[No public or meaningful name is available]

Neurotoxicity

Description of key information

OECD 422 guideline GLP study (rat, oral gavage)

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: short-term oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Valtris Specialty Chemicals Ltd, lot/batch number W051290.
- Purity, including information on contaminants, isomers, etc.: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in original container.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Test material concentrations were shown by testing to be stable in the corn oil vehicle after 24 hours. Dose formulations were prepared and used daily.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal breeding facility at Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: [yes/no] yes
- Age at study initiation: (P) 11-12 wks
- Mean Group Weight at study initiation: (P) Males: 388-392 g; Females: 246-249 g
- Housing: Rats were housed in groups of 2 or 3 rats/sex/cage during premating period. During the mating period, rats were housed in groups of 2 rats/cage (one male plus one female) and mated female rat was caged individually. Enrichment material was provided to all rats. Nesting material was provided at near parturition (from gestation day 14). During the study, rats were housed in solid floor polypropylene rat cages. Each cage was fitted with a stainless-steel top grille having provision for a polypropylene water bottle with a stainless steel drinking nozzle. The bottom of the cages was layered with clean sterilised rice (paddy) husk as the bedding material. Cages were placed on a 5 tier racks. Cages and enrichment material were changed minimum twice a week. Cages were arranged in such a way that possible effects due to cage placement were minimised.
- Diet (e.g. ad libitum): Rats were fed ad libitum with standard rodent diet (Teklad Certified Global 16% Protein Rodent Maintenance Diet, Batch 2016SC-120519MA procured from Envigo Laboratories, Inc., USA).
- Water (e.g. ad libitum): Rats were provided reverse osmosis hi-tech sweet water (RO) water ad libitum (filtered through RO water filtration system) in polypropylene bottles.
- Acclimation period: Seven days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 degrees
- Humidity (%): 65-68
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: April 9, 2020 To: July 10, 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was mixed with the appropriate volume of corn oil vehicle to prepare dose formulations. The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing and during the dosing. Dose formulations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Solubility

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and homogeneity of the test item in the vehicle were analyzed once before initiation of treatment and twice during the treatment period using a validated GC-FPD method. The mean percent recovery obtained for the test doses were within the acceptance level of ±15% of the nominal concentration demonstrating that the exposure concentrations were as intended in the study plan and the %CV was less than 10%, indicating that the test doses were homogeneously prepared.

Duration of treatment / exposure:

Dosing of both sexes was initiated 2 weeks prior to the mating and continued during the mating period. After mating, the male rats were further dosed up to and including the day before scheduled sacrifice on study Day 32. Female rats were dosed during pregnancy and up to post-partum day 14.

Rats belonging to recovery groups were kept for 15 days after the first scheduled sacrifice of dams, without treatment.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

450 mg/kg bw/day

No. of animals per sex per dose:

Main control and treated groups: 15 males and 15 females
Recovery groups (control and high dose only): 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a 28-day dose range finding study (JRF Study N° 410-1-04-24390) where significant effects on organ weights were observed at 500 and 1000 mg/kg b. wt./day.
- Fasting period before blood sampling for clinical biochemistry: Food was withheld overnight.

Observations and clinical examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during treatment period; once daily during recovery period.
- Cage side observations: mortality, morbidity, visible clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Neurobehavioral: prior to initiation of treatment and weekly thereafter.
- Functional Observational Battery: 5 rats/sex/group randomly selected near the end of the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: Males - On the first day of dosing and weekly thereafter. Females - On the first day of dosing and at weekly intervals during the pre-mating and the mating periods. During the gestation period, female rats were weighed on gestation days 0, 7, 14, 20, and 25. During the lactation period, female rats were weighed within 24 hours of parturition (day ‘0’ post-partum/lactation day), and on post-partum days 4, 7, and 14.

FOOD CONSUMPTION: Yes
- Time schedule: The food consumption was determined by differentiating the weight of food input and leftover.
Food weights of male rats were determined weekly during the pre-mating and post-mating periods.
In female rats, during the pre-mating period, food weights were recorded at weekly intervals. During the gestation period, food weights were measured on days 0, 7, 14, and 20. During the lactation period, food weights were measured on days 0, 4, 7, and 14.
Food consumption was not measured during the mating period.
Food weights of male and female rats belonging to recovery groups were determined weekly throughout treatment and recovery periods.

WATER CONSUMPTION: No

CLINICAL PATHOLOGY (hematology, clinical chemistry, thyroid hormones, urine): Yes
- Time schedule: At terminal sacrifice, blood was collected from all surviving rats under anaesthesia (isoflurane) by orbital plexus puncture. Rats were deprived of food overnight (allowed water ad libitum) prior to blood collection. Blood samples were collected for haematology (in vials containing 4% EDTA), coagulation parameters PT and APTT (in vials containing 3.2% sodium citrate), clinical chemistry, and thyroid hormone (T3, T4, TSH) analysis (in vials without anticoagulant).

At the time of terminal sacrifice, urine samples were collected overnight from five rats (adult)/sex/group in graduated collecting tubes.

Specific biochemical examinations:

NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY: No

CHOLINESTERASE ACTIVITY: No

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Parameters examined: Sensory reactivity measurements (approach response, touch response, click response, pupil response, tail-pinch response, and air righting reflex); hind limb foot splay; grip strength; and motor activity. Motor activity of each rat was monitored using an automated photobeam activity system equipped with a computer analyser. Rats were monitored for three consecutive 10 minutes intervals (total 30 minutes for each rat) allowing for examination of both exploratory and acclimation activity levels. The motor activity parameters i.e., total activity, ambulatory activity and fine activity were evaluated and reported.
- Description of procedures: performed on five randomly selected rats/sex/group towards the end of the study.

NEUROBEHAVIORAL OBSERVATION: Yes
- The following parameters were evaluated prior to initiation of treatment and at weekly intervals thereafter. On the day of Functional Battery Observation, NBO was performed before the start of FOB.
- Parameters examined: Home cage observation (posture, clonic movement, tonic movement); handling observations (ease of removing animal from cage, handling reactivity, palpebral closure, lacrimation, eye examination, piloