4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

Endpoint summary

Description of key information

Repeated Dose toxicity: Subacute (28 day) study oral (gavage), rat (Wistar) m/f (OECD TG 407, GLP): NOAEL = 1000 mg/kg bw/d, no adverse effects observed up to the limit dose

No specific target organ toxicity was identified, no classification as STOT RE Cat. 2 was triggered.

Repeated Dose toxicity: Range-finding study (14 days), oral (gavage), rat (Wistar) m/f (GLP): NOAEL = 1000 mg/kg bw/d, no adverse effects observed up to the limit dose

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2016 - 15 September 2016 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

Range-finding study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Commission Directive 96/54/EC (Method B7)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, 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. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C, in the dark; used/formulated in light

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

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 or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Age at study initiation: approximately six to eight weeks old
- Weight at study initiation: At the start of treatment the males weighed 220 to 246g, the females weighed 171 to 188g.
- Fasting period before study: no
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were 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.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2014C 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: The animals were acclimatized for seven days during which time their health status was assessed

ENVIRONMENTAL CONDITIONS
- Temperature (°C) / Humidity (%): The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.
- Air changes (per hr): The rate of air exchange was 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. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records.

IN-LIFE DATES: The in-life phase of the study was conducted between 04 May 2016 (first day of treatment) and 01 June 2016 (necropsy).

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered once daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 8 mL/kg of Propylene Glycol.
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:

propylene glycol

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0, 3.75, 37.5, 125 mg/ml
- Amount of vehicle (if gavage): 8 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

See attachments

Duration of treatment / exposure:

twenty-eight consecutive days

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

limit dose

No. of animals per sex per dose:

5 / sex /dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels and dose volume were chosen in consultation with the Sponsor based on available toxicity data including a 14 Day range-finding study. In that study, administration of the test item to male and female rats up to a dose level of 1000 mg/kg bw/day was well tolerated. There did not appear to be any adverse effect of treatment with the test item on body weight performance or dietary intake in animals of either sex. Additionally, there were neither any clinical signs of toxicity for the animals on the study nor any macroscopic findings at necropsy and a dose level of 1000 mg/kg bw/day was therefore considered a suitable high dose for this study together with 30 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
- 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 or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing during the working week. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS:
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).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively

WATER CONSUMPTION:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Animals fasted: No
- How many animals: all
- Parameters examined:
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)
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: Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29.
- Animals fasted: No
- How many animals: All
- Parameters examined: The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)
Triglycerides (Tri)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity
Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and 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 one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour 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 grip strength 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. 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
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

IMMUNOLOGY: Yes
Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the plasma from each animal was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples will be discarded following the issue of final report.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a
suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic
abnormalities were recorded.
Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the plasma
from each animal was stored frozen at approximately -20 °C. No treatment-related effects on
the pituitary-thyroid axis were identified, therefore these samples will be discarded following
the issue of final report.
Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing
period, were dissected free from fat and weighed before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (post-fixation) Thyroid/Parathyroid (post-fixation)
Prostate and Seminal Vesicles Uterus with Cervix
(with coagulating glands and fluids)

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered
10% formalin, except where stated:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Colon
Duodenum
Epididymides ♦
Esophagus
Eyes *
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)#
Lymph nodes (mandibular and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries
Pancreas
Pituitary
Prostate
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles (with coagulating glands and fluids)
Skin
Spinal cord (cervical, mid thoracic and lumbar)
Spleen
Stomach
Testes ♦
Thymus
Thyroid/Parathyroid
Trachea
Urinary bladder
Uterus & Cervix
Vagina

* Eyes fixed in Davidson’s fluid
♦ Preserved in modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were dispatched to the histology processing Test Site for processing. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 1000 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined. Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the liver and thyroid glands from animals in the low and intermediate dose groups.

Statistics:

Data Evaluation
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarized in tabular form.

