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Neurotoxicity

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Description of key information

Assessment of neurotoxicity.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Effect on neurotoxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: sub-chronic inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Mar 2018 to 07 Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 424 (Neurotoxicity Study in Rodents)
Deviations:
yes
Remarks:
See "Any other information on materials and methods incl. tables" below.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)
Deviations:
yes
Remarks:
See "Any other information on materials and methods incl. tables" below.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Receipt: On 27 Feb 2018, Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were 6 weeks old and weighed between 133 and 251 g at the initiation of exposure.

Animal Identification: Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system).

Environmental Acclimation: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of exposure. During acclimation, animals were acclimated to restraint procedures required for exposure.

Selection, Assignment, and Disposition of Animals: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals at extremes of body weight range were not assigned to groups. The disposition of all animals was documented in the Study Records.

Husbandry
Housing: On arrival, animals were group housed (up to 3 animals of the same sex) in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. Animals were separated during designated procedures/activities (see Appendix 1 - Study Protocol and Deviations). Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), exposure level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions: Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water: Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during designated procedures. Water bottles were provided, if required. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health.

Veterinary Care: Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.
Route of administration:
inhalation: aerosol
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2 - <= 2.5 other: microns
Geometric standard deviation (GSD):
2.01
Details on exposure:
Acclimation to Restraint in Nose-Only Exposure Holding Tubes
To screen animals for poor tolerance of restraint and to limit the potential effects on respiration of the novel environment/conditions of restraint, the animals were subjected to restraint in nose-only exposure tubes. Animals were acclimated to restraint tubes four times (1 acclimation/day) prior to their first day of animal exposure. Animals were acclimated to restraint in nose-only exposure restraint tubes by increasing the restraint time over the acclimation period (1st day-1 hour, 2nd day-2 hours, 3rd day-4 hours and 4th day-6 hours; times are approximate). Following the restraint period, each animal were observed for clinical signs of injury or stress.

Animal Selection and Randomization
At the conclusion of the acclimation period, animals judged to be suitable test subjects and meeting acceptable body weight requirements were assigned to the study at random using a computer program. At that time, the animal numbers and corresponding body weights will be entered into the WIL Toxicology Data Management System (WTDMS). A printout containing the animal numbers and individual group assignments were generated based on body weight stratification into a block design. The animals will then be arranged into the groups according to the printout. The control group and 3 test substance groups will consist of 10 male and 10 female rats each. An additional 10 rats/sex were assigned to the control and high dose groups were assigned to the 28-day recovery phase.
Following randomization,if it is necessary to replace individual animal(s) prior to the initiation of dosing (due to being found dead, euthanized in extremis, exhibiting abnormal clinical signs, reduced food consumption, or body weight losses). Individual replacement animals will be selected from the remaining unassigned animals and assigned arbitrarily (not computer randomized) to the study based on comparable body weights (if possible) with respect to the animal(s) that was/were replaced. The reason(s) for replacement were appropriately documented in the study records.

Inhalation Exposure Methods
The test substance, Disflamoll TKP-P and Kronitex TCP (1:1 mixture), or filtered-air were administered as daily, 6-hour, nose-only inhalation exposures.
Exposures were conducted using either 2-tier (7.9-L) or 4-tier (14.1-L), stainless steel, conventional nose-only systems (CNOS) (designed and fabricated by Charles River) with grommets in the exposure ports to engage animal holding tubes. One exposure system was dedicated to each group: 1 for the filtered-air control group and 1 for each test substance group.
Air supplied to the nose-only systems was provided from the Inhalation Department breathing quality, in-house compressed air source and a HEPA- and charcoal-filtered, temperature- and humidity-controlled supply air source. All nose-only system exhaust passed through a canister filter prior to entering the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA filtration units.
All animals were housed in an animal colony room during non-exposure hours. Prior to each exposure, the animals selected for exposure were placed into nose-only exposure restraint tubes in the colony room and transported to the exposure room. Animals were then placed on the nose-only systems, exposed for the requisite duration, and returned to their home cages in the animal colony room. The animals were rotated on a daily basis among the ports of the tiers of the nose only exposure system. Food and water were withheld during the exposure periods.
The mean temperature and mean relative humidity of the exposure atmospheres were to be between 19°C and 25°C and 30% and 50%, respectively. Oxygen content of the exposure atmospheres was measured during the method development phase and was 20.9% for all groups.

Exposure Atmosphere Generation Methods
Control Exposure System
The control exposure system (Group 1) was operated as follows. Humidified, supply air was delivered to the nose-only system (NOS) from the facility in-house, humidified supply air source using a rotometer.

Test Substance Exposure Systems
A liquid droplet aerosol atmosphere of the test substance was generated as follows. The test substance was aerosolized using a glass Collison nebulizer. Test substance was added to each nebulizer, as necessary. In-house, compressed air was supplied to the nebulizer to create atomization of the test substance. In order to produce a stable exposure atmosphere at the target concentrations, a portion of the aerosol output from each nebulizer was directed towards facility exhaust, as needed. To prevent aerosolized test substance from entering the rotameter, a filtration system preceded each rotameter using a liquid trap followed by a breathing circuit filter.

Methods of Characterization of Exposure Atmospheres
Aerosol Particle Size Measurement
Aerosol particle size measurements were conducted using a 7-stage stainless-steel cascade impactor. Pre-weighed, 22-mm stainless steel discs were used as collection substrates. A pre-weighed, 25-mm glass fiber filter was used as the final collection substrate. Aerosol particle size measurements were conducted once per week for each test substance system. All samples were collected at an approximate sample flow rate of 1.9 to 2.1 LPM for 5, 2, and/or 0.5 minutes. Sample flow rates were measured using a Mini-Buck Calibrator. Following sample collection, the filters were re weighed and the particle size was calculated based on the impactor stage cut-offs.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal Exposure Concentrations
A nominal exposure concentration was calculated daily by dividing the total amount of test substance consumed during the exposure period by the total volume of air that passed through the exposure system.

Actual Exposure Concentrations
Aerosol exposure concentrations were measured using standard gravimetric methods. Samples were collected on pre-weighed glass-fiber filters held in an open-face filter holder that was positioned in the animal breathing zone of each conventional nose-only systems (CNOS). Following each sample collection, the filters were reweighed and the mass concentration (mg/m³) was calculated by dividing the filter weight difference by the sample collection volume. Samples were collected from test substance exposure systems at least 6 times during each exposure and from the control exposure systems at least once weekly during an exposure period.
Duration of treatment / exposure:
Control (filtered air) and test substance aerosol atmospheres were administered for 90 consecutive days
Frequency of treatment:
Control (filtered air) and test substance aerosol atmospheres were administered as a 6-hour, nose-only exposure.
Dose / conc.:
0 mg/m³ air (nominal)
Remarks:
Filtered air
Dose / conc.:
100 mg/m³ air (nominal)
Remarks:
Disflamoll TKP-P and Kronitex TCP (1:1 Mixture)
Dose / conc.:
300 mg/m³ air (nominal)
Remarks:
Disflamoll TKP-P and Kronitex TCP (1:1 Mixture)
Dose / conc.:
1 000 mg/m³ air (nominal)
Remarks:
Disflamoll TKP-P and Kronitex TCP (1:1 Mixture)
No. of animals per sex per dose:
Control group (1) and Group 4: 40 animal (20 male/20 female)
Groups 2 & 4: 20 animals (10 male/10 female)
Control animals:
yes, concurrent vehicle
Details on study design:
Justification for Test System and Number of Animals
The Crl:CD(SD) rat is recognized as appropriate for subchronic neurotoxicity studies and has been proven to be susceptible to the effects of neurotoxicants. In addition, Charles River Ashland has neurotoxicity historical control data in the Crl:CD(SD) rat.
The number of animals selected for this study was based on the US EPA Health Effects Test Guidelines OPPTS 870.6200, Neurotoxicity Screening Battery, August 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 424, Neurotoxicity Study in Rodents, July 1997.

Justification of Route and Exposure Levels
The route of administration was nose-only inhalation as ECHA requested a 90-day repeated dose neurotoxicity study in rats via nose-only inhalation. Nose-only exposure method was chosen as a means to reduce the potential for dermal and/or oral dosing due to a result from grooming. The 6-hour period of restraint was necessary to achieve the exposure duration set forth in the OECD and OPPTS testing guidelines for repeat exposure inhalation toxicity studies.
The exposure concentrations were determined from results of a previous 14-day inhalation range-finding study in rats. In the previous study, male and female Sprague Dawley rats were exposed to the test substance by nose-only inhalation exposure for 14 consecutive days at target concentrations of 200, 800, and 2000 mg/m3. Slightly lower mean body weights were observed in the 800 and 2000 mg/m3 group males at the end of the 14-day exposure period and decreased brain cholinesterase activity was observed in the 2000 mg/m3 group males and females at the scheduled necropsy. Additionally, test substance-related higher mean lung and liver weights were noted in the 2000 mg/m3 group males and females. Microscopic findings included minimal to mild mononuclear inflammation and increased alveolar macrophages in the lungs in the 800 and 2000 mg/m3 group males and the 2000 mg/m3 group females. Based on the increased lung weights and microscopic findings in the lungs, a high concentration of 1000 mg/m3 was selected for use in the current study. Low- and mid-exposure levels of 100 and 300 mg/m3, respectively, were chosen to assess the dose-response of the test substance following a minimum of 90-days of nose-only inhalation exposure.
Observations and clinical examinations performed and frequency:
Viability: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Observations: Animal were removed from cage, and a detailed clinical observations was performed weekly throughout the study. During the exposure periods, these observations were performed prior. On exposure days, clinical observations were also recorded 30–60 minutes postexposure.During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

Body Weights: Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy.

Food Consumption: Food consumption was quantitatively measured weekly throughout the study.

Food Evaluation: Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.
Specific biochemical examinations:
Neuropathology: Neuropathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified for microscopic examination (see Any other information for details) were evaluated from 6 randomly selected animals/sex in the control and high-dose groups. The following 50 subanatomic sites of the brain were examined and recorded as normal, or lesions described. All brain sections were examined in their entirety and if lesions were detected in sites other than those designated, they were added to the subanatomic brain tissue list.

Brain Cholinesterase Activity: The brains from 4 rats/sex/group at the primary and recovery necropsies (those not selected for neuropathology tissue collection and/or examination) were collected, weighed, and snap frozen for analysis. Brains were diluted in 10 volumes (based on weight of the brain recorded at necropsy) of ice cold 1% Triton X-100 and were homogenized using an Omni TH homogenizer. Brain homogenates were centrifuged under refrigerated conditions. Brain samples were maintained in an ice water bath following processing to ensure minimal reactivation of any inhibited cholinesterase and were analyzed using an assay based on a modification of the Ellman reaction. Following analysis, samples were stored in a freezer set to maintain -55°C to 85°C.
Neurobehavioural examinations performed and frequency:
FOB Assessments: FOB assessments were recorded for 10 animals/sex/group during the pretest period, during Study Weeks 1, 3, 7, and 11 (conducted prior to daily exposure), and during the last week of the recovery period (Study Week 16). The FOB used at Charles River is based on previously developed protocols. Testing was performed at approximately the same time each day by the same trained technicians, when possible, who did not know the animal’s group assignment. Testing of treatment groups was balanced across the day of testing, to the extent possible. The FOB was performed in a sound attenuated room equipped with a white noise generator.

Motor Activity: Motor activity was assessed for 10 animals/sex/group during the pretest period, during Study Weeks 1, 3, 7, and 11 (conducted prior to daily exposure), and during the last week of the recovery period (Study Week 16). The same animals were tested at each interval by biologists using a personal computer controlled system that utilizes a series of infrared photobeams surrounding an amber, plastic rectangular cage (18.5 in. x 9.5 in. x 8 in.) to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The motor activity assessment was performed in a sound attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5 minute epochs over a period of 60 minutes, and the data were reported in 10 minute subintervals. Total motor activity was defined as a combination of fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of 2 or more consecutive photobeams).

Learning and Memory: Beginning Study Week 12, up to 10 animals/sex/group, each assigned to the primary necropsy, were assessed for swimming ability and learning and memory using a water filled 8-unit T maze. In addition, during the last week of the recovery period (Study Week 16), 10 animals/sex in the control and high-exposure groups were evaluated using the T-maze. Different animals were tested at each interval. Animals were placed in the maze and were required to traverse the maze and escape by locating a submerged platform. For the learning and memory phases, the time required to traverse the maze and the numbers of entries into an error zone for all trials were recorded. The time to escape (all phases) and the number of errors (Phases 2 and 3) were evaluated.
Phase 1 was an evaluation of swimming ability and motivation to escape from the maze. Animals were allowed 2 minutes to complete each trial; animals that failed to escape within that time were placed on the platform for up to 20 seconds. Phase 2 evaluated sequential learning, and Phase 3 evaluated each animal for its memory to solve the maze when challenged in path A. Animals were allowed 3 minutes to solve the maze in Phases 2 and 3; animals that failed to escape within that time were placed on the platform for up to 20 seconds. Animals were allowed to rest for at least 1 hour between trials during Phases 2 and 3.

Open Field Anxiety: The adopted open-field assessments were carried out to potentially measure anxiety like behavior. The underlying hypothesis is that anxious rats tend to spend a higher proportion of time in the periphery of an enclosure, whereas non-anxious rats will spend time in all areas of an enclosure. The adopted open-field assessments were conducted during Study Week 11 using 10 animals/sex/group assigned to the primary necropsy and near the end of the recovery period (Study Week 17) using 10 animals/sex in the control and high-dose groups assigned to the recovery necropsy. Anxiety assessments were performed in a room equipped with a white noise generator. Animals assigned to testing were evaluated prior to the daily exposure. Animals were placed individually in an opaque, open top enclosure (1 m x 1 m with 0.38 m walls; internal measurements) with 4 near evenly spaced incandescent light bulbs suspended above the enclosure. Brightness levels were adjusted until the intensity of light at the center of the base of each enclosure was 60 lux ± 10. The area of the enclosure was divided into 2 zones, the central zone (0.7 m x 0.7 m) and the peripheral zone (0.15 m from the periphery on all sides). The ANY-maze® (Stoelting Co., Wood Dale, IL) video tracking software and system was used to measure the time spent and the number of entries into each zone. The number of occurrences of additional behaviors (rearing, wall-touch, and grooming) were also recorded. The testing session was 5 minutes in duration.
Sacrifice and (histo)pathology:
Unscheduled Deaths: No animals died during the course of the study.

Scheduled Euthanasia: At each necropsy, 6 animals/sex/group were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and perfused in situ with fixative (4% paraformaldehyde solution in 0.1M phosphate buffer). Remaining animals at each necropsy (4 animals/sex/group) were euthanized by carbon dioxide inhalation and discarded without examination following brain collection for cholinesterase activity assessment. No necropsy was performed.

Necropsy: The central and peripheral nervous system tissues were dissected. Any observable gross changes, abnormal coloration, or lesions of the brain and spinal cord were recorded.

Organ Weights and Measurements: Fixed brain weight and brain dimensions (length [excluding olfactory bulbs] and width) were recorded for 6 animals/sex/group perfused in situ.

Tissue Collection and Preservation: Representative samples of the tissues identified in Table form (see ANy other information for details) were collected from 6 animals/sex/group perfused in situ, unless otherwise indicated.

Histology: Tissues were processed at PAI Durham. Neuropathological assessments of central and peripheral nervous system tissues from 6 perfused animals/sex in the control and high-dose groups at the primary necropsy were conducted as follows:
The brain was sectioned into 8 total sections (olfactory bulbs plus 7 coronal sections), according to Bolon et al. Brain sections were processed and embedded in paraffin, sectioned at 5 micrometers thick, and stained with hematoxylin and eosin (H&E), Luxol fast blue/cresyl violet (LFB/CV), Bielchowsky’s silver, and Fluorojade B. In addition, brain sections were immunohistochemically (IHC) stained for glial fibrillary acidic protein (GFAP) and Iba-1.
Transverse and oblique (longitudinal) sections of cervical, thoracic, and lumbar spinal cord were processed and embedded in paraffin (one block for each anatomic level), sectioned at 5 micrometers thick, and stained with H&E, LFB/CV, Bielschowsky’s silver, and Fluorojade B. In addition, spinal cord sections were IHC stained for GFAP and Iba-1.
Trigeminal ganglia/nerves and dorsal root ganglia (with associated dorsal and ventral nerve roots) were processed and embedded in glycol methacrylate (GMA), sectioned at 2 3 micrometers thick, and stained with H&E, LFB/CV, and Bielschowsky’s silver. The left and right side of the trigeminal ganglia/nerves were embedded separately. In addition, the cervical and lumbar dorsal root ganglia (with associated nerve roots) were embedded separately.
Longitudinal sections of peripheral nerves (sciatic at mid-thigh and sciatic notch, tibial, sural, and peroneal nerves) were processed and embedded in GMA (left and right sides kept separate), sectioned at 2-3 micrometers thick, and stained with H&E. Cross-sections of the peripheral nerves were osmicated, processed and embedded in Spurrs resin (left and right sides kept separate), ultramicrotomed at 500–700 nm thick, and stained with toluidine blue.
The eyes, optic nerves, and gastrocnemius muscle were processed and embedded in paraffin, sectioned at 5 micrometers thick, and stained with H&E.
Statistics:
Each mean was presented with the standard deviation (S.D.) and/or the number of animals or cages (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Full details of statistical analysis are detailed below under "Any other information on materias and methods inc. tables".

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A higher incidence of dried red material around the nose was observed in the 1000 mg/m3 group males at the 30-minute post-exposure observations when compared to the control group. This finding was considered test substance–related but not adverse as it was noted sporadically during the study and did not persist to the weekly examinations performed prior to daily exposures.
All other clinical findings in the test substance-exposed groups at the 30 minute post exposure observations or the weekly examinations were noted with similar incidence in the control group, were limited to single animals, were not noted in an exposure-related manner, and/or were common findings for laboratory rats of this age and strain.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower mean body weight gains in the 1000 mg/m3 group males (occasionally statistically significant when compared to the control group) were noted beginning as early as Days 7–14, which continued until the primary necropsy (Week 13). A lower mean body weight gain was noted in this group when the overall exposure period (Days 0–91) was evaluated. These decreases resulted in statistically significantly lower mean body weights for the 1000 mg/m3 group males (up to 11.2% lower than the control group) beginning on Day 42 and continuing throughout the exposure period. During the recovery period, mean body weight gains in the 1000 mg/m3 group males were similar to the control group values, but mean body weights remained up to 9.9% lower (statistically significant) due to the deficits noted during the exposure period. These changes in body weight and body weight gain in males at 1000 mg/m3 were considered test substance–related and adverse.
There were no test substance-related effects on mean body weights or body weight gains in the 100 and 300 mg/m3 group males or the females. Occasional statistically significant differences from the control group were isolated, transient, and considered unrelated to test substance exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean g/animal/day food consumption in the 1000 mg/m3 group males was generally slightly lower than the control group values throughout the exposure period (food consumption statistically significant for Days 49–56 and 77–84 and when the entire exposure period was evaluated [Days 0–91]). The differences were slight (≤3 g/animal/day); however, these results corresponded to the lower mean body weight gains noted for males in this group. Therefore, the slightly lower mean food consumption in the 1000 mg/m3 group males was attributed to the test substance. During the recovery period, mean food consumption continued to be slightly lower than the control group (statistically significant during Days 112–119).
There were no test substance-related effects on mean food consumption in the 100 and 300 mg/m3 group males or the 100, 300, and 1000 mg/m3 group females. Sparingly statistically significant differences from the control group were isolated, transient, and considered unrelated to test substance exposure.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Mean g/animal/day food efficiency in the 1000 mg/m3 group males was generally slightly lower than the control group values throughout the exposure period (food efficiency statistically significant for Days 35–42 and when the entire exposure period was evaluated [Days 0–91]). Therefore, the slightly lower mean food efficiency in the 1000 mg/m3 group males was attributed to the test substance. During the recovery period, mean food efficiency was generally similar to the control group.
There were no test substance-related effects on mean food efficiency in the 100 and 300 mg/m3 group males or the 100, 300, and 1000 mg/m3 group females. Sparingly statistically significant differences from the control group were isolated, transient, and considered unrelated to test substance exposure.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Home Cage Observations
Home cage parameters were unaffected by test substance exposure. There were no statistically significant differences between the control and test substance–treated groups (by sex) at any evaluation (Week 1, 3, 7, 11, and 16 [Groups 1 and 4]).

Handling Observations
A higher incidence of slightly soiled or very soiled (crusty) fur appearance was observed in the 1000 mg/m3 group females at the Week 7, 11, and 16 (recovery) evaluations when compared to the control group values; the difference at Week 16 was statistically significant. These findings were considered test substance–related but not adverse as they generally did not persist to the next weekly detailed examinations and were not observed in males at this exposure level.
There were no other test substance–related effects on handling parameters at any exposure level or statistically significant differences between the control and test substance–treated groups (by sex) at the Week 1, 3, 7, 11, and 16 (Groups 1 and 4) evaluations.

Open Field Observations
Open field parameters were unaffected by test substance exposure. There were no statistically significant differences between the control and test substance–treated groups (by sex) at the Week 1, 3, 7, 11, and 16 (Groups 1 and 4) evaluations, with the following exceptions. A statistically significantly higher mean urination count was noted for the 1000 mg/m3 group females at Week 3; however, this value was lower than the pretest value for this group and was within the Charles River Ashland historical control data. Therefore, this difference was not considered related to test substance exposure. A statistically significantly higher mean rearing count was noted at Week 7 for females in the 300 mg/m3 group; this difference was not attributed to the test substance based on the lack of a similar effect in the 1000 mg/m3 group females.

Sensory Observations
Sensory parameters were unaffected by test substance exposure. There were no statistically significant differences between the control and test substance–treated groups (by sex) at the Week 1, 3, 7, 11, and 16 (Groups 1 and 4) evaluations.

Neuromuscular Observations
Neuromuscular parameters were unaffected by test substance exposure. There were no statistically significant differences between the control and test substance–treated groups (by sex) at the Week 1, 3, 7, 11, and 16 (Groups 1 and 4) evaluations, with the following exception. A statistically significantly higher mean hindlimb grip strength value was noted in the 100 mg/m3 group males at Week 11 when compared to the control group value. A similar effect was not observed for males in the higher exposure groups, the direction of change (increase) was not considered toxicologically relevant, and there was no dose response; therefore, this difference was not attributed to the test substance.

Physiological Observations
Physiological parameters were unaffected by test substance exposure. There were no statistically significant differences between the control and test substance–treated groups (by sex) at the Week 1, 3, 7, 11, and 16 (Groups 1 and 4) evaluations, with the following exceptions. A statistically significantly lower mean catalepsy value was noted for females in the 300 mg/m3 group at Week 7. A similar change was not observed in females at any other time point or at an exposure level of 1000 mg/m3 or in males at any exposure level; therefore, this difference was not attributed to the test substance. In addition, a statistically significantly lower mean body weight was noted for males in the 1000 mg/m3 group at Week 16, which corresponded with the lower mean body weights observed in this group throughout the exposure and recovery periods.

Motor Activity
Within-session repeated measures analyses of variance were conducted across the subintervals of each test session for total and ambulatory counts and for overall interval means (representing the entire 60-minute session activity) during each test session. Motor activity patterns (mean ambulatory and total motor activity counts) were unaffected by test substance exposure. Statistically significantly lower mean total and ambulatory counts were noted for females in the 1000 mg/m3 group during the Week 3 evaluation at 51–60 minutes; however, these results were transient and did not affect the overall pattern of habituation. In addition, statistically significantly higher mean ambulatory counts were noted for males in the 1000 mg/m3 group during the Week 16 testing at 31–40 minutes; however, this was transient and occurred during the recovery period, and similar results were not noted during the dosing period. There were no other statistically significant differences between the control and test substance-exposed groups when values obtained from the 6 subintervals (0–10 minutes, 11–20 minutes, 21–30 minutes, 31–40 minutes, 41–50 minutes, and 51–60 minutes) and the overall 60 minute test session were evaluated during Weeks 1, 3, 7, 11, and 16 (Groups 1 and 4). No remarkable shifts in the pattern of habituation occurred in any of the test substance exposed groups when the animals were evaluated on Weeks 1, 3, 7, 11, and 16 (Groups 1 and 4).

Biel Maze Swimming Trials
Swimming ability on Day 1 of the Biel maze assessment (Weeks 12 and 16 [Groups 1 and 4]) was similar between the control, 100, 300, and 1000 mg/m3 groups. There were no biologically meaningful trends for the times to criterion (mean time to locate the submerged platform) during the learning and memory trials between the males and females in the test substance exposed groups and the control group beginning on Days 84 or 112 (Groups 1 and 4). The mean numbers of errors committed during the various phases of evaluation were generally similar in all test substance exposed groups and the control group. The mean escape time and number of errors for males in the 1000 mg/m3 group during the Week 16 overall memory trials (Trials 11 and 12) were statistically significantly lower than the control group; however, a decrease in escape time and number of errors is not considered toxicologically relevant. Furthermore, these differences were only noted during the recovery period and similar results were not observed during treatment period. No other statistically significant differences were noted following repeated measures analysis.

Adopted Open Field Testing
Open field anxiety parameters were unaffected by test substance exposure. There were no statistically significant differences between the control and test substance–treated groups (by sex) at the Week 11 and 17 (Groups 1 and 4) evaluations, with the following exceptions. Mean central zone and peripheral zone entry counts for the 300 mg/m3 group females at Week 11 were statistically significantly higher than the control group values. These differences were not attributed to the test substance based on the lack of a similar effect in the 1000 mg/m3 group females and the lack of a dose response.

Brain Cholinesterase Activity
Mean brain cholinesterase activities in the 1000 mg/m3 group females were slightly lower than the control group values when evaluated at the primary (Week 13) and recovery (Week 17) necropsies (12.0% and 8.9% lower, respectively); the differences were statistically significant. The lower mean brain cholinesterase activity in the 1000 mg/m3 group females was considered test substance–related but nonadverse based on the small magnitude of the differences from the control group, absence of corresponding behavioral effects in the females, and lack of a similar effect on brain cholinesterase activity in males at this exposure level. The literature suggests that the biological significance of statistically significant changes in cholinesterase inhibition less than 20% should be judged on a case-by-case basis and assessment should include the pattern of changes in enzyme levels and the presence or absence of accompanying clinical signs and/or symptoms. As previously stated, there were no behavioral effects noted in the females throughout the course of the study and therefore, the lower mean brain cholinesterase activity in the 1000 mg/m3 females at the primary necropsy is not considered adverse.
Mean brain cholinesterase activities in the 100 and 300 mg/m3 group females and the 100, 300, and 1000 mg/m3 group males were similar to the control group values at the Week 13 and/or 17 (Groups 1 and 4) necropsies, with the following exception. Mean brain cholinesterase activity in the 1000 mg/m3 group males at the recovery necropsy was 37.2% lower than the control group value; this change was not statistically significant due to high inter-animal variability which was noted for these males at this time point and a similar effect was not observed at the primary necropsy; therefore, no relationship to the test substance was evident.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test substance-related brain weight changes were noted, and no test substance-related changes in brain length or brain width were noted at the primary necropsy. There were lower group mean body weights in the treated males at primary necropsy, but the changes were not statistically significant. These changes included a 4.0%, 7.0% and 14.1% lower group mean final body weight in the 100 mg/m3, 300 mg/m3 and 1000 mg/m3 dose groups, respectively. These lower body weights had no effect on brain weights or gross brain measurements.
No test substance-related brain weight changes were noted and no test substance-related changes in brain length or brain width were present when the 1000 mg/m3 groups were compared to the control groups at the recovery necropsy. There was an 8.5% lower (not statistically significant) group mean final body weight in the 1000 mg/m3 males at recovery when compared to the control group of males
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related gross findings were noted at the primary or recovery necropsies.
Neuropathological findings:
no effects observed
Description (incidence and severity):
No test substance-related microscopic findings were noted at the primary necropsy.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
300 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
1 000 mg/m³ air (nominal)
Based on:
test mat.
Sex:
female
Key result
Dose descriptor:
NOAEL
Remarks:
Neurotoxicity
Effect level:
1 000 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
no

Analyzed Exposure Concentrations

Results of the characterization of exposure atmospheres are summarized below.

 

Nominal Concentrations

The overall mean nominal concentrations for each test substance exposure system are presented in the following table.

Overall Mean Nominal Exposure Concentrations

Exposure System:

2

3

4

Target Concentration (mg/m3):

100

300

1000

Mean Nominal Concentration (mg/m3):

197

627

1523

Standard Deviation:

106.0

69.6

183.6

Number of Exposures:

57*

92

92

*= On various occasions throughout the study, for Exposure System 2, the mean siphon flow was greater than or equal to the mean generation airflow. In those instances, a nominal concentration was unable to be calculated on these exposure days.

 

Actual Exposure Concentrations

The overall mean aerosol concentrations for each exposure system are presented in the following table.

Overall Mean Actual Exposure Concentrations

Exposure System:

1

2

3

4

Target Concentration (mg/m3):

0

100

300

1000

Mean Concentration (mg/m3):

0

104

304

993

Standard Deviation:

0.0

7.7

9.5

73.4

Number of Sampling Days:

14

92

92

92

 

Aerosol Particle Size Determination

The overall mean aerosol particle size for each test substance exposure system is presented in the following table.

Mean Aerosol Particle Size

Exposure System:

2

3

4

Mean MMAD (microns):

2.5

2.4

2.0

Mean GSD:

1.98

2.07

1.98

Number of Samples:

14

14

14

 

Brain Cholinesterase Activity

Summary of Cholinesterase Activity

Group Number

Actual Gravimetric Concentration (mg/m3)

 

Brain Cholinesterase Activity (Mean/± S.D./% of Control)a

Males

Females

Week 13

Week 17

Week 13

Week 17

1

0

Mean

49084

55568

54640

69817

S.D.

2303.4

16848.3

1157.3

3044.5

% of Control

100

100

100

100

2

104

Mean

53458

NA

53306

NA

S.D.

3155.4

NA

2819.5

NA

% of Control

108.9

NA

97.6

NA

3

304

Mean

49489

NA

53106

NA

S.D.

4859.8

NA

2326.5

NA

% of Control

100.8

NA

97.2

NA

4

993

Mean

57265

34924

48064**

63068*

S.D.

1051.0

16826.5

3235.5

3413.2

% of Control

96.3

62.8

88.0

91.1

NA = not applicable; S.D. = standard deviation; *= p<0.05 and **= p<0.01 when mean brain cholinesterase (U/L) was compared to the control group using Dunnett’s test.

aPercent brain cholinesterase activity (% of control) was calculated as the mean cholinesterase activity of each test substance-exposed group divided by the mean cholinesterase activity of the concurrent control group.

Conclusions:
Based on the lower mean body weights and body weight gains for 1000 mg/m3 group males, with corresponding effects on mean food consumption and food efficiency, an exposure level of 300 mg/m3 was considered to be the no-observed-adverse-effect level (NOAEL) for male systemic toxicity. Based on the lack of adverse effects at any exposure level in females, the exposure level of 1000 mg/m3, the highest exposure level evaluated, was considered the NOAEL for female systemic toxicity. In the case of male and female subchronic neurotoxicity evaluation of Disflamoll TKP-P and Kronitex TCP (1:1 Mixture), when administered via 6–hour nose-only inhalation exposures for 90 consecutive days to Crl:CD(SD) rats, the NOAEL was considered to be 1000 mg/m3, the highest exposure level evaluated.
Executive summary:

The objective of this study was to determine the potential neurotoxic effects of the test substance when administered daily by nose-only inhalation to Sprague Dawley rats for 90 consecutive days (7 days per week). The neurotoxic potential of the test substance was evaluated using a neurotoxicity screening battery (consisting of functional observational battery, locomotor activity, and neuropathological assessments).

The study design was as follows:

 

Experimental Design

Group Number

Treatment

Target Exposure Concentration (mg/m3)

Number of Animalsa

Males

Females

1

Filtered Air

0

20

20

2

Disflamoll TKP-P and Kronitex TCP (1:1 Mixture)

100

10

10

3

Disflamoll TKP-P and Kronitex TCP (1:1 Mixture)

300

10

10

4

Disflamoll TKP-P and Kronitex TCP (1:1 Mixture)

1000

20

20

aTen animals/sex/group were assigned to the primary necropsy. The remaining 10 animals/sex/group in Group 1 and 4 were assigned to a minimum of 28 days of recovery.

 

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, functional observational battery, motor activity, learning and memory, adopted open-field, brain cholinesterase activity, brain weights and measurements, and neuropathology.

 

All animals survived to the scheduled necropsies. There were no adverse test substance-related clinical observations or effects on food consumption, behavioral assessments (functional observational battery, motor activity, learning and memory, and adopted open-field), or brain cholinesterase activity.

 

A higher incidence of dried red material around the nose was noted in the 1000 mg/m3 group males at the 30–60-minute post-exposure observation recording and appearance of slightly soiled to very soiled fur was noted in the 1000 mg/m3 group females during the functional observational battery assessments at exposure Weeks 7, 11, and 16 (recovery). These findings were considered test substance‑related, but non-adverse, as they did not persist to the next detailed weekly clinical examinations and were each limited to a single sex.

 

Lower mean body weight gains were noted in the 1000 mg/m3 group males generally throughout the exposure period, resulting in lower absolute mean body weights (up to 11.2% lower, when compared to the control group males) which began on Day 42 and continued throughout the remainder of the exposure and recovery periods. Correspondingly, slightly lower mean food consumption and food efficiency were generally noted for these males throughout the exposure and recovery (food consumption only) periods. These changes in body weight, body weight gain, food consumption, and food efficiency in males at 1000 mg/m3 were considered a test substance–related adverse toxic effect. No test substance‑related effects on body weight, body weight gain, food consumption, or food efficiency were noted for males at 100 and 300 mg/m3 or females at any exposure level.

 

Slightly lower mean brain cholinesterase activities were noted in the 1000 mg/m3 group females at Weeks 13 and 17 (recovery) compared to the control group females. These changes were considered test substance–related but non-adverse based on the small magnitude of the differences from the control group (≤12.0%), absence of corresponding behavioral effects in the females, and lack of a similar effect on brain cholinesterase activity in males at this exposure level.

 

No test substance-related gross or microscopic neuropathology findings were noted. No test substance-related brain weight changes or changes in brain length or brain width were noted. 

 

Based on the lower mean body weights and body weight gains for 1000 mg/m3 group males, with corresponding effects on mean food consumption and food efficiency, an exposure level of 300 mg/m3was considered to be the no-observed-adverse-effect level (NOAEL) for male systemic toxicity. Based on the lack of adverse effects at any exposure level in females, the exposure level of 1000 mg/m3, the highest exposure level evaluated, was considered the NOAEL for female systemic toxicity. In the case of male and female subchronic neurotoxicity evaluation of Disflamoll TKP-P and Kronitex TCP (1:1 Mixture), when administered via 6–hour nose-only inhalation exposures for 90 consecutive days to Crl:CD(SD) rats, the NOAEL was considered to be 1000 mg/m3, the highest exposure level evaluated.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 000 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
K1

Effect on neurotoxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

On the basis of the evidence available, it is considered that TCP, as registered is not a potential neurotoxic substance, as the ortho elements responsible for this type of effect are not sufficiently present. No classification and labelling is applicable.

Furthermore, review of the available data, components of TCP are not present in aircraft cabin air in concentrations that could cause concern. The partial pressure in the alveolar gas mixture of any TCP contamination of the cabin air is so low that it is unlikely to cross the alveolar membrane at levels that would cause any effects.

Additional information

Neurotoxicity of phosphate esters

 Two types of neurotoxicity are associated with some phosphate ester products. An acute neurotoxicity with rapid onset can be seen after single doses and is based on acetylcholinesterase inhibition. This is a reversible biochemical alteration.

 The second type of neurotoxicity associated with a few phosphate esters is “delayed neuropathy”, which has been shown to occur after high dose exposures to certain phosphate esters with specific characteristic structures. This form of neurotoxicity usually results in irreversible alterations to peripheral nerves. This is often referred to as organophosphate-induced, delayed neurotoxicity (OPIDN), and can occur in humans and several animal species, after exposure to these agents.

 The relationship between the chemical structure of many pure triarylphosphates and potency in causing OPIDN has been extensively studied, and there is a rather comprehensive knowledge of the relative neurotoxic activities of these compounds (Henschler, 1958, 1959; Neumann and Henschler, 1957; Henschler and Bayer, 1958; Johannsen, et al, 1977; Metcalf, 1982; Bondy, et all 1960; Johnson, 1975).

 Mode of action and structure/activity relationship of phosphate esters.

 The mode of action of organophosphates to induce delayed neuropathy is still not completely understood; however, the requirement for the inhibition of a non-specific carboxyl esterase seems to be established. This enzyme is termed “neuropathy target esterase” (NTE). NTE is a high molecular weight membrane protein present in neurons, which appears to have an unknown role in neuronal metabolism. The extent of inhibition of NTE by individual compounds correlates with their potential to induce OPIDN. It is known that certain phosphate esters cause a rapid “aging” of the phosphorylated enzyme resulting in an irreversible inhibition of NTE. Aging of the phosphorylated NTE seems to be a prerequisite for the development of OPIDN. A large number of triarylphosphates and tricresylphosphates (TCP) have been investigated for their potential to cause OPIDN. These assessments conducted over many years indicate that only triarylalkylphosphates with specific chemical structures may cause OPIDN:

 - Certain substituents in the ortho position have the potential to result in neurotoxicity.

 - Potential for neurotoxicity decreases with increasing size of the orthosubstituents

 - Arylphosphates with methyl substituents in the meta- or para-position do not have a potential for neurotoxicity.Only p-ethyl substituted arylphosphates have a potential for neurotoxicity due to a specific biotransformation.

 Technical arylalkyphosphate ester products often are complex mixtures containing many individual isomers and isomer content is heavily influenced by the chemicals used as starting materials and the reaction conditions. Considerable efforts were made to adjust the reaction conditions and the starting materials to reduce the level of potentially neurotoxic compounds (ortho-cresyl and ortho-ethylphenyl phosphate isomers) in the commercial products. Changes in production techniques have reduced the content of tri-o-cresylphosphate in the tricresylphosphate batches produced today to < 0.07% thus generating products with very low potential for neurotoxicity.

 

Substance Specific Investigations:

 Given the proposed neurotoxic effects noted with tricresylphosphates (TCP), the substance has been the subject of many investigations, and it is scientifically proven that the neurotoxic components in TCP are produced from ortho-alkyl-substituted phenol or xylenol present in the reaction mixture used for the synthesis. Orthomethyl (cresyl) or ortho-ethyl phenols lead to highly toxic components, whereas ortho-substituted xylenols lead to less toxic components (Henschler and Bayer, 1958).

 As such, based on assessment, it is known that the groups of ortho-substituted isomer (ortho-TCP) are responsible for neurotoxicity observed in species, with the meta and para isomers thought to contribute little to neurotoxicity. This opinion is enforced by the World Health Organisation (WHO) whom have conducted an in-depth assessment of the risks posed by TCP, and specifically a discussion on the toxicity associated with the various isomers.  The WHO report is referenced as:

 

WHO Library Cataloguing in Publication Data

 Tricresyl phosphate.

 Environmental health criteria ; 11

 1.Tritolyl phosphates - adverse effects 

2.Tritolyl phosphates –toxicity

I.Series

 ISBN 92 4 157110 1(NLM Classification: QV 627)

ISSN 0250-863X

 The full report being available at:

 http://www.inchem.org/documents/ehc/ehc/ehc110.htm#SectionNumber:1.7

 The report states that of the three isomers of TCP, ortho-TCP is by far the most toxic in acute and short-term exposure, and that it is the only isomer that produces delayed neurotoxicity. This evaluation is supported by a sufficient amount of literature references detailed within the WHO report.

 The publication “Toxicological Risks of Selected Flame-Retardant Chemicals (2000)” (prepared by theSubcommittee on Flame-Retardant Chemicals, Committee on Toxicology, Board on Environmental Studies and Toxicology, National Research Council” available at:

 http://www.nap.edu/openbook.php?record_id=9841&page=387

 ISBN-10:0-309-07651-X ISBN-13: 978-0-309-07651-7)

 

 further assesses the differences in the different isomers and states as follows:

 

9.1 Historical background

 Of the tricresyl phosphate isomers, the ortho (ortho-TCP) is by far the most toxic and alone gives rise to the major neurotoxicity in man. It is considered that the toxicity of the commercial products depends on the concentration of the ortho isomer, but the mixedo-cresyl esters in these products are also toxic and contribute to the neurotoxic action.

 Furthermore, in the published review notes by Professor Dietrich Henschler: Meeting: T Van Beveren & S Michaelis: Wurzberg, Germany: 18/3/09 – Report by Susan Michaelis/ GCAQE Quotes from 1958/59: [1,2]

 There is a significant discussion on ortho-TCP, which states that:

 ‘Only tricresyl phosphates with ortho cresyl radicals were found to have toxic paralytic effects”.

 As such, all of the evidence available in the public domain discusses that it is the ortho-isomer that is ultimately responsible for neurotoxic effects noted within TCP from the study data available.

 Ortho-isomer metabolism.

 Ortho-TCPis metabolized via three pathways. The first is the hydroxylation of one or more of the methyl groups to hydroxymethyl, which is responsible for the formation of mono- and di-hydroxymethylortho-TCPand o-hydroxy-benzyl alcohol. This reaction is known to be catalysed by the microsomal mixed-function oxidase system (Eto et al., 1967). The hydroxymethylortho-TCPis cyclized to form saligenin cyclic o-tolyl phosphate with spontaneous release of o-cresol, this being catalysed by the reaction of plasma albumin or other components (Eto et al., 1967). The cyclic phosphate metabolite is relatively unstable and is rapidly hydrolysed to inactive metabolic products (Eto et al., 1967). The second pathway is the dearylation of one or more of the o-cresyl groups ofortho-TCP, resulting in the formation of o-cresol, di- o-cresyl phosphate,  o-cresyl phosphate, and phosphoric acid. The third pathway is further oxidation of hydroxymethyl to aldehyde and carboxylic acid. These oxidation reactions are most likely to be mediated by alcohol and aldehyde dehydrogenases.

 Ortho-TCPis the only isomer that produces delayed neurotoxicity.These effects are considered to be attributable to the metabolic pathway identified utilising the cyclized route that is specific to this isomer. It is this pathway that is proposed to produce the neurotoxic esterase inhibition effects that are known to be associated with this isomer.  In rats injected intraperitoneally withortho-TCP, this esterase inhibitor was located mainly in the intestine and liver. The neurotoxic metabolite was isolated from the intestine and liver of rats followingortho-TCPadministration and was identified as saligenin cyclic o-tolyl phosphate [2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one] (M-1); M2 and M3 are also possible metabolites. The saligenin cyclic o-tolyl phosphate was also found in chickens and in cats. Although quantitative data are not available, indirect evidence suggests that cats metabolize this neurotoxic compound more efficiently than chickens. Two intermediate metabolites, di-(o-cresyl) mono-o-hydroxymethylphenyl phosphate [mono-hydroxymethylortho-TCP] and di-(o-hydroxymethylphenyl) mono- o-cresyl phosphate, [di-hydroxymethylortho-TCP], transform to saligenin cyclic o-tolyl phosphate, which is relatively unstable and is rapidly hydrolysed to inactive metabolic products.

 

The pathway is described in the document below entitled “Statement on TCP Ortho-isomer metabolism”.

 A large number of triarylphosphates and tricresylphosphates (TCP) have been investigated for their potential to cause OPIDN. These assessments conducted over many years indicate that only triarylalkylphosphates with specific chemical structures may cause OPIDN. Johnson (1975) compared the metabolic pathways of the three isomeric forms of TCP. The main observations, which concerned several organophosphorus esters, were as follows:

 Certain substituents in the ortho position have the potential to result in neurotoxicity

Provided that the o-alkyl group has at least one hydrogen on the alpha-carbon atom, cyclic derivatives can be obtained that are often highly neurotoxic on the basis of the above metabolic pathway.

Potential for neurotoxicity decreases with increasing size of the orthosubstituents

At the para position, a substituent requires two hydrogen atoms on the alpha-carbon atom in order to produce a cyclic neurotoxic metabolite inhibiting NTE. Sterically this is difficult to action. Only p-ethyl substituted arylphosphates seem to have the potential to react in this manner.

Substituents at the meta position may be metabolized but do not yield inhibitory products.

 TCP producers in current times have taken significant steps to reduce the amount of ortho-alkyl-substituted phenols in reaction blends, resulting in TCP synthesized from only meta- and para-cresols that does not cause OPIDN. Commercial TCP is a now a mixture containing the meta TCP (TMCP) and para TCP (TPCP) isomers. The ortho-TCP isomer occurs only as a contaminant in commercial mixtures and usually at very low concentrations (<0.07%).  Comparison of the neurotoxic activities of TCPs produced over the past 50 years shows that those produced in the 1940's and 1950's were more than 400 times as toxic as the low toxicity TCPs produced today.

 Commercial TCP, as subject to the registration is composed of structural isomer compounds as follows:

 tri-m-tolyl phosphate EC no.: 209-241-8 at >= 10.0 — <= 35.0 % (w/w)

Di-m Cresyl p Cresyl Phosphate at >= 28.0 — <= 45.0 % (w/w)

Di-p Cresyl m Cresyl Phosphate at >= 11.0 — <= 36.0 % (w/w)

Tri-cresyl-phosphate impurities containing mono-ortho- tolyl, di-ortho-tolyl, and tri-ortho tolyl groups < 0.15% (w/w)

tri-o-tolyl phosphate EC no.: 201-103-5 at 0.0 — < 0.07% % (w/w)

Plus various unidentified low level impurities.

 Since at least the late 1990s the compositional specification of cresol for the production of tri-cresolphosphate has been set to contain no more that 0.05% (w/w) [500 ppm] ortho-cresol.

 Assuming equal reactivity of m-cresol, p-cresol and o-cresol to form today’s commercial TCP, then stoichiometrically the maximum content of the ortho form in TCP cannot exceed*:

         

If all as mono-ortho < 0.16 % (w/w)

If all as di-ortho < 0.08 % (w/w)

If all as tri-ortho < 0.05 % (w/w)

 Calculations as follows: 

Molecular Weight of cresol (meta, para, and ortho) is 108 gr/M

1000 gr of cresol would contain a maximum of 0.5 gr o-cresol

0.5 gr ortho cresol = 0.5/108 =0.0046 Moles of o-cresol

MW of TCP (m-, p- and o-) = 368 gr/M

1000 gr of cresol at 100% yield will form: (1000/(3 x 108)) = 3.09 Moles TCP

3.09 Moles of TCP = 3.09 x 368 = 1136 gr TCP

 o-cresol at 0.0046 Moles will form a maximum of:

If all as mono-o-TCP:

0.0046 Moles of mono-o-cresyl TCP= 0.0046 x 368 = 1.7 gr mono-o-TCP =0.15% (w/w).

If all as di-o-TCP:

0.0023 Moles of di-o-cresyl TCP = 0.85 gr di-o-TCP = 0.075% (w/w).

If all as tri-o-TCP:

0.00153 Moles of tri-o-cresyl TCP =  0.56 gr tri-o-TCP = 0.05% (w/w).

 The concentrations of o-cresol containing TCP on the market will be lower than calculated above since in the manufacturing process for TCP, chemical analysis of excess unreacted cresol (following the reaction step) is rich in o-cresol content. Therefore the reaction of o-cresol to form TCP is not favoured as compared to the reactions with m-cresol and p-cresol. This is presumably because of steric hindrance of the hydroxyl group by the methyl group in the ortho position. Furthermore, the recovered stream of excess cresols (being rich in o-cresol) is not recycled. Therefore the true concentrations of any and all o-TCPs on the market are lower than the values calculated above as these calculations were made based on equal reactivity of all three cresol isomers

 On this basis, it can be concluded that commercially available TCP contains very low ortho isomer content

It is therefore not possible for metabolites to form that are associated with causing a neurotoxic response in toxicologically relevant amounts.

