produced no findings suggestive of liver disorders, and high levels of total cholesterol were observed in females and an increase in cholesterol has been reported to be correlated to an increase in drug metabolising enzymes, which suggested that these findings were the result of adaptation reflecting induction of drug metabolising enzymes by administration of the test substance.

Regarding the effect on the kidneys, both high absolute weights and relative weights were observed in males in the 50 and 250 mg/kg bw/day groups, and an increase in PAS stain-negative hyaline droplets in the renal proximal tubular epithelium was observed as a corresponding histological change. In addition, high relative weight of the kidneys was observed, and appearance of PAS stain-positive hyaline droplets and fatty degeneration of the renal proximal tubular epithelium were observed in females in the 250 mg/kg bw/day group. The PAS-negative hyaline droplets in the proximal tubules of the kidneys observed in males were confirmed by immunostaining to be alpha2u-globulin deposits, and a specific so-called alpha2u-globulin nephropathy was confirmed in male rats. The PAS stain-positive hyaline droplets observed in the kidneys of females in the 250 mg/kg bw/day group, on the other hand, were observed to show signs of reabsorption of a protein other than alpha2u-globulin, fatty degeneration was also present, and this was determined to be a toxic effect on the kidneys. In addition, regarding the low levels of chlorine observed in the clincical chemistry evaluation in females, individual values were within the reference range of the background data, but the change was thought to involve the effect on the kidneys described above.

Regarding the effect on the thyroid gland, both high absolute weights and relative weights were observed in females in the 250 mg/kg bw/daygroup, and histological tests revealed follicular epithelial hyperplasia in males and females. In addition, high levels of total cholesterol were observed in females in the 250 mg/kg bw/day group as an apparently related change. Regarding the follicular epithelial hyperplasia, since rats lack thyroxine binding globulin, little T3 and T4 is bound to plasma proteins and their half-lives are short. Blood levels are known to be more easily reduced than in humans. The follicular epithelial hyperplasia observed during the present study may have involved a decrease in blood T3 and T4 concentrations caused by induction of drug metabolising enzymes in the liver as described above, stimulation of TSH secretion by a negative feedback mechanism, and hyperplasia of the thyroid follicular epithelium.

Spermatogenetic cycle tests: Significantly low spermatogonia count at stage II-III in the 250 mg/kg bw/day group, but histopathological tests found no changes in the testes. 
Regarding the females observed in the 250 mg/kg bw/day group whose pups all died during the nursing period, no noteworthy changes were observed compared with other animals whose nursing went smoothly.
Regarding the one pair each in the control group and 10 mg/kg bw/day group and two pairs in the 250 mg/kg bw/day group in which gestation was not achieved: atrophy of the seminiferous tubules, interstitial cell hyperplasia in the testes and a decrease in spermatozoa in the ducts of the epididymides in one animal in the 250 mg/kg bw/day group revealed to have testes and epididymides of reduced size, but no other changes were observed in the reproductive organs or pituitary glands.
For further details on results regarding reproductive and developmental parameters please refer to chapter 7.8 Toxicity to reproduction
Please refer also to Table 24-1 / Table 24-2 / Table 24-3 / Table 25-1 Table 25-2 / Table 26 in the attachement for further details.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects observed at this concentration.
Key result
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
Based on the centrilobular hepatocellular hypertrophy in males and females of the 50 mg/kg bw/day group during repeated administration of 2,5-bis(2,4,4-trimethylpentan-2-yl)benzene-1,4-diol to parent animals, the no observed adverse effect level (NOAEL) for repeated dose toxicity was estimated to be 10 mg/kg bw/day for both males and females. Based on the toxic effects on the liver of male and female animals and on the kidney of female animals the substance is classified for STOT RE, 2, H373 (organs: liver, kidney).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 22th March 1996
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29th July 1996
Deviations:
yes
Remarks:
substance administration only until nursing day 4, no blood samples taken from pups, no determination of serum T4 levels, no counting of nipples / aerolae in male pups
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone
EC Number:
212-990-3
EC Name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone
Cas Number:
903-19-5
Molecular formula:
C22H38O2
IUPAC Name:
2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Tsukuba Breeding Centre (955 Kamibayashi, Ishioka-shi, Ibaraki-ken, Japan)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 weeks
- Weight at study initiation: Males: 390 g (mean) / 351 - 427 g (range); Females: 234 g (mean); 212 - 253 g (range)
- Fasting period before study: No
- Housing: In individual stainless steel mesh cages [260W x 380D x 180H (mm)] arranged on five-tier stainless steel racks; females that achieved copulation were housed in polycarbonate cages [265W x 426D x 200H (mm)] containing nesting material (white flake, Charles River Laboratories Japan) on day 0 of gestation, and their offspring were allowed to share the cage after delivery
- Diet: Solid feed lab MR stock, Nosan Corporation; ad libitum
- Water: Sterilised tap water filtered through a cartridge filter with a pore size of 1 µm and then irradiated with ultraviolet rays; ad libitum
- Acclimation period:12 days (including quarantine and observation of the oestrus cycle)

DETAILS OF FOOD AND WATER QUALITY:
The results of the analyses of contaminants in the feed and nesting material were all confirmed to be within the permissible range at the laboratory set with reference to standards such as the “Limits on Contaminants in Feed and Solvents under the United States Environmental Protection Agency Toxic Substances Control Act" (1979), and drinking water was confirmed to conform to the water quality standards under the Water Works Law.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.6 - 23.0
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:

- VEHICLE
- Concentration in vehicle: 10 mg/kg bw/day group: 2 mg/mL, 50 mg/kg bw/day group: 10 mg/mL, and for 250 mg/kg bw/day group: 50 mg/mL
- Amount of vehicle: 5 mL administration solution/kg bw
- Lot/batch no: OG-17 (Kozakai Pharmaceutical Co. Ltd)

The administration solutions prepared were subdivided into amounts for 1 day and stored tightly stoppered and protected from light in a cool place (2 - 6°C) until use. Since the test substance was confirmed to be stable after storage for 7 days in cool place and protected from light and for 1 day at room temperature and protected from light, it was used within 8 days of preparation. The first and last administration solutions to be prepared were analysed and were confirmed to have been prepared at the designated concentrations.
Details on mating procedure:
- M/F ratio per cage: 1 / 1
- Length of cohabitation: Until copulation was confirmed or up to a maximum of 2 weeks
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): In polycarbonate cages [265W x 426D x 200H (mm)] containing nesting material (white flake, Charles River Laboratories Japan) on day 0 of gestation, and their offspring were allowed to share the cage after delivery
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
To confirm that the test substance administration solutions had been prepared at the designated concentrations, the first and last prepared solutions of 2,5-bis(2,4,4-trimethylpentan2-yl)benzene-1,4-diol in olive oil prepared (for 10 mg/kg bw/day group: 2 mg/mL, for 50 mg/kg bw/day group: 10 mg/mL, and for 250 mg/kg bw/day group: 50 mg/mL) were each analysed three times. The results confirmed that the test substance administration solutions had been prepared at the designated concentrations.
Duration of treatment / exposure:
Duration of administration was from 14 days prior to the start of mating for both males and females and for 42 days in the case of males. In the case of females, administration continued during mating and gestation and until day 4 of nursing after delivery, with the duration ranging from 42 to 46 days. In the female recovery group, the duration of administration was 42 days as with the males.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 12 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Test substance groups: 12 animals/dose
Control group males: 12 animals (for the recovery group: 5 animals each were randomly selected from the control group and the maximum dose group on day 42 of administration)
Control group females: 12 animals; in addition: two satellite groups consisting of a control group and a recovery group (not mated) with five females in each group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a dose setting study, 0 (solvent only), 30, 100, 300 and 1000 mg/kg bw/day of the substance was repeatedly orally administered for 14 days to four male and four female rats per group, and observation of general condition, measurement of body weight and feed intake, urinalysis, haematological and blood biochemical tests, necropsy and organ weight measurement were performed. The results showed that in the 100 mg/kg bw/day and higher groups, low haematocrit was observed in males and females, low absolute weight of prostate was observed in males, and low haemoglobin levels and high absolute and relative weight of liver were observed in females. In addition, in the 300 mg/kg bw/day and higher groups, loose stools and high platelet count were observed in males and females, high activated partial thromboplastin time and ALT, low absolute and relative weight of prostate were observed in males, and increased feed intake and low red blood count were observed in females. In addition to these changes, in the 1000 mg/kg bw/day group, bIotted fur in the lower abdomen with urine, increased salivation and feed intake, dilation of the caecum and low chlorine were observed in males and females, low levels of neutral lipids, low relative weight of heart and high relative weight of liver were observed in males, and high γ-GTP and low urine pH were observed in females. On the basis of these results, 250 mg/kg bw/day, a concentration at which repeated dose toxicity was definitely expected to occur, was set as the maximum dose in the present study, and 10 mg/kg bw/day, a concentration at which no toxic effects were expected to be observed, was set as the minimum dose. A dose of 50 mg/kg bw/day falling between these two doses was also set, making three doses in total.

- Post-exposure recovery period in satellite groups: 14 days after the end of administration.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were for mortality / morbidity and to examine their behaviour twice a day (in the morning and afternoon) from the start of administration until the date of necropsy. Gestation, delivery and nursing status were examined carefully.
- Cage side observations checked: Mortality, behaviour, gestation / delivery and nursing status

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
On the day prior to the start of administration and then once a week, animals were examined upon removal from the cage and outside the cage in an aluminium open field (370W x 560D x 40 H mm) for reactivity on removal from the cage, reactivity on handling, muscle tone (relaxed to rigid), skin (colour), fur, piloerection, eye discharge, palpebral closure, exophthalmos, lachrymation, smudge around mouth/nose (dirt), salivation, soiled fur in the lower abdomen with urine, soiled fur around anus with faeces, vocalisation, breathing, body position, convulsion, tremor, exploration (arousal), alertness, locomotor activity (activity), walk (wobbly), abnormal behaviour (self-biting, walking backwards etc.), stereotypy (excessive grooming, repeated turning movement etc.), failure of consciousness (confusion, catalepsy, coma), limb tone, urination and defecation.

BODY WEIGHT: Yes
- Time schedule for examinations:
In males on administration day 1 (test substance administration start date, immediately prior to test substance administration), 7, 14, 21, 28, 35 and 42, the necropsy date and in the case of the recovery group, also on day 49 (recovery day 7) and 56 (recovery day 14) and the necropsy date. In females, body weight was measured on administration day 1, 7 and 14, gestation day 0, 7, 14 and 20, lactation day 0 and 4, and the necropsy date. In the female recovery groups set up as satellite groups, mating was not performed and body weight was therefore measured on the same days as in males.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Yes; the amount of feed consumed in each cage over the 24 h until the following day was measured on the body weight measurement days. However, the final feed intake measurement day was administration day 41 in males, lactation day 3 in females and recovery day 13 in the recovery groups.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, but as g food/rat/day
- 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 (if drinking water study): No

OTHER: Haematology, clinical chemistry, urinalysis, neurobehavioural examinations; please refer to chapter Repeated dose toxicity for details
Oestrous cyclicity (parental animals):
In females, following the quarantine and acclimatisation period, Giemsa-stained vaginal smears were prepared from the administration start date until confirmation of copulation and examined by microscope to assess the stage of the oestrus cycle. In addition, the oestrus cycle was calculated based on the assessment of oestrus cycle from the administration start date until the mating start date. One oestrus cycle was treated as the time from the day on which signs of oestrus were observed until the day prior to observation of signs of the next oestrus. If signs of oestrus were continuous, calculations were made from the day on which the first signs of oestrus were observed.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight / epididymis weight of all males in each group, spermatogenetic cycle inspections in males of the control and 250 mg/kg group
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number (total number of dead and live pups) and sex (anogenital distance) of pups, body surface abnormalities including within the oral cavity, checks of general condition and for deaths were made every day. Weight of the males and females in each litter of surviving pups was measured on lactatoin day 0 (birth date) and 4 (sacrifice date), mean weight per animal was calculated

GROSS EXAMINATION OF DEAD PUPS: Yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males for panned sacrifice were bled out under ether anaesthesia on the day following administration day 42
- Maternal animals: Females for planned sacrifice were bled out under ether anaesthesia on nursing day 5
- Recovery groups: Animals for planned sacrifice were bled out under ether anaesthesia the day following recovery day 14

GROSS NECROPSY
Following sacrifice, the surface of the body, mucous membranes at the incision and internal organs were examined macroscopically. In addition, the liver, kidneys, adrenal glands, thymus, spleen, brain, heart, pituitary gland, thyroid gland, prostate, seminal vesicles, testes and epididymides of all males and females in each group were weighed (absolute weight), and the ratio to body weight (relative weight) was calculated based on body weight on the day of sacrifice. Weights for symmetrical organs were combined weights and the pituitary gland and thyroid gland were weighed after fixing. In females, number of corpora lutea on the ovaries and number of implantations in the uterus were examined and the implantation rate [(number of implantations/number of corpora lutea) x 100] was calculated.

HISTOPATHOLOGY: Yes
Histopathology was performed for all males and females in the control group and 250 mg/kg bw/day group. The following organs were examined: brain (including cerebrum, cerebellum and pons), pituitary gland, thyroid gland (including parathyroid glands), thymus, trachea and lungs (immersed in fixing solution after infusion), stomach, intestines (from the duodenum to the rectum, including Peyer patch), heart, liver, spleen, kidneys, adrenal glands, urinary bladder, testes, epididymides, prostate, seminal vesicles, vagina, uterus, spinal cord (cervical region, thoracic region, lumbar region), sciatic nerve, bone and bone marrow (femur), lymph nodes (submandibular lymph nodes, mesenteric lymph nodes), mammary glands.
The results showed that administration of the test substance had affected the liver, kidneys and thyroid gland in males and females, and the liver, kidneys and thyroid gland were therefore inspected in males and females in other groups, including the recovery groups. The pituitary gland and reproductive organs were also inspected in the pairs of males and females that did not achieve gestation. Macroscopically abnormal sites in any group were inspected in all animals. Inspections were performed by preparation of paraffin embedded sections in accordance with the usual method, H-E staining and microscopy. In addition, PAS staining was performed on the testes of animals for spermatogenetic cycle inspections, and PAS staining and alpha2u-globulin immunostaining was performed to identify deposits in the kidneys.

Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on lactation day 4
- These animals were subjected to postmortem macroscopic examinations as follows:

GROSS NECROPSY
- Major organs in the chest and abdominal region were examined macroscopically around the time of death in the case of animals found dead and after euthanisation by inhalation of carbon dioxide in other cases

Statistics:
Mean values and frequencies obtained were used to test for significant differences from the control group (ratio of not more than 5%):
(1) Parametric data: Bartlett variance test for comparisons of multiple groups. If variance was uniform: analysis of variance with a one-dimensional layout. If the results revealed a significant difference, comparative tests of each group against the control group by the Dunnett test method. If the variance was not uniform, test method using non-parametric data (body weight, body weight increase, feed intake, organ weights, number of corpora lutea, number of implantations, duration of achievement of copulation, duration of gestation, number of pups delivered, number of live pups, number of dead pups). An F test was used for comparisons between two groups. If the results revealed uniform variance, Student t test, and if the results revealed non-uniform variance, an Aspin-Welch t test.
(2) Non-parametric data: Kruskal-Wallis variance test for comparisons of multiple groups. If the results revealed a significant difference, the control group and each group were compared by the Dunnett test method (spermatogenetic cycle tests, number of implantations, viability, delivery rate, oestrus cycle, live birth survival rate, sex ratio). Mann-Whitney U test for comparisons between two groups (histopathological test findings).
(3) Categorical data
Fisher's exact test (incidence rate, copulation rate, conception rate and birth rate for animals with abnormalities in detailed clinical observations, observation of general condition and pathological tests). Mann-Whitney U test for graded data from histopathological test findings.
Reproductive indices:
- Copulation rate: [(number of copulated animals/number of animals kept together) x 100]
- Conception rate (%): [(number of conceiving females/number of copulated females) x 100]
- Duration of gestation: (number of days from gestation day 9 until the day of confirmation of completion of delivery)
- Birth rate (%): [number of females delivering live offspring/number of gestating females) x 100]
Offspring viability indices:
- Delivery rate: [(total number of pups born/implantation rate) x 100]
- Sex ratio of pups: (number of male pups born/total number of pups born); on lactation day 0
- Sex ratio of live pups: (number of live male pups born/total number of live pups born); on lactation day 0 and 4
- Viability (%): [(number of live pups upon confirmation of delivery/total number of pups born) x 100]
- Survival rates (%): [(number of surviving pups on nursing day 4/number of live pups upon confirmation of delivery) x 100]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the test substance administration period: Mild loose stools in 12/12 males and 9/12 females in the 250 mg/kg bw/day group and 5/5 females in the 250 mg/kg bw/day recovery group. Loose stools were observed transiently after test substance administration and the animals recovered prior to test substance administration the following day.
Mild soiled fur in the lower abdomen with urine was observed in 11/12 males and 7/12 females in the 250 mg/kg bw/day group and 5/5 females in the 250 mg/kg bw/day recovery group. In addition, salivation was observed in 11/12 males and 8/12 females in the 250 mg/kg bw/day group and 3/5 females in the 250 mg/kg bw/day recovery group. Salivation was observed in 1/12 females in the 50 mg/kg bw/day group. The salivation was transient and involved mild wetness around the mouth immediately after test substance administration. No changes in general condition were observed in the 10 mg/kg group. In addition, during the recovery period, no changes in general condition were observed in any group.
Please refer also to chapter Repeated dose toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
One female in the 250 mg/kg group died on gestation day 21. Another female of the same group was moribund and therefore sacrificed on gestation day 22 (administration day 39).
Dead animal: A tendency for suppression of body weight increase was observed during gestation as this female had not eaten when food intake was measured on gestation day 20 (test substance administration day 38); the animal was emaciated, and adopted the recumbent position and died the following day (gestation day 21).
Moribund animal: A similar tendency for suppression of body weight increase was observed during gestation in another female. Also this female had not eaten when food intake was measured on gestation day 20 (test susbstance administration day 38) and reddish lachrymation was observed from gestation day 21 (test substance administration day 38). The following day, the animal was observed in the prone position with low skin temperature, and moribund sacrifice was therefore performed.

Histopathological tests on dead animals revealed a high incidence of the changes due to administration of the test substance described under Histopathology, particularly changes in the kidneys, and death was inferred to have occurred as a result of this combined with stress from the end of gestation until the start of delivery.
Please refer also to chapter Repeated dose toxicity.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the test substance administration period: Significantly decreased body weight gain in males and significantly higher body weight gain during the period prior to mating in females in the 250 mg/kg bw/day group. Significantly higher body weight (compared to controls) on administration day 21 and 28 and body weightgain during the test substance administration period were observed in the female recovery group. No significant changes were observed in males or females in the 10 and 50 mg/kg bw/day groups.
During the recovery period: Significantly low body weight in males for the recovery group from the end of administration and throughout the recovery period, but no changes in body weight gain during the recovery period. However, the body weights of the animals selected for the recovery period were coincidentally lower than those for animals sacrificed at the end of the administration period. In the female recovery group, significantly low body weight gain was observed during the recovery period, and body weight, which had been rather higher than in the control group during the test substance administration period, now approached that in the control group, and there was no longer any difference in recovery days 7-14.
Please refer also to chapter Repeated dose toxicity.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
During the test substance administration period: In the 250 mg/kg bw/day group, significantly higher food intake in males on test substance administration day 14, 28, 35 and 41 and in females on test substance administration day 14 and gestation day 14, and lower food intake were observed in females on nursing day 3. In the female recovery group, significantly higher food intake was observed in the 250 mg/kg bw/day group on administration day 14. Although significantly low levels of food intake were observed in males in the 10 mg/kg bw/day group on administration day 35, these appeared to be incidental changes. No significant changes were observed in males or females in the 50 mg/kg bw/day groups.
During the recovery period: Significantly higher food intake was observed in males of the 250 mg/kg bw/day group on recovery day 0. No significant changes were observed in the female recovery group
Please refer also to chapter Repeated dose toxicity.
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):
Tests on animals sacrificed at the end of the administration period: significantly low levels of haemoglobin and MCH in males in the 50 and 250 mg/kg bw/day group and significantly low levels for red blood count and haematocrit in males in the 250 mg/kg bw/day group. In females, significantly low levels of haemoglobin were observed in the 50 mg/kg bw/day group, and significantly low levels of MCH and a tendency for low levels of haemoglobin were observed in the 250 mg/kg w/day group. Furthermore, in males, for white blood cell percentages, significantly low levels of neutrophils and significantly high levels of lymphocytes were observed in the 50 mg/kg bw/day group and significantly low levels of neutrophils and a significantly high white blood count were observed in the 250 mg/kg bw/day group. In addition, significantly high platelet count and significantly low prothrombin time were observed in females in the 250 mg/kg bw/day group.
Tests on animals sacrificed at the end of the recovery period revealed a decrease in the amount of variation from the control group and a tendency for recovery, although a significant difference remained in terms of low levels of MCHC and high platelet count in females.
Individual values, however, were all within the reference range of the background data and no changes were observed in haematopoietic tissue or peripheral blood reticulocyte count, and no changes were observed that were suggestive of hyper-destruction of red blood cells in the reticuloendothelial system such as the spleen.
Please refer also to chapter Repeated dose toxicity.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Significantly low levels of calcium in males and significantly high levels of γ-GTP, total protein, albumin and total cholesterol and significantly low levels of creatinine and chlorine in females of the 250 mg/kg bw group. Significantly high levels of calcium were observed in the 10 mg/kg bw/day group, but no dose correlation was observed. No significant changes in any test item were observed in males or females in the 50 mg/kg bw/day group.
Tests on animals sacrificed at the end of the recovery period revealed significantly high levels of total protein, albumin and total cholesterol and significantly low levels of creatinine and chlorine in females, but a decrease in the amount of variation from the control group and a tendency for recovery were observed in all cases in comparison with female animals sacrificed at the end of the test substance administration period. In addition, significantly low levels of alkaline phosphatase and significantly high levels of calcium and inorganic phosphorus were observed in females, but all changes were slight.
Please refer also to chapter Repeated dose toxicity.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Tests during the test substance administration period: significantly low levels of both specific gravity in the 10 mg/kg bw/day group and potassium in the 50 mg/kg bw/day group. Individual values, however, were all within the reference range of the background data from the research institute (specific gravity: mean 1.065 / range 1.042-1.087, potassium: mean 2.81 / range 0.63-4.99 mEq/18 hr) and, in addition, the changes had no dose correlation.They were therefore considered to be incidental. No significant changes were observed in the 250 mg/kg bw/day group. No significant changes were observed in animals during the recovery period.
Please refer also to chapter Repeated dose toxicity.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory function tests: No changes were observed for any test item in any test substance administration group in tests during the test substance administration period and recovery period.
Grip strength and amount of spontaneous exercise: No significant changes in grip strength were observed in measurements on the final test substance administration date. Regarding amount of spontaneous exercise, a significant decrease was observed in 60 min measurements in males in the 10 and 250 mg/kg bw/day groups. In the recovery group, a significant decrease was observed in 60 min measurements in tests on the final test substance administration date in males in the 250 mg/kg bw/day group. No significant changes were observed in 30 min measurements and in the amount of spontaneous exercise in the 50 mg/kg bw/day group. Since all values were within the reference range apart from those for one animal in the 250 mg/kg bw/day group, this was considered to be an incidental change.
Please refer also to chapter Repeated dose toxicity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Among animals sacrificed at the end of the administration period: changes attributable to administration of the test substance in the liver, kidneys and thyroid gland.
- Liver: Centrilobular hepatocellular hypertrophy in 3/12 males and 5/12 females in the 50 mg/kg bw/day group and 5/7 males and 11/12 females in the 250 mg/kg bw/day group (significant difference in number of incidences compared to the control group in females in the 50 mg/kg bw/day group and males and females in the 250 mg/kg bw group). The degree of the change tended to be more severe in the 250 mg/kg group than in the 50 mg/kg group for both males and females. No hepatocellular hypertrophy was observed in the liver amon animals sacrificed at the end of the recovery period.
- Kidneys: PAS stain-negative hyaline droplets in the renal proximal tubular epithelium in all groups, including the control group, but the number increased in the 50 and 250 mg/kg bw/day groups (there was a tendency for enlargement, and a comparison of graded data with the control group revealed a significant difference in the 250 mg/kg bw/day group). These PAS stain-negative hyaline droplets were confirmed to be alpha2u-globulin immunostain-positive. In females: fatty degeneration of the renal proximal tubular epithelium (5/12 animals) and appearance of PAS stain-positive hyaline droplets in the renal proximal tubular epithelium (12/12 animals) in the 250 mg/kg bw/day group (significant difference from the control group). Hyaline droplets in males had recovered to the same level as the control group, the incidence rate of hyaline droplets and fatty degeneration of the renal proximal tubular epithelium in females had decreased to 1/5 and 2/5 animals, respectively, and a tendency for recovery was observed at the end of the recovery period.
- Thyroid gland: Diffuse follicular epithelial cell hyperplasia in 7/7 males and 12/12 females in the 250 mg/kg bw/day group (significant difference from the control group in both males and females). In the 250 mg/kg bw/day group, thyroid follicles had decreased in size, epithelial cells had become cuboidal or cylindrical, and colloids within the follicles tended to decrease. No follicular epithelial cell hyperplasia in the thyroid gland was observed among animals sacrificed at the end of the recovery period.

Regarding the death of one and moribund sacrifice of one female animal in the 250 mg/kg bw/day group: In addition to the observation of the changes in the animals sacrificed at the end of the administration period, severe changes in the kidneys in both animals, formation of hyaline casts and erythrocyte casts in the moribund animal, and thymic cortical atrophy in both animals.

In addition to these changes, fatty degeneration of hepatocytes in the perilobular zone (males: 0, 10, 50, 250 mg/kg bw/day groups, females:10 mg/kg bw/day group) was observed in animals such as those with yellowing of the liver revealed by necropsy to be unrelated to administration of the test substance, and dilation of the renal tubules in the kidneys, interstitial oedema in the prostate, inflammation of the seminal vesicles and dilation of the lumen, calculus in the urinary bladder and inflammation of the transitional epithelium were observed in the animal in the 10 mg/kg bw/day group with renal enlargement, prostatomegaly, distension of the seminal vesicles and distension of the urinary bladder. In addition, microgranuloma (males: 0, 10, 50 mg/kg bw/day groups, females: 0, 50, 250 mg/kg bw/day groups, recovery group males: 250 mg/kg bw/day group, females: 0, 250 mg/kg bw/day groups), focal necrosis (males: 0, 50, 250 mg/kg bw/day groups, females: 50 mg∕kg bw/day group, recovery group males: 0 mg/kg bw/day group) and granuloma that appeared to be a sign of repairs (males: 10 mg/kg bw/day group, females:10, 50, 250 mg/kg bw/day groups) in the liver; so-called eosinophilic bodies with a connection to PAS stain-negative hyaline droplets in the renal proximal tubular epithelium (males: 10 mg/kg bw/day group), basophilic renal tubules (males: 0, 10, 50, 250 mg/kg bw/day groups, females: 10, 50, 250 mg/kg bw/day groups, recovery group males: 0, 250 mg/kg bw/day groups, females: 0 mg/kg bw/day group), cortical lymphocyte infiltration (males: 10, 50 mg/kg bw/day groups, recovery group males: 250 mg/kg bw/day group), cortical fibrosis (males: 0 mg/kg bw/day group, females: 10 mg/kg bw/day group), mineralisation of the medullary tubules (females: 10 mg/kg bw/day group), hyaline casts (males: 0, 10, 50, 250 mg/kg bw/day groups, recovery group females: 0 mg∕kg bw/day group, females: 250 mg/kg bw/day group), solitary cyst (males: 0, 10, 250 mg/kg bw/day groups) and regenerating renal tubules (males:10 mg/kg bw/day group) in the kidneys; foam cell accumulation (males: 0, 250 mg/kg bw/day groups, females: 0 mg/kg bw/day group) and arterial wall mineralisation (males: 0, 250 mg/kg bw/day groups, females: 0, 250 mg/kg bw/day groups) in the lungs; myocardial degeneration and fibrosis (males: 0, 250 mg/kg bw/day groups, females: 0, 250 mg/kg bw/day groups) in the heart; dilation off the gastric fundal gland (males: 0, 250 mg/kg bw/day groups, females: 0, 250 mg/kg bw/day groups) in the glandular stomach; vacuolation of cytoplasm in cortical cells (males: 250 mg/kg bw/day group) and cortical focal fatty metamorphosis (females: 0 mg/kg bw/day group) in the adrenal glands; interstitial lymphocyte infiltration and sperm granuloma (males: 250 mg/kg bw/day group) in the epididymides; interstitial lymphocyte infiltration (males: 250 mg/kg bw/day group) in the prostate; and follicular cysts (females: 250 mg/kg bw/day group) in the ovaries were observed at a low incidence rate, but these changes are all spontaneously occurring lesions in rats, and no increase in incidence rates due to administration of the test substance was observed.

Extramedullary haematopoiesis and brown pigmentation in the spleen were both observed at high incidence rates in males and females, but no difference in the incidence rate or severity of changes was observed between the 250 mg/kg bw/day group and the control group. The changes were therefore deemed to have no relationship to administration of the test substance in any case.

Regarding the centrilobular hepatocellular hypertrophy, this was not associated with regressive changes in hepatocytes, blood biochemical tests produced no findings suggestive of liver disorders, and high levels of total cholesterol were observed in females and an increase in cholesterol has been reported to be correlated to an increase in drug metabolising enzymes, which suggested that these findings were the result of adaptation reflecting induction of drug metabolising enzymes by administration of the test substance.

Regarding the effect on the kidneys, both high absolute weights and relative weights were observed in males in the 50 and 250 mg/kg bw/day groups, and an increase in PAS stain-negative hyaline droplets in the renal proximal tubular epithelium was observed as a corresponding histological change. In addition, high relative weight of the kidneys was observed, and appearance of PAS stain-positive hyaline droplets and fatty degeneration of the renal proximal tubular epithelium were observed in females in the 250 mg/kg bw/day group. The PAS-negative hyaline droplets in the proximal tubules of the kidneys observed in males were confirmed by immunostaining to be alpha2u-globulin deposits, and a specific so-called alpha2u-globulin nephropathy was confirmed in male rats. The PAS stain-positive hyaline droplets observed in the kidneys of females in the 250 mg/kg bw/day group, on the other hand, were observed to show signs of reabsorption of a protein other than alpha2u-globulin, fatty degeneration was also present, and this was determined to be a toxic effect on the kidneys. In addition, regarding the low levels of chlorine observed in the clincical chemistry evaluation in females, individual values were within the reference range of the background data, but the change was thought to involve the effect on the kidneys described above.

Regarding the effect on the thyroid gland, both high absolute weights and relative weights were observed in females in the 250 mg/kg bw/daygroup, and histological tests revealed follicular epithelial hyperplasia in males and females. In addition, high levels of total cholesterol were observed in females in the 250 mg/kg bw/day group as an apparently related change. Regarding the follicular epithelial hyperplasia, since rats lack thyroxine binding globulin, little T3 and T4 is bound to plasma proteins and their half-lives are short. Blood levels are known to be more easily reduced than in humans. The follicular epithelial hyperplasia observed during the present study may have involved a decrease in blood T3 and T4 concentrations caused by induction of drug metabolising enzymes in the liver as described above, stimulation of TSH secretion by a negative feedback mechanism, and hyperplasia of the thyroid follicular epithelium.

Spermatogenetic cycle tests: Significantly low spermatogonia count at stage II-III in the 250 mg/kg bw/day group, but histopathological tests found no changes in the testes. 
Regarding the females observed in the 250 mg/kg bw/day group whose pups all died during the nursing period, no noteworthy changes were observed compared with other animals whose nursing went smoothly.
Regarding the one pair each in the control group and 10 mg/kg bw/day group and two pairs in the 250 mg/kg bw/day group in which gestation was not achieved: atrophy of the seminiferous tubules, interstitial cell hyperplasia in the testes and a decrease in spermatozoa in the ducts of the epididymides in one animal in the 250 mg/kg bw/day group revealed to have testes and epididymides of reduced size, but no other changes were observed in the reproductive organs or pituitary glands.
For further details on results regarding reproductive and developmental parameters please refer to chapter 7.8 Toxicity to reproduction
Please refer also to chapter Repeated dose toxicity.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No significant difference in mean oestrus cycle was observed between the test substance administration groups and the control group.
During the inspection period, sustained signs of anoestrus were observed transiently from inspection day 1-6 in 1/17 animals in the control group and from inspection day 13-15 in1/17 animals in the 250 mg/kg bw/day group, but copulation was subsequently confirmed after mating, and normal gestation, delivery and nursing were observed. Data on the one animal in the control group was only available for one oestrus cycle during the inspection period, so it was excluded from calculations of mean oestrus cycle for the group.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Spermatogenetic cycle tests revealed significantly low spermatogonia count at stage II-III in males of the 250 mg/kg bw/day group, but since the change was very slight, there were no significant changes at any other stages, and histopathological tests found no changes in the testes, it appeared unlikely that this was caused by administration of the test substance.
Please refer also to Table 26 in the attachement for further details.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in number of days required until copulation, copulation rate and conception rate were observed in any of the test substance administration groups.
No animals failed to achieve copulation in any group, and failure to achieve gestation was observed in one animal in the control group, one animal in the 10 mg/kg bw/day group and two animals in the 250 mg/kg bw group.
No significant changes in number of corpora lutea, number of implantations and implantation rate were observed in any of the test substance administration groups.
No significant changes in duration of gestation and birth rate were observed in any of the test substance administration groups.
One animal in the 10 mg/kg bw/day group and two animals in the 250 mg/kg bw/day group failed to achieve gestation. Atrophy of the seminiferous tubules and interstitial cell hyperplasia in the testes and a decrease in spermatozoa in the ducts of the epididymides were observed in the male in one pair in the 250 mg/kg bw/day group, and this appeared to be the cause of infertility, but no findings to explain the failure to achieve gestation were observed in any other animals. However, since also one animal in the control group failed to achieve gestation, this was determined to be unconnected to administration of the test substance. In addition, the changes to the testes and epididymides observed only occurred in one animal and were determined to be spontaneously occurring.
One female in the 250 mg/kg bw/day group died prior to delivery on gestation day 21, and another female became a moribund sacrifice on gestation day 22. Twelve macerated foetuses were observed in the uterus of the dead animal, and 14 dead foetuses were observed in the uterus of the moribund animal. In addition, another female in the same group was observed to have delivered 14/15 pups dead or to have cannibalised them on the day of completion of delivery (nursing day 0). Although the animal displayed nursing behaviour towards the one surviving pup, it was cannibalised on nursing day 1. Nothing out of the ordinary was observed in the delivery and nursing status of other dams in the 250 mg/kg bw/day group or in other test substance administration groups including the control group. One female in the 10 mg/kg bw/day group displayed normal delivery and nursing behaviour, but milk spots visible through the abdominal wall in offspring were small, and milk secretion was therefore determined to be low.
Please refer also to Table 28 in the attachement for further details.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects observed at this dose level.
Dose descriptor:
LOAEL
Remarks:
systemic toxicty
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
(reproduction)
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effect observed at this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
(reproduction)
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: Effects secondary to maternal toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities were observed in the general condition of the pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
No significant differences were observed in total number of births, delivery rate and live birth index. Low total number of live offspring, number of male live offspring and viability index were observed onlactation day 4 in the 250 mg/kg bw/day group.
One female dam in the 250 mg/kg bw/day group was observed to have delivered 14/15 pups dead or to have cannibalised them on the day of completion of delivery (lactation day 0). Although the animal displayed nursing behaviour towards the one surviving pup, it was cannibalised on lactation day 1.
Please refer also to Table 28 in the attachement for further details.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Low body weight in males and females in the 10 mg/kg bw/day group on lactation day 4. Low body weight on lactation days 0 and 4 in the 250 mg/kg bw/day group. No significant changes were observed in the 50 mg/kg bw/day group. The significantly low body weight in males and females observed in the 10 mg/kg bw/day group was due solely to the low body weight of the offspring of the female animal in the 10 mg/kg bw/day group with low milk secretion, which reduced the body weight of the group as a whole. No significant difference was observed when this case was excluded from the statistical treatment. That is also shown when comparing the individual values per litter as they were all within the reference range of the demographic data from the research institute (male: mean 11.6 g, range 8.7-14.5 g, female: mean 11.3 g, range 8.5-14.1 g) apart from the male and female offspring obtained from female animal with low milk production (male: mean 6.3 g, female: mean 6.5 g). It therefore appeared unlikely that administration of the test substance had any effect on this.
Please refer also to Table 28 in the attachement for further details.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No significant differences were observed in the sex ratio per litter.
Please refer also to Table 28 in the attachement for further details.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
One pup in the 10 mg/kg bw/day group was observed to have kidneys of reduced size (incidence rate 0.5%). As there was no dose-correlated trend and it was only observed in 1 pup, the changes were considered to be within the spontaneously occurring range. No pups with organ abnormalities or variations were observed in the 250 mg/kg bw/day group.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormalities in external appearance in any of the test substance administration group. Cervical remnant of the thymus was observed as an organ lesion in one pup in the control group (incidence rate 0.6%), two pups in the 10 mg/kg bw/day group (incidence rate 1.0%), and one pup in the 50 mg/kg bw/day group (incidence rate 0.7%), but the incidence rate was low in every case and there was no dose-correlated trend for increase, so the changes were considered to be within the spontaneously occurring range.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

It appears possible that administration of the test substance could have a suppressing effect on the growth and development of foetuses in the uterus, and this was inferred to have caused a decrease in the number of live pups and mild underdevelopment after birth.

Effect levels (F1)

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Key result
Dose descriptor:
NOAEL
Remarks:
(development)
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects at this dose level
Key result
Dose descriptor:
LOAEL
Remarks:
(development)
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Remarks on result:
other: Secondary effects due to maternal toxicity

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
other: reduced viability, reduced body weight and body weight gain
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the centrilobular hepatocellular hypertrophy in males and females of the 50 mg/kg bw/day group during repeated administration of 2,5-bis(2,4,4-trimethylpentan-2-yl)benzene-1,4-diol to parent animals, the no observed adverse effect level (NOAEL) for repeated dose toxicity was estimated to be 10 mg/kg bw/day for both males and females. Further, since low number of live offspring on lactation day 4, a reduced vialbiltiy index and a reduced body weight on lactation days 0 and 4 were observed in pups of the 250 mg/kg bw/day group, the NOAEL for toxicity to reproduction was estimated to be 50 mg/kg bw/day. This is higher than the estimated NOAEL for maternal toxicity of 10 mg/kg bw/day and the observed effects on reproduction are therefore considered to be secondary effects caused by the maternal toxicity.

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