Statistical Analysis
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Throughout the dosing period, there were no clinical signs considered to be related to the toxicity of the test item.
Sporadic instances of increased post-dose salivation were evident in animals of either sex treated with 300 or 1000 mg/kg bw/day from Days 11 or 12 and up to Day 27 of dosing in a dose-related manner. Such observations are common in this type of study and may reflect an irritant nature of the test item and/or formulation; these observations were considered to be of no toxicological significance.
One male and female from the 1000 mg/kg bw/day dose group exhibited clinical signs of noisy respiration soon after dosing on Day 22. These observations were no longer present at approximately one hour post-dose check, and in the absence of similar clinical signs in any other animals, this finding was considered likely to be due to the dosing procedure rather than an indication of test item toxicity.
There were no clinical signs for any of the animals receiving the test item at a dose level of 30 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect of treatment with the test item at any dose level on body weight development in animals of either sex.
At 300 or 1000 mg/kg bw/day, males occasionally showed slightly higher group mean body weight gains in relation to controls but without attaining statistical significance. This resulted in slightly higher overall body weight gain for these animals. An increase in body weight is generally considered to be of no toxicological significance and taken into account the small magnitude of these increases, this finding was considered to be of no toxicological significance. Minor fluctuations in weekly body weight gains were also apparent in females, but overall body weight gains for these animals were comparable with controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item up to a dose level of 1000 mg/kg bw/day on food consumption or food conversion efficiency for animals of either sex.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment with the test item up to a dose level of 1000 mg/kg bw/day on food consumption or food conversion efficiency for animals of either sex.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Gravimetric measurement of water consumption during Week 3 of the treatment period identified sporadic instances of slightly higher water intake for females receiving 300 or 1000 mg/kg bw/day. There was no dose-relationship and the corresponding values in males were comparable with controls and as such these intergroup differences were considered likely to be due to normal biological variation.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected at any dose level in animals of either sex.
At the end of the dosing period, group mean reticulocytes in males treated with 1000 mg/kg bw/day were statistically significantly higher than controls (p<0.05) with these males also exhibiting a statistically significant shortening in prothrombin time (p<0.05). Males from the 300 or 1000 mg/kg bw/day dose groups showed statistically significant increases in platelet counts when compared with controls (p<0.01). There was no clear dose-relationship for any of these parameters and all individual values for the corresponding animals remained within the historical control data ranges. Hematology evaluations for the females did not reveal any statistically significant intergroup differences and these findings were deemed to be of no toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected at any dose level in animals of either sex.
When compared with controls, males treated with 300 or 1000 mg/kg bw/day showed statistically significantly lower bile acid concentrations (p<0.05) in a dose-dependent manner but all individual values were within the historical control data ranges. Females from these dose groups also showed statistically significantly lower albumin/globulin ratios in comparison with controls (p<0.05). A dose-relationship was evident, but all individual values remained within the historical control data ranges and the associated parameters in animals of either sex were also comparable with controls. These intergroup differences may be associated with minor perturbations in metabolism as a result of the adaptive liver changes but were regarded of no toxicological importance.
At 300 and 1000 mg/kg bw/day, group mean alanine aminotransferase activities in males were statistically significantly lower than controls (p<0.01). It is worth noting, however, that individual alanine aminotransferase activities in 2/5 control males exceeded the background data range which is likely to have exaggerated these intergroup differences. The changes were minor and deemed to be of no toxicological relevance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessments
There were no changes in the assessed behavioral parameters considered to be related to treatment with the test item at any dose level.
Functional Performance Tests
There were no treatment-related changes in functional performance at any dose level. Grip strength evaluations during the last week of dosing revealed statistically significantly higher forelimb strength for males treated with 1000 mg/kg bw/day in relation to controls (p<0.05). This was only evident in 1/3 tests with most individual values for the affected parameter remaining within the historical control data ranges. There were no apparent clinical signs of neurotoxicity for any of the animals throughout the dosing phase and the corresponding values in females were comparable with controls, this observation was considered to be due to normal biological variation.
Sensory Reactivity Assessments
Treatment with the test item up to a dose level of 1000 mg/kg bw/day did not result in any changes in relation to controls.

Immunological findings:

no effects observed

Description (incidence and severity):

no adverse effects noted

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

An evaluation of absolute and body weight-related organ weights did not identify any statistically significant intergroup differences in animals of either sex receiving the test item up to a dose level of 1000 mg/kg bw/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic findings at any dose level in animal of either sex.
Macroscopic abnormalities at terminal necropsy were confined to the 300 mg/kg bw/day dose group and included one male showing spleen with mottled appearance, one female with small heart and another female with small/mottled liver and small/pale ovaries. Similar findings were not present in any animals of either sex receiving the test item at a dose level of 1000 mg/kg bw/day and were considered to be incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment-related histopathology findings were as following:
Liver
Centrilobular hepatocyte hypertrophy at a minimal level was present in 2/5 Groups 2 and 3 males, all Group 4 males and 3/5 Group 4 females. An increased mitotic rate was noted in one Group 3 male, but due to the isolated nature of this incident it was deemed to be incidental.
Thyroid Glands
Follicular cell hypertrophy at a minimal level was present in 2/5 Group 2 and all Group 3 and 4 males. In females, it was present in 1/5 animals in Groups 2, 3 and 4 and was considered to be incidental.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

immunology

mortality

organ weights and organ / body weight ratios

water consumption and compound intake

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No adverse effects noted up to the limit dose of 1000 mg/kg

Key result

Critical effects observed:

no

Conclusions:

The study was conducted under GLP according to OECD guideline 407 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).
The oral (gavage) administration of the test item to male and female Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated and did not result in any toxicological findings. Based on the in-life results and histopathology findings, it is considered that a dose level of 1000 mg/kg bw/day could be assigned a No Observed Adverse Effect Level (NOAEL) for systemic toxicity within the confines of this study.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12Hdibenzo[d,g][1,3,2]dioxaphosphocin under GLP and is compatible with the following regulatory guidelines:

- Commission Directive 96/54/EC (Method B7).

- The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

-Commission Regulation (EC) No 440/2008 of 30 May 2008, 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 five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 or 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Propylene Glycol).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were no clinical signs of toxicological significance at any dose level considered to be related to the test item.

Behavioral Assessment

Behavioral assessment scores across the treated animals of either sex remained similar to controls.

Functional Performance Tests

Functional performance testing did not identify any treatment-related changes.

Sensory Reactivity Assessments

Sensory reactivity evaluation did not identify any effect of treatment with the test item.

Body Weight

Treatment with the test item up to a dose level of 1000 mg/kg bw/day did not result in any adverse changes in body weight development in animals of either sex.

Food Consumption

There was no detrimental effect of treatment with the test item at any dose level on dietary intake or food conversion efficiency for animals of either sex.

Water Consumption

There was no effect of treatment with the test item on water intake for animals of either sex.

Hematology

No toxicologically significant effects were detected at any dose level in animals of either sex.

Blood Chemistry

No toxicologically significant effects were detected at any dose level in animals of either sex.

Necropsy

Macroscopic examination at terminal necropsy did not reveal any treatment-related findings in animals of either sex up to a dose level of 1000 mg/kg bw/day.

Organ Weights

No treatment-related effects were detected at any dose level in animals of either sex.

Histopathology

Treatment-related histopathology findings were as following:

Liver

Centrilobular hepatocyte hypertrophy at a minimal level was present in 2/5 Groups 2 and 3 males, all Group 4 males and 3/5 Group 4 females.

Thyroid Glands

Follicular cell hypertrophy at a minimal level was present in 2/5 Group 2 and all Groups 3 and 4 males.

 

