Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to REACH Annex VIII column 1 (8.6.1), the following study for repeated dose toxicity is required: Short-term repeated dose toxicity study (28 days), one species, male and female, most appropriate route of administration, having regard to the likely route of human exposure. Alternatively, this endpoint can be covered scientifically reasonable with an OECD 422 study, as the exposure duration is prolonged and the animals are due to their pregnancy potentially more susceptible. There is a suitable Klimisch 1 GLP OECD 422 guideline study available, assessing the toxicological properties of Phenol, 2,2'-methylenebis-(6-cyclohexyl-4-methyl) after oral gavage over more than 28 days, i.e. approx. 6-7 weeks. In general, the oral route is the most suitable one to assess systemic effects in humans, which is the main aim of this endpoint. The dermal or inhalative route is only scientifically relevant in case of considerable exposure, any route-specific toxicological mode of action or local effects, whereas sufficient information on the latter can be gained via irritation tests (REACH No. 8.1. or 8.2). According to REACH Annex VIII column 2 (8.6.1), the appropriate route shall be chosen on the following basis: Testing by the dermal route is appropriate if: (1) inhalation of the substance is unlikely; and (2) skin contact in production and/or use is likely; and (3) the physicochemical and toxicological properties suggest potential for a significant rate of absorption through the skin.
Although inhalation of 2,2’-methylenebis[6-cyclohexyl-p-cresol] to any toxicologically relevant amount is unlikely, the latter conditions do not apply. Due to the inherent rather low toxicity of 2,2’-methylenebis[6-cyclohexyl-p-cresol], a high exposure to the substance would be required, which is excluded due to the taken workplace safety precautions. Even if exposure arises, exposition of workers would be magnitudes below any possible dose levels which could reveal any effects in animal models. Further, the physicochemical and toxicological properties do not suggest potential for a significant rate of absorption through the skin. Skin absorption is influenced by several factors, i.a.:
- Molecular weight: Less than 100 favors dermal uptake. Above 500 the molecule may be too large. With a molecular weight of 392.57 g/mol, absorption is in theory possible, but not highly favoured.
- LogPow: for substances having a logPow above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and will limit absorption across the skin. Uptake into the stratum corneum itself may be slow. Since the substance has a logPow of 6.3, dermal absorption may practically not occur.
- Water solubility: The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore, if the water solubility is below 1 mg/L, dermal uptake is likely to be low. As stated above, log Pow is 6.3, and additionally, the substance was found to be insoluble in water, i.e. having a water solubility of 34 µg/l. Also here, dermal absorption may practically not occur due to the fact that the substance is insoluble in water.
- Skin irritation / corrosion: If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration. 2,2’-methylenebis[6-cyclohexyl-p-cresol] is not classified as irritant to the skin. Also no pathological changes on the treated skin of mice were noted in the available LLNA, no symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. Therefore, no additional penetration enhancement must be considered. In consequence, the available OECD 422 study (oral exposure route) is sufficient to cover this endpoint, no repeated dose testing via dermal route needs to be performed and can consequently be waived due to animal welfare.
Cross-referenceopen allclose all
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-11-20 - 2016-07-11 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Remarks:
Range-finding study
Related information:
Composition 1
Reason / purpose:
reference to same study
Related information:
Composition 1
Reason / purpose:
reference to other study
Remarks:
supporting study
Related information:
Composition 1
Reason / purpose:
reference to other study
Remarks:
supporting study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.422: "Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test" (adopted 22 March 1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008
Version / remarks:
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).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
Species:
rat
Strain:
Wistar
Remarks:
Han™:RccHan™:WIST strain
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: At the start of treatment the males weighed 310 to 357g, the females weighed 190 to 224g.
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during the final week of gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. The animals were housed in a single air-conditioned room within the Rodent Facility.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.), ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage, ad libitum.
- Acclimation period: On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed.