 Evaluation of study data in support of the registration:

 Of the studies available to the registrant in support of the registration, the results for assessment of neurotoxicity on TCP are variable but conclusive for current TCP materials, with the following values detailed within the dossier:

 Key - Mallet E J (2003) [K1]; Acute Delayed Neurotoxicity Study of Durad 125, Durad 125-S, Durad 125 SD (I), and Durad 125 SD (2) in Adult Hens; NOAEL > 2000 mg/kg of body weight

 

Supporting: Daughtrey W, Biles R, Jortner B, Erich M (1996) [K2] - Subchronic Delayed Neurotoxicity Evaluation of Jet Engine Lubricants Containing Phosphorus Additives; NOEC >= 1000 mg/kg/day (formulated product containing the substance at 3% w/w)

 Supporting - Walsh M I, Caldwell, D J, Narayanan L (1995) [K3]; Inhalation Toxicity Of Vapor Phase Lubricants; NOEC <= 5 mg/L in air

 Supporting - Fowler, M. J. et al. (2004) [K2]; Effects of neuropathic and non-neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon-like processes by differentiating mouse N2a neuroblastoma cells; The ortho isomer and its metabolites have a more potent neurodegenerative effect in vitro than the nonneuropathic isomer TPCP.

 Supporting - Banerjee, B. D. et al. (1992) [K2]; Effect of Tricresyl Phosphate on Humoral and Cell-Mediated Immune Responses in Albino Rats; NOEC > 100 ppm

 Disregarded - Cascieri, T Jr (1977) [K3]; Kronitex TCP triaryl phosphate neurotoxicity study in hens; effects noted in75 - 300 mg/kg oral dose range ; no information on ortho-TCP content.

 Disregarded - Freudenthal R I, Rausch L, Gerhart J M, Barth M L, Mackerer C R, Bisinger E C (1993) [K3]; Subchronic neurotoxicity of oil formulations containing either tricresyl phosphate or triorthocresyl phosphate; NOEL 20 mg/kg/day (formulated product containing the substance at 1% w/w, so correlation is difficult)

 The following summaries are also included, referenced from US National Toxicology Program (NTP) and the UK MSA (the Environment Agency):

 Henschler (1958, cited in IUCLID 2001) described ‘tricresyl phosphate poisoning’ as related to peripheral neurotoxicity and identified the ortho-isomer, tri-o-cresyl phosphate, as the principal neurotoxic component. The article also details the degenerative changes that occur in response to different amounts of tri-o-cresyl phosphate present in tricresyl phosphate.

 Bischoff (1977, cited in IUCLID 2001) also reviewed the neurotoxicity of tri-o-cresyl phosphate and provided a detailed picture of the neurological and histological changes that occur, identifying the hen as a sensitive model with which to evaluate the neurotoxic potential of the phosphate esters.

 

A number of studies by the NTP have also investigated aspects of the neurotoxic potential of tricresyl phosphate using a mixed isomer preparation of 79 per cent tricresyl phosphate esters consisting of 21 per cent tri-m-cresyl phosphate, four per cent tri-p-cresyl phosphate and less than one per cent tri-o-cresyl phosphate (plus other unidentified tricresyl phosphate esters).

 Hindlimb grip strength in male mice receiving 360 and 1,450 mg/kg and male and female mice that received 730 and 5,800 mg/kg in the NTP 16-day gavage study were significantly lower than those of the controls at the end of the study. However, the observed neurobehavioural changes in animals in the top three dose groups were not attributed by the authors to a direct neurotoxic response (NTP 1994).

 In a 13-week oral study conducted in mice, groups of ten male and ten female animals received tricresyl phosphate in corn oil by gavage at doses of 0, 50, 100, 200, 400 or 800 mg/kg bodyweight. Hindlimb grip strength in male mice receiving 200 mg/kg or more was significantly lower than that of controls at the end of the study. Multifocal degeneration of the spinal cord occurred in males and females receiving 100 mg/kg or more, and multifocal degeneration of the sciatic nerve was observed in male mice receiving 200 mg/kg or more and in females dosed with 100 mg/kg or more (NTP 1994). However, an equivalent study in rats, in which groups of ten male and ten female rats similarly received tricresyl phosphate in corn oil by gavage at doses of 0, 50, 100, 200, 400 or 800 mg/kg bodyweight, revealed no evidence of neurotoxicity (NTP 1994).

 In the 13-week feeding study in rats fed diets containing 0, 900, 1,700, 3,300, 6,600 or 13,000 ppm tricresyl phosphate – estimated to deliver 0, 55, 120, 220, 430 or 750 mg/kg bodyweight (males) and 0, 65, 120, 230, 430 or 770 mg/kg bodyweight (females) discussed above, no biologically significant changes in neurobehavioural parameters were noted. However, in a similar 13-week study in mice fed diets containing 0, 250, 500, 1,000, 2,100 or 4,200 ppm tricresyl phosphate (estimated to deliver 0, 45, 110, 180, 380 or 900 mg/kg bodyweight (males) and 0, 65, 130, 230, 530 or 1,050 mg/kg bodyweight (females)) changes in grip strength were noted in groups of animals receiving 2,100 or 4,200, although the significance of this finding was said, by the authors, to have been confounded by reduced bod weight of these animals, and axonal degeneration occurred in male and female mice exposed to 2,100 and 4,200 ppm and females exposed to 1,000 ppm (NTP 1994).

 In the two-year feeding study in rats, animals were fed diets containing 0, 75, 150 or 300 ppm of tricresyl phosphate – estimated to deliver average daily doses of 0, 3, 6 or13 mg/kg (males) and 0, 4, 7 or 15 mg/kg (females), respectively. An additional group of 95 male and 95 female rats were fed diets containing 600 ppm of tricresyl phosphate for 22 weeks and then received only control diet. After 3, 9, and 15 months exposure, up to 15 males and 15 females per group were evaluated for forelimb and hindlimb strength, then necropsied and evaluated for histopathological lesions. Hindlimb grip strengths in 300 ppm male rats and in males and females exposed to 600 ppm were significantly lower than controls at the 3-month interim evaluation, but no significant changes in neurobehavioural parameters were seen among any groups of rats at the 9- and 15- month evaluations (NTP 1994).

 Similarly, in a two-year mouse dietary study at levels of 0, 60, 125 or 250 ppm of tricresyl phosphate – estimated to deliver average daily doses of 0, 7, 13 or 27 mg/kg (males) and 0, 8, 18 or 37 mg/kg (females), respectively, after 3, 9, and 15 months exposure, up to 15 males and 15 females per group were evaluated for forelimb and hindlimb strength, then necropsied and evaluated for histopathological lesions. Hindlimb grip strength in 250 ppm female mice was significantly lower than in controls at the 3-month interim evaluation, but no other neurobehavioural changes were noted (NTP 1994).

In a recent sub-chronic inhalation neurotoxicity study, negligible effects associated with neurotoxicity were observed. Based on the lower mean body weights and body weight gains for 1000 mg/m3 group males, with corresponding effects on mean food consumption and food efficiency, an exposure level of 300 mg/m3 was considered to be the no-observed-adverse-effect level (NOAEL) for male systemic toxicity. Based on the lack of adverse effects at any exposure level in females, the exposure level of 1000 mg/m3, the highest exposure level evaluated, was considered the NOAEL for female systemic toxicity. In the case of male and female subchronic neurotoxicity evaluation of Disflamoll TKP-P and Kronitex TCP (1:1 Mixture), when administered via 6–hour nose-only inhalation exposures for 90 consecutive days to Crl:CD(SD) rats, the NOAEL was considered to be 1000 mg/m3, the highest exposure level evaluated.

 Thus, overall, studies in rodents have not definitively established the neurotoxicity of tricresyl phosphate, except in the presence of tri-o-cresyl phosphate, following repeated exposure for periods of up to two years.

A number of studies on the neurotoxicity of tricresyl phosphate in the hen are briefly noted in the IUCLID (1998) but details of test material and methodology employed, and status with regard to international guidelines and GLP, are inadequately reported so their robustness cannot be assessed. In one paper, treatment with 0.21 mg/kg bw/d or 0.26 mg tri-p-cresyl phosphate/kg bw/d for 18 to 20 days was reported not to affect serum cholinesterase or cause signs of ataxia or paralysis, while five doses of 5,000 mg tri-m-cresyl phosphate or tri-p-cresyl phosphate/kg bw/ d given over ten days, also elicited no effect (Hine et al. 1943, cited in IUCLID 1998).

 

Aircraft cabin air quality

 In recent years, TCP exposure has been subject of discussions relating to possible adverse impacts on the health and well-being of air crew resulting from exposure to substances in cabin air. Concerns were expressed, for example by the British Airline Pilots Association (BALPA), that the intermittent “fume events” on aircraft – in particular on two types of aircraft (Boeing 757 and BAe 146 aircraft.)– may have long-term health impacts, in relation to TCP. As discussed above, ortho-TCP is known to be the main protagonist for neurotoxic effects; however for the issue of other isomers of TCP, some concerns have also been raised. While since on or about 1997 TCP contains very low levels of ortho TCP isomers, and since the cabin air syndrome issue has been specifically raised by the CoRAP this is further investigated.

 As such, full investigations have been made into this aspect of potential exposure and associated effects. The studies are summarised in section 7.10.5 Exposure related observations in humans: other data within the dossier, and a synopsis of the results is as follows:

 Supporting – Exposure related observations in humans: other data. COT (2007); Cabin Air Environment, Ill-Health in Aircraft Crews and the Possible Relationship to Smoke / Fume Events in Aircraft

 Results: On the basis of their review, the COT concluded that it was not possible to determine whether a causal association exists between cabin air exposures (general or following incidents) and ill-health (acute or chronic) among flight crew. The inability to reach such a conclusion was based on the lack of studies specifically designed to address this question systematically. Members considered that while there is a large body of anecdotal and descriptive evidence on possible associations of health symptoms with cabin air quality, such data do not meet the standard of a properly designed and performed epidemiology study necessary to reach definitive conclusions.

 Supporting - Exposure related observations in humans: other data. Schindler BK et al (2012) [K2]; Occupational exposure of air crews to tricresyl phosphate isomers and organophosphate flame retardants after fume events

Results: None of the 332 urine samples contained metabolites of ooo-, oom- or oop-TCP. One urine sample contained m- and p-TCP metabolites close to the LOD (0.5 ug/l). With respect to 332 analysed samples, an occupational exposure of air crews to TCP isomers and particularly neurotoxic o-TCP after fume events was not evident. Based on the results and the failure of o-TCP detection in air during fume events, the reported health effects in air crews can hardly be attributed to o-TCP exposure.

 

Supporting - Exposure related observations in humans: other data. Crump D et al (2011) [K2]; Report for DfT by the Institute of Environment and Health (Cranfield Ref No YE29016V) Aircraft Cabin Air Sampling Study

Results: The maximum 5 minute mean concentration or ortho-TCP recorded by sorbent tube sampling was 0.02 mg m-3. This concentration was measured during the climb phase of a flight in Part 2 of the study. ortho-TCP concentrations during all other phases of this flight were below the quantification limit of the test (as was the travel blank).This value is also below the derived DNEL for the substance, No detectable amount of ortho-TCP or other TCPs were found in over 95% of the cabin air samples. Highest levels of ortho-TCP, and other TCPs noted in the study occurred during climb, pre-landing and take-off respectively. With respect to the conditions of flight that were experienced during this study, there was no evidence for target pollutants occurring in the cabin air at levels exceeding available health and safety standards and guidelines.

 Supporting - Exposure related observations in humans: other data. TNO (2013) [K2]; Assessment of TCP in aircraft cabin air.

Results: The average TCP levels were 7 ng/m3, well below the derived DNEL value for the substance. The most toxic type, ortho-TCP was entirely absent.. No health risk levels have been set for other TCPs. On average, the TCP levels measured by TNO were 14,000 times lower than the health risk level set for ToCP. In only one instance was an atypical value of 155 ng/m3 measured, but this is still 650 times lower than the stipulated ToCP norm.

Supporting - Exposure related observations in humans: Bagshaw (2013) [K2] Health Contaminants in Cabin Air (v2.5)

Results: The report concludes that investigations of aircraft cabin air world-wide have failed to detect levels of TCP above well-established and validated occupational exposure limit values. The partial pressure in the alveolar gas mixture of any TCP contamination of the cabin air is so low that it is unlikely to cross the alveolar membrane. Genetic or particular susceptibility to a particular adverse effect of certain chemicals on the part of an individual does not alter the need for there to have been a sufficient chemical exposure to cause the injury or damage. For the reasons set out above, the possible exposure levels to ToCP on aircraft are so low relative to what is required to create a toxic effect through inhalation that a toxic injury is simply not medically feasible with current understanding.

Overall: TCP and its isomers are not considered to be present within aircraft cabin air at sufficient levels to cause concern.

 

Conclusion on Neurotoxicity

The review of the toxicology of different organophosphate esters shows widely different toxicity profiles for representative compounds of these classes with a high structural diversity. While some phosphate esters, based on their specific chemical structure and reactivity, may cause toxic effects at low exposure concentration,others have a very low potential for toxicity and require very high doses to induce effects. Based on the available toxicology information and the modes of action, however, tolerable exposure concentrations for humans without health risks may be defined for many of these compounds.

 

The second type of neurotoxic response for phosphate ester products, delayed neuropathy or OPIDN, has been associated with ortho-containing isomers of TCP (TOCP). The neurotoxicity of certain of these formulations is well studied. Induction of OPIDN in relevant animal test systems requires extremely high doses to cause a significant inhibition of the crucial enzyme NTE. Due to the high doses needed, and the requirement to have at least 70% inhibition of NTE to induce delayed neuropathy, it is highly unlikely that any normal use pattern for TCP will result in human exposures sufficient to cause OPIDN.

Certain animal species (e.g., cats, dogs, cows, and chickens) are susceptible to OPIDN-related paralysis, whereas others (e.g., rats and mice) are less susceptible to the ataxia but very susceptible to the pathological changes.  Species susceptibility to delayed neurotoxicity induced by TOCP shows an inverse correlation with the rate of metabolic conversion to the neurotoxic metabolite. Because of its high susceptibility to ataxia, the adult chicken has been used as an experimental model to study OPIDN.

As discussed above, TOCP is metabolized to the more potent neurotoxic agent, saligenin cyclic o-tolyl phosphate, which is at least five times more neurotoxic than TOCP after oral administration to chickens: a metabolite level of 40 mg/kg caused ataxia equivalent to that resulting from 200 mg TOCP/kg (Bleiberg & Johnson, 1965).

Factors such as age, sex, and strain figure prominently in the expression of OPIDN. The young of most species are non-susceptible to TOCP-induced delayed neuropathy (Johnson & Barnes, 1970), which could be due to poor absorption of TOCP. However, experiments (Olson & Bursian, 1988) have suggested that factors (e.g., route of administration) other than absorption are more critical to this lack of susceptibility.

There have been many reported cases of human poisoning, mostly from accidental or irresponsible contamination of food- stuffs.  Occupational poisoning, usually resulting from dermal exposure, has also been reported. The ortho isomer of TCP is the responsible toxic agent. Though  short-term symptoms of ingestion might involve vomiting, abdominal pain, and diarrhoea, characteristically delayed, longer-term symptoms are neurological, frequently leading to paralysis and pyramidal signs (spasticity, etc.). There is considerable variation in the sensitivity of individuals to TOCP; severe symptoms were reported with a TOCP dose of 0.15 g in one individual, while others were unaffected by 1 to 2 g. There is also considerable variation in the rate of recovery from poisoning, some patients recovering completely and others still severely affected years later, after apparently similar exposure. First-aid treatment involves the induction of vomiting or pumping of the stomach. The patient should be hospitalized as soon as possible. Atropine or 2-PAM may be used as an effective antidotal treatment against cholinergic symptoms. Long-term, antispastic drugs may be useful, though physical rehabilitation is the cardinal therapy.

Due to the low stability of commercially available TCP in the environment, its rapid biotransformation and the demonstrated high thresholds for OPIDN-induction, repeated exposures by workers and consumers will not result in an accumulation of these compounds in humans and large safety factors between actual exposures (doses) and occupational exposures, exist.

In summary, the different modes of action resulting in the vast differences in neurotoxicity are associated with specific structural features and chemical reactivity of different phosphate products. Current production of TCP precludes the presence of such specific structures that are associated with neurotoxicity. 

Justification for classification or non-classification

The above studies have all been ranked reliability 1 thru 3 according to the Klimish et al system. This ranking was deemed appropriate because some of the reports do not detail a specific method; however these documents dose levels and responses in some detail, so is deemed appropriate for use in the support of a formal registration. Data from the literature reports similar results in that the criteria for neurotoxicity are associated with the ortho-isomer, which is not present at sufficient levels in current manufactured products to cause effects.

 

Justification for classification or non classification

The above results triggered no classification under the CLP Regulation (EC No 1272/2008).

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