Conclusion

The oral (gavage) administration of the test item to male and female Wistar Han™:RccHan™:WIST strain rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated and did not result in any toxicological findings. Based on the in-life results and histopathology findings, it is considered that a dose level of 1000 mg/kg bw/day could be assigned a No Observed Adverse Effect Level (NOAEL) for systemic toxicity within the confines of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Study was conducted on the registered substance itself acc. OECD TG 407 under GLP. Hence, the tonnage-driven data requirements under REACH are fully met, and the database is of high quality.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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. There is a suitable Klimisch 1 GLP OECD 407 guideline study available, assessing the toxicological properties of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin after oral gavage over 28 days. 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), testing by the inhalation route is appropriate if exposure of humans via inhalation is likely taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.
The vapour pressure of the substance, extrapolated from experimental data is 4.3×10^-7 hPa at 20 °C, 6.2×10^-7 hPa at 25 °C and 3.5× 10^-6 hPa at 50 °C.
So, the exposure to 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin via inhalation of vapour can be disregarded as no vapour will be formed during handling. As it is a solid, no droplets of inhalable size will be formed. Also, the substance is not distributed as e.g. aqueous solution, so the formation of inhalable droplets or aerosols can furthermore be excluded. There are no particles of inhalable size formed because the substance is manufactured and used in a non solid/granular form (pastilles). So, direct dust exposure is excluded during handling, hence not fulfilling the above-mentioned criteria for the necessity of testing via inhalation route.
Further, no route-specific toxicity can be expected, and it is considered more reasonable to focus on the assessment of systemic toxicity, which can be best performed using the oral application route. In consequence, the available OECD 407 study (oral exposure route) is sufficient to cover this endpoint, no repeated dose testing via inhalation route needs to be performed and can consequently be waived due to animal welfare.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

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. There is a suitable Klimisch 1 GLP OECD 407 guideline study available, assessing the toxicological properties of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin after oral gavage over 28 days. 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 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin to any toxicologically relevant amount is unlikely, the latter conditions do not apply. Due to the inherent low toxicity of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin, a very 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. Based on the current knowledge, the substance is not classified, and any theoretical exposure which could lead to any effects in humans, even with applying appropriate uncertainty factors, would be magnitudes below the actual one. 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 438.54 g/mol, absorption is in theory possible, but not 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 7.1, 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 7.1, and additionally, the substance was found to be insoluble in water. 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. 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin is not classified as irritant to the skin. Also no pathological changes on the treated skin of male and female rats were found in a acute dermal toxicity test at a dose of 2000 mg/kg. Further, the substance does not need to be classified as irritating to the eye, which is in general considered to be more sensitive than the skin. Therefore, no additional penetration enhancement must be considered. In consequence, the available OECD 407 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.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Based on the minor severity (actually lack) of the observed effects and the absence of any other test item related effects, it is rather impossible to hypothesize a concrete mode of action.

There were no deaths or effects on body weight development, or on food and water consumption during the study. Throughout the dosing period, there were no clinical signs considered to be related to the toxicity of the test item. No toxicologically significant effects were detected at any dose level in animals of either sex with regard to haematology, clinical biochemistry or behaviour. An evaluation of absolute and body weight-related organ weights did not identify any statistically significant intergroup differences in animals of either sex receiving the test item up to a dose level of 1000 mg/kg bw/day. There were no treatment-related macroscopic findings at any dose level in animal of either sex.

Treatment-related histopathology findings were as following:

Liver: Centrilobular hepatocyte hypertrophy at a minimal level was present in 2/5 Groups 2 and 3 males, all Group 4 males and 3/5 Group 4 females. An increased mitotic rate was noted in one Group 3 male, but due to the isolated nature of this incident it was deemed to be incidental.

Thyroid Glands: Follicular cell hypertrophy at a minimal level was present in 2/5 Group 2 and all Group 3 and 4 males. In females, it was present in 1/5 animals in Groups 2, 3 and 4 and was considered to be incidental.

The oral (gavage) administration of the test item to male and female Wistar rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated and did not result in any toxicological findings. Based on the in-life results and histopathology findings, it is considered that a dose level of 1000 mg/kg bw/day could be assigned a No Observed Adverse Effect Level (NOAEL) for systemic toxicity within the confines of the study.

No definitive human relevance framework can be described due to the lack of any other effects securing any postulation, and no conclusion on biological plausibility can be drawn.

Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the lack of relevance of the observed effects does also not indicate any high hazard for humans and so does not trigger any further examinations.

Justification for classification or non-classification

According to Regulation 1272/2008, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values which take into account the duration of exposure and the dose/concentration which produced the effect(s). 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 values for classification into category 2 are 10 < C ≤ 100 mg/kw bw/d in a subchronic study, corresponding to ≤ 300 mg/kg in subacute studies. The oral administration of the test item to rats, at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated and did not result in any toxicological findings. Hence, no classification according to Regulation 1272/2008 is triggered.