DETAILS OF FOOD AND WATER QUALITY:
The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES:
From: 28 January 2016 (first day of treatment)
To: 14 March 2016 (final day of necropsy)
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 25, 75, 250 mg/ml
- Amount of vehicle (if gavage): 4 ml / kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Item Preparation and Analysis
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by laboratories' Analytical Services. Results show the formulations to be stable for up to 21 days. Formulations were initially prepared weekly and following further stability fortnightly and stored at approximately 4 °C in the dark.
Samples of test item formulation were taken and analyzed on three occasions for concentration of the test item at Analytical Services. The results indicate that the prepared formulations were within 10% of the nominal concentration.
Duration of treatment / exposure:
43 or 44 days (males)
until day 5 post partum (females)
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 / sex / dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on preliminary range-finding study, in accordance with OECD TG 422
- Rationale for animal assignment (if not random): The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week and at weekends (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was measured daily throughout the study (with the exception of the pairing phase).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological and blood chemical investigations were performed on five males selected from each test and control group prior to termination (Day 42 for males) and five selected females from the control, low or intermediate dose groups and four females with litters and four females that showed a total litter loss from the high dose group. (Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: No
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb) Erythrocyte count (RBC) Hematocrit (Hct)
Erythrocyte indices:
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic):
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by 'Innovin' and Activated partial thromboplastin time (APTT) was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: See above
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations / Dose groups that were examined: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males from each dose level and five selected females treated with 100 and 300 mg/kg bw/day, prior to termination, together with an assessment of sensory reactivity to various stimuli. Functional performance tests and sensory reactivity assessment was also performed on four pregnant females retaining offspring to Day 4 post partum and females showing a total litter loss at 1000 mg/kg bw/day.
- Battery of functions tested: sensory activity / grip strength / motor activity
Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation. This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).
Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).
Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Vocalization, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex.

IMMUNOLOGY: No

OTHER: Reproductive Performance
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights
The following organs were dissected free from fat and weighed before fixation from five selected males from each dose group and five selected females from the control, low and intermediate dose groups and four females with litters and four females that showed a total litter loss from the high dose group. Some tissues were weighed from all remaining animals(A):
Adrenals, Brain, Epididymides(A), Heart, Kidneys, Liver, Ovaries(A), Pituitary (post-fixation)(A), Prostate and Seminal Vesicles(A), Spleen, Testes(A), Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix)(A)

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from five selected males and five selected females from each dose group. For females treated with 1000 mg/kg bw/day four females with litters and four females showing total litter loss and all tissues preserved in buffered 10% formalin, except where stated. Some tissues were preserved from all remaining animals (A):
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland(A)
Colon
Duodenum
Epididymides(A) •
Esophagus Eyes *
Gross lesions(A)
Heart
Ileum (including peyer's patches)
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Lymph nodes (mandibular and mesenteric)
Mammary gland(A)
Muscle (skeletal)
Ovaries(A)
Pancreas
Pituitary(A)
Prostate(A)
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles(A)
Skin
Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes(A) •
Thyroid/Parathyroid
Trachea
Thymus
Urinary bladder
Uterus & Cervix(A)
Vagina(A)

* Eyes fixed in Davidson's fluid
• preserved in Modified Davidsons fluid
# lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative

The tissues from five selected control and 1000 mg/kg bw/day dose group animals and any animals dying during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of liver and thyroid glands (both sexes), pituitary (males only), spleen, thymus, lungs and the kidneys (females only) from animals in the low and intermediate groups
Statistics:
Due to limitations of this free-text field, see "any other information on materials and methods".
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs of toxicity for males or surviving females treated with 100, 300 or 1000 mg/kg bw/day.
One control male and one male treated with 1000 mg/kg bw/day were observed to have open wounds which later formed scabs between Days 20 to 33 and 17 to 33, respectively. An additional male treated with 1000 mg/kg bw/day showed signs of a swollen hind limb from Day 37 onwards. These observations were considered to be physical injuries and not related to treatment.
One male, each treated with 300 or 100 mg/kg bw/day had signs of noisy respiration on Days 1 to 5 and Day 8 (respectively). In the absence of a similar effect at 1000 mg/kg bw/day, these observations were considered to be incidental and of no toxicological importance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated with 300 mg/kg bw/day was found dead on Day 3 post partum. There were no clinical signs of toxicity prior to death and the macroscopic findings included an enlarged spleen and liver together with red colored fluid in the thoracic cavity. It was noted this female had a long gestation period of 24 days.
One female treated with 1000 mg/kg bw/day was killed in extremis due to showing symptoms of difficulty during parturition; clinical signs for this animal included labored respiration, decreased respiratory rate, pilo-erection, lethargy, hypothermia, hunched posture, pallor of the extremities and dehydration. The macroscopic findings included a pale brain, liver, mammary gland and pancreas and the small and large intestine (caecum, colon, duodenum, ileum and jejunum) had black colored contents. It was noted that this female would have had a gestation length of 24 days.
There were no further unscheduled deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day throughout the treatment period.
Fluctuations in weekly group mean body weight gains were evident in treated males, in females treated with 1000 mg/kg bw/day during maturation and in females treated with 100 mg/kg bw/day during lactation, achieving statistical significance. There was generally no dose-dependance and, on a number of occasions group mean values for treated animals exceeded controls, therefore the intergroup differences were considered not to be of toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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.
During gestation, treated females showed an increase in food intake throughout this phase, attaining statistical significance for females treated with 300 and 1000 mg/kg bw/day. An increase in food consumption is generally considered not to represent an adverse effect of treatment. Food intake for females during lactation was generally similar, however females treated with 100 mg/kg bw/day showed a statistically significant reduction between Days 1 and 4 post partum. In the absence of a dose related response the intergroup difference was considered of no toxicological significance.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water intake was measured gravimetrically for each cage throughout the study with the exception of the mating period.
There was a significant increase in water consumption for animals of either sex from all treatment groups throughout the study for males and for females during pre-pairing, gestation and lactation phases. During pre-pairing, the overall increase in water intake for males was 15%, 15% and 34% (100, 300 and 1000 mg/kg bw/day, respectivity) and for females the overall increase was 25%, 59% and 73% (100, 300 and 1000 mg/kg bw/day, respectively). A statistically significant increase in water consumption was only evident in females treated with 1000 and 300 mg/kg bw/day throughout gestation and lactation.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treated males from all dose 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. However, these findings may be associated with the histopathological findings of hemopoiesis in the liver and spleen.
Females treated with 1000 mg/kg bw/day showed a reduction in neutrophil counts when compared to controls, attaining statistical significance. There was no dose relationship present and individual values were within the background control ranges.
There were no toxicologically significant effects detected in females treated with 300 or 100 mg/kg bw/day.
Males treated with 300 mg/kg bw/day showed a statistically significant increase in monocytes. Although the majority of the individual values were above the background control ranges no such effects were evident in 1000 mg/kg bw/day males or in corresponding females. Therefore in the absence of a true-dose related response the intergroup difference was considered of no toxicological significance.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males from all treatment groups showed statistically significant increases in cholesterol attaining statistical significance in all cases, without a dose related response. The majority of individual values were within the background control ranges however in view of the microscopic changes evident in the liver, an association to treatment cannot be excluded.
Females treated with 1000 or 300 mg/kg bw/day showed statistically significant increases in total protein. A dose relationship was evident but individual values were all within the background control ranges. However in view of the microscopic changes in the liver, an association to treatment cannot be excluded.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments: There were no treatment-related effects detected.
Functional Performance Tests: There were no treatment-related changes in functional performance.
At 1000 mg/kg bw/day females showed a statistically significant increase in hind limb grip strength whilst females from all treatment groups showed a statistically significant increase in fore limb grip strength. Due to a lack of consistency and no dose relationship, these findings were considered to be incidental. At 1000 mg/kg bw/day the two sub-groups of females were assessed. There were no differences between the two female sub-groups in the different physiological states that were considered to be significant.
Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.
At 1000 mg/kg bw/day the two sub-groups of females were assessed. There were no differences between the two female sub-groups in the different physiological states that were considered to be significant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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, attaining statistical significance, excluding females treated with 100 mg/kg bw/day. This can be associated with the centrilobular hepatocellular hypertrophy in these animals.
Kidney: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in kidney weights both absolute and relative to terminal body weight with a dose related response. This may be associated with the histopathological findings identified.
Thymus: Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in thymus weight both absolute and relative to terminal body weight with a dose related response.
Thyroid/Parathyroid: Animals of either sex showed a statistically significant increase in thyroid/parathyroid weights in 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. Although a true dose response was not evident and statistical significance was only achieved at 300 mg/kg bw/day, in view of the histopathological changes identified, an association to treatment can not be excluded.
At 1000 mg/kg bw/day the two sub-groups of females did not show any significant differences between the two physiological states.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Early decedents
One female treated with 300 mg/kg bw/day that was found dead on Day 3 post partum showed an enlarged liver and spleen and a fluid filled thoracic cavity.
One female treated with 1000 mg/kg bw/day that was killed in extremis during 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, one of which was also mottled. A further one male (No.74) had a small prostate and seminal vesicles. Three females from this dose group and two females treated with 300 mg/kg bw/day had enlarged spleens.
One male (No.30) treated with 100 mg/kg bw/day was observed to have small epididymides and the testes were noted to be small and flaccid. This male did not produce a pregnancy in its female partner. One female at this level was noted to have fluid filled uterus and cervix. In the absence of a similar effect at 1000 mg/kg bw/day, these intergroup differences were considered to be incidental.
There were a number of animals of either sex observed to have reddened lungs including control animals. This was considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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. In both sexes treated with 100 or 300 mg/kg bw/day centribular hepatocellular hypertrophy was evident from minimal to mild 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 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 levels 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 1000 mg/kg bw/day males. At 100 or 300 mg/kg bw/day the incidence was comparable to controls.
Spleen: Increased hematopoiesis was present in 7/9 1000 mg/kg bw/day females (mild to marked); 6/6 300 mg/kg bw/day females (mild to marked) and in 4/5 100 mg/kg bw/day females (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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
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: In total, 1 (No. 37), 5 (No. 61, 64, 68, 69 and 71) and 4 (No. 86, 88, 89 and 96) females showed total litter loss from 100, 300 and 1000 mg/kg bw/day dose groups, respectively. A further one female (No. 87) showed evidence of a pregnancy but no litter was observed. 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.
Premature Decedents: Female 64 had an enlarged liver and spleen, plus a fluid filled thorax at necropsy. There were numerous histopathological changes and septicemia was present. This is considered to be the cause of death. Although the initial cause is not clear it is likely to have originated in the uterus (degeneration was present) and this may have been a complication of pregnancy and parturition. Therefore, this is considered not to be related to treatment. Female 91 was killed due to issues with parturition and showed non-specific changes; most were considered to be agonal and its death could not be unequivocally related to treatment.
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.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
other: based on the severity and adaptive response nature of the findings rather than an absence of findings.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive toxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
immune system
Organ:
spleen
thymus
pituitary gland
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The study was conducted under GLP according to OECD guideline 422 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as sufficiently reliable to assess the repeated dose oral toxicity in rats (subacute; reproductive effects are discussed in the respective section).

Predominantly for the 1000 and 300 mg/kg bw/day dose groups microscopic examinations revealed treatment related changes in the liver, thyroid, pituitary, spleen, thymus, lungs and kidneys. The minimal centribular hepatocellular hypertrophy, minimal follicular cell hypertrophy in the thyroid gland and the mild increased hematopoiesis observed in the 100 mg/kg bw/day dose group were regarded as adaptive responses.
More specifically, the hypertrophy in the liver observed in animals from all dose groups was mainly centrilobular but due to the severity was spreading outwards towards the mid-zonal area. The histological changes noted in the thyroid gland (enlargement of follicles/ hypertrophy), pituitary (vacuolation males only) and the liver (hypertrophy) may be linked. Again, these findings are suggestive of an adaptive response to mixed function oxidase induction in the liver with concomitant increased incidence of follicular cell hypertrophy in the thyroid gland. In the pituitary, the enlarged vacuolated cells may reflect hypertrophy of thyroid-stimulating hormone-producing cells (thyrotrophs); a common finding following administration of liver enzyme inducers where the underlying mechanism is considered to be increased hepatic clearance of thyroid hormones (causing hypertrophy of follicular cells) followed by a compensatory increase in the pituitary secretion of TSH. These histopathological changes can correlate with the increase in liver and thyroid gland weights observed at necropsy. The vacuolation in the liver of animals in all groups treated with the test item may be due to a perturbation of metabolic activity however a direct effect of the test item cannot be ruled out.
The increase in hematopoiesis in the spleen and liver of some females treated with the test item is of unknown etiology as there were no notable changes in the hematological picture but a direct effect of the test item cannot be ruled out. These finding can correlate to the increase in spleen weights observed at necropsy.
The reduction in thymic involution in treated females correlates with the increased weight noted but may be related to the reduced number of viable litters produced.
There were three animals with minimal or mild changes in the kidneys - basophilic (degenerating/regenerating) tubules one of which also had tubular dilation. These types of changes are seen at a low incidence as common background changes but none were apparent in control animals of either sex in this study. Whilst any significance within the study is equivocal, it was noted that there was an increase in kidney weight at necropsy.
At 1000 mg/kg bw/day there was an increase in the number of females with alveolar macrophage accumulation in the lungs and the significance of this finding is unclear.

Slight changes in hematological values (erythrocyte count, hemoglobin and hematocrit) and histopathological evidence of mild splenic/hepatic changes were considered to be non-adverse in nature at 100 mg/kg bw/day. It was considered that these changes are adaptive responses related to the administration of the test item at the lowest dose level as compared to the adverse effects noted for the two higher dose groups of 300 and 1000 mg/kg bw/day.
Collectively these findings, showing only mild differences compared to controls in the absence of a dose response relationship were not considered to be adverse. Therefore, the No Observed Adverse Effect Level was determined to be 100 mg/kg bw/day for systemic toxicity.

According to Regulation 1272/2008, Table 3.9.1, Categories for specific target organ toxicity-repeated exposure, Category 2 is required for substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. Guidance dose/concentration values are provided below (see 3.9.2.9) in order to help in classification.
Guidance Value Ranges (dose/concentration) to assist in Category 2 classification are i.a. 10 < C ≤ 100 mg/kg body weight/day for a Oral (rat) study over 90 days. For subacute studies, the limit value is 300 mg/kg bw. The Regulation states further, that the guidance values and ranges mentioned in paragraphs 3.9.2.9.6 and 3.9.2.9.7 are intended only for guidance purposes, i.e. to be used as part of the weight of evidence approach, and to assist with decisions about classification. They are not intended as strict demarcation values. Substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement; assessment shall take into consideration not only significant changes in a single organ or biological system but also generalised changes of a less severe nature involving several organs.
As stated above, there were effects in various organs noted, i.e. liver, thyroid, pituitary, spleen, thymus, lungs and kidneys. The increase in the number of females with alveolar macrophage accumulation in the lungs in the 1000 mg/kg is both of unclear toxicological significance and way above the guidance value for classification. Hence, Classification as STOT RE Cat. 2 (lungs) is not triggered.

Females treated with 1000 and 300 mg/kg bw/day showed statistically significant increases in kidney weights both absolute and relative to terminal body weight with a dose related response. This may be associated with the histopathological findings identified. 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. Nevertheless those effects are only present at elevated dose levels which are not considered to justify a classification as STOT RE Cat. 2 (kidneys), and further the significance within the study is considered equivocal.

The reduction in thymic involution in treated females correlates with the increased weight noted but may be related to the reduced number of viable litters produced. In this case, this is considered as a secondary effect not directly related to the treatment, no classification is required.

As stated above, the effects noted in the other organs, i.e. centribular hepatocellular hypertrophy, follicular cell hypertrophy in the thyroid gland, vacuolation in pituitary (males), the increased hematopoiesis, and the increase in spleen weights are linked. They need to be regarded as adaptive response. Here, it cannot be absolutely distinguished to which dose the effect is not considered as toxic rather than adaptive, and at which dose exactly it has to be considered adverse or severe. Further, there are multiple organs involved which are connected, and it cannot be definitively distinguished whether there is a direct effect from the substance or secondary ones. The noted effects should be hence considered as a general systemic response rather than a single organ toxicity effect.

In consequence, clear evidence for classification as STOT RE Cat. 2 is not given, and classification can hence be omitted.
Executive summary:

Introduction

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).

 

Methods

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.

 

Results

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.

 

Laboratory Investigations

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.

 

Pathology

Necropsy:

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.

 

Organ Weights

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.

 

Histopathology

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.

 

Conclusion

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.

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
partition coefficient
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no GLP
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 117 (Partition Coefficient (n-octanol / water), HPLC Method)
Deviations:
no
GLP compliance:
no
Type of method:
HPLC method
Partition coefficient type:
octanol-water
Test material information:
Composition 1
Analytical method:
high-performance liquid chromatography
Key result
Type:
log Pow
Partition coefficient:
6.3
Remarks on result:
other: Temperature and pH were not specified in the study.
Conclusions:
The log Pow of 2,2'-methylenebis[6-cyclohexyl-p-cresol] was determined in an OECD 117 study with acceptable restrictions. The reverse phase HPLC method enables partition coefficients to be estimated in the log Pow range between 0 and 6, but can be expanded to cover the log Pow range between 6 and 10 in exceptional cases. The log Pow was determined as 6.3, and so falls into applicability of the employed method.
Executive summary:

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.

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
water solubility
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-09-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 105 (Water Solubility)
Deviations:
not specified
GLP compliance:
no
Type of method:
flask method
Test material information:
Composition 1
Key result
Water solubility:
0.034 mg/L
Temp.:
20 °C
pH:
>= 4.9 - <= 6.4
Remarks on result:
other: average of solubilities determined after 24, 48, and 72h shaking
Water solubility:
0.021 mg/L
Temp.:
20 °C
pH:
5.4
Remarks on result:
other: average of solubilities determined after 24h shaking
Water solubility:
0.039 mg/L
Temp.:
20 °C
pH:
6.4
Remarks on result:
other: average of solubilities determined after 48h shaking
Water solubility:
0.041 mg/L
Temp.:
20 °C
pH:
4.9
Remarks on result:
other: average of solubilities determined after 72h shaking
Conclusions:
The water solubility of the test item (CAS 73912-21-7) was determined according to OECD 105 with accetable restrictions, using the flask method, which may be considered as a scientifically reasonable approach, and hence the result of 0.034 mg/L at 20°C can be considered to be reliable. This value is below the limit value of 0.1 mg/L, leading to the classification of the test item as insoluble.
Executive summary:

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).

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Conducted prior to GLP implementation similar to OECD 404 on the registered substance itself with some deficiencies in documentation and performance, i.e. only two animals were used instead of three, substance was applied on the ear, not the trunk, evaluation criteria not defined.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
not applicable
GLP compliance:
no
Remarks:
test conducted prior to GLP implementation
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Housing: individually
Type of coverage:
semiocclusive
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 500 mg
Duration of treatment / exposure:
24 h
Observation period:
7 days
Number of animals:
2
Details on study design:
TEST SITE
- Area of exposure: inner face of the ear
- Type of wrap if used: band aid

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with water and soap/vegetable oil
- Time after start of exposure: 24 h
Irritation parameter:
other: irritating properties
Basis:
mean
Remarks on result:
other: not irritating
Interpretation of results:
not irritating
Conclusions:
The study was conducted prior to GLP implementation similar to OECD 404 on the registered substance itself with some deficiencies in documentation and performance. Nevertheless, with the given properties of the test item, the methodological deficiencies are unlikely to have any influence on the results. The given information on test performance allows the conclusion that the method was scientifically reasonable performed. Also, the result (not irritating) is confirmed by the other available studies on skin and in vivo eye irritation, clearing any doubts regarding the reliability of the result. Hence, Phenol, 2,2'-methylenebis-(6-cyclohexyl-4-methyl) does not need to be regarded as dermal irritant.
Executive summary:

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.

 

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-11-02 - 2016-02-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented OECD GLP guideline study without deviations on the registered substance itself.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitisation: Local Lymph Node Assay (adopted 22 July 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No. 440/2008, B.42: “Skin Sensitisation: Local Lymph Node Assay”, dated 06 July 2012
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D65189 Wiesbaden
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 17.5 - 21.6 g
- Housing: Conventionally, each dose group (5 mice/group) in one cage, Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top, Bedding: granulated soft wood bedding
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): approx. 45-65%
- Photoperiod (hrs dark / hrs light): 12/12 (artificial light 6.00 a.m. - 6.00 p.m.)
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Acetone: Purity: 99.98%; Olive oil: highly refined, low acidity
Concentration:
0, 10%, 25%, 50%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was a 50% solution in acetone/olive oil (4+1, v/v). Vortexing was used to formulate the test item.
- Irritation: To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of systemic toxicity. On day 2 and 3, the animal treated with 50% test item concentration showed a very slight erythema of the ear skin (Score 1). The animal treated with 25% test item concentration did not show any signs of local skin irritation.
Thus, the test item in the main study was assayed at 10, 25, and 50% (w/w). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Test Item Preparation
The test item was placed into an appropriate container on a tared balance and acetone/olive oil (4+1, v/v) was added.
The different test item concentrations were prepared individually.
The preparations were made freshly before each dosing occasion.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical Analysis
The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
The Dean-Dixon-Test and the Grubb’s test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw). No outlier value was detected.
Positive control results:
S.I. values per Test item concentration % in acetone:olive oil (4+1, v/v):
0%: 1.00
5%: 1.62
10%: 4.23
25%: 17.56
Parameter:
SI
Remarks on result:
other: Vehicle Control Group (acetone/olive oil (4+1, v/v)): 1.00 10% : 1.32 25% : 1.10 50% : 1.20
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle Control Group (acetone/olive oil (4+1, v/v)): 1458.5 10% : 1928.7 25% : 1611.1 50% : 1744.1
Interpretation of results:
GHS criteria not met
Conclusions:
The study was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the sensitizing potential of the test item in mice. The test item did not induce signs of general toxicity.
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices 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).
So, the test item was identified as a non-sensitizing agent in the Local Lymph Node Assay. Hence, no classification as skin sensitizer is triggered.
Executive summary:

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.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion