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EC number: 223-773-8 | CAS number: 4066-02-8
The present GLP study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development, presented in the respective section) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test" (adopted 22 March 1996).
This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to seven weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).
Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.
Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.
During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.
Extensive functional observations, hematology and blood chemistry evaluations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from the control, low and intermediate dose groups on Day 4 post partum. Due to a high number of total litter losses evident in the high dosage group, four females with litters and four females with a total litter loss were investigated to allow comparison between the two sets of females which were in a different physiological state on Day 4 post partum.
Adult males were terminated on Days 43 or 44, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.
Mortality: One female treated with 300 mg/kg bw/day was found dead on Day 3 post partum and one female treated with 1000 mg/kg bw/day was killed in extremis due to difficulty during parturition. There were no further unscheduled deaths.
Clinical Observations: There were no clinical signs of toxicity for males or the surviving females treated with 100, 300 or 1000 mg/kg bw/day.
Behavioral Assessment: There were no treatment-related effects detected.
Functional Performance Tests: There were no treatment-related changes in the functional performance.
Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.
Body Weight: There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day.
Food Consumption: There were no adverse effects on food consumption or food conversion efficiency for males throughout the study or for females during pre-mating, gestation or lactation at dose levels of 100, 300 or 1000 mg/kg bw/day.
Water Consumption: Water intake was measured gravimetrically for each cage throughout the study with the exception of the mating period. An increase in water consumption was detected for animals of either sex from all treatment groups for the majority of the treatment period.
Hematology: Treated males from all groups showed a statistically significant reduction in hemoglobin, hematocrit and erythrocyte count; a dose relationship was present in all cases. With the exception of one male from the high dose group, all individual values were within the background control ranges. Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in neutrophil count when compared to controls attaining statistical significance. No toxicologically significant effects were detected in females treated with 300 or 100 mg/kg bw/day.
Blood Chemistry: Males from all dose groups showed a statistically significant increase in cholesterol when compared to controls but without a dose relationship. At 1000 or 300 mg/kg bw/day females showed a statsistically significant increase in total protein when compared to controls with a dose related response.
Early decedents: One female treated with 300 mg/kg bw/day that was found dead on Day 3post partumshowed an enlarged liver and spleen and a fluid filled thoracic cavity. A further female treated with 1000 mg/kg bw/day that was killedin extremisduring parturition had black coloured contents in the caecum, colon, duodenum, ileum and jejunum. The brain, liver, mammary gland and pancreas were also pale.
Scheduled necropsy : At 1000 mg/kg bw/day seven males had enlarged livers of with one of these appearing mottled. Three females from this dose group and two females treated with 300 mg/kg bw/day were also observed with enlarged spleens. There were higher instances of small, weak and no milk in the stomach in offspring from litters of the high and intermediate dose groups.
Liver: Animals of either sex from all treatment groups showed a dose related increase in liver weight both absolute and relative to terminal body weight. Statistical significance was achieved excluding females treated with 100 mg/kg bw/day.
Kidney: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increase in kidney weights both absolute and relative to terminal body weight with a dose related response.
Thymus: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in thymus weights both absolute and relative to terminal body weight with a dose related response.
Thyroid/Parathyroid: Animals of either sex from all treatment groups showed statistically significant increases in thyroid/parathyroid weight both absolute and relative to terminal body weight. Females receiving 100 mg/kg bw/day did not attain statistical significance.
Spleen: Females from all treatment groups showed an increase in spleen weights both absolute and relative to terminal body weight.
Liver: Centrilobular hepatocellular hypertrophy, moderate or mild was present in all males treated with 1000 mg/kg bw/day and in eight females from this treatment group. Both sexes treated with 100 or 300 mg/kg bw/day showed from minimal to mild centrilobular hepatocellular hypertrophy and showed a dose dependant trend. Diffuse vacuolation (fat type), minimal or mild was present in 4/8 males and 5/9 females at 1000 mg/kg bw/day with periportal vacuolation being evident in another female. It was also present at a minimal level in both sexes at 100 or 300 mg/kg bw/day. Hematopoiesis was present in 3/9 1000 mg/kg bw/day females; 4/5 100 mg/kg bw/day females and 4/6 300 mg/kg bw/day females. The severity of hematopoiesis was increased at 300 mg/kg bw/day compared to 100 mg/kg bw/day.
Thyroid Gland: Mild follicular cell hypertrophy was present in all males and 8/9 females treated with 1000 mg/kg bw/day. At 100 or 300 mg/kg bw/day it was present at minimal or mild severity in males and females.
Pituitary: There was a minor increase in the amount of vacuolation in pars distalis (anterior lobe) of the pituitary gland in males at 1000 mg/kg bw/day. At 100 or 300 mg/kg bw/day the incidence was comparable to controls.
Spleen: Increased hematopoiesis was present in 7/9 females at 1000 mg/kg bw/day (mild to marked); 6/6 females at 300 mg/kg bw/day females (mild to marked) and in 4/5 females at 100 mg/kg bw/day (mild or moderate).
Thymus : There was a reduction in the amount of involution noted in treated females from all dose groups when compared to control animals. Involution/atrophy is seen as a normal background change in pregnant and lactating animals therefore not generally recorded thus this may be linked to lower numbers of animals producing and feeding litters.
Lungs: There was a minor increase in the incidence of alveolar macrophage accumulation (focal) in the lungs of females at 1000 mg/kg bw/day only.
Kidneys: Basophilic tubules minimal or mild were present in 3/9 1000 mg/kg bw/day females with one also having tubular dilation. This was present at a minimal level in 1/5 100 mg/kg bw/day and 1/6 300 mg/kg bw/day females and at this level is not considered to be related to treatment. Moderate nephropathy was present in one 300 mg/kg bw/day female and although this is unusual in animals of this age it is not unknown to occur as a background change.
Non-Pregnancy: A number of females were not pregnant after mating (40, 42, 44 and 47 from 100 mg/kg bw/day; 67 from 300 mg/kg bw/day and 95 from 1000 mg/kg bw/day). Female 42 was paired with male 30 which had severe testicular atrophy and this would have prevented pregnancy. All other females had no obvious reason for failure of mating and appeared to be cycling normally.
Total Litter Loss: Several animals in the study showed total litter loss. Histologically other than the general findings indicated above the only notable differences between control and treated females were fewer animals lactating (linked to lack of live pups) and 3 without implantation sites on the section considered to be linked to the reduction noted. There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.
Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity and reproductive toxicity was considered to be 100 mg/kg bw/day, based on the severity and adaptive response nature of the findings rather than an absence of findings.
The log Pow of 2,2'-methylenebis[6-cyclohexyl-p-cresol] was determined using reverse phase HPLC according to OECD guideline 117, with acceptable restrictions, to be 6.3.
The water solubility of 2,2'-methylenebis[6-cyclohexyl-p-cresol] was determined to be 0.034 mg/L at 20°C using the shake flask method according to OECD guideline 105 (no GLP).
In a primary dermal irritation study similar to OECD 404, two New Zealand White rabbits were dermally exposed to 500 mg of unchanged Phenol, 2,2'-methylenebis-(6-cyclohexyl-4-methyl) for 24 hours to the inner side of the ear. Animals then were observed for 7 days. In this study, Phenol, 2,2'-methylenebis-(6-cyclohexyl-4-methyl) is not a dermal irritant.
In the study according to OECD 429 under GLP the test item formulated in acetone/olive oil (4+1, v/v) was assessed for its possible skin sensitising potential.
For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.
The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.
In this study Stimulation Indices (S.I.) of 1.32, 1.10, and 1.20 were determined with the test item at concentrations of 10, 25, and 50% (w/w) in acetone/olive oil (4+1, v/v), respectively.
The test item was not a skin sensitiser under the test conditions of this study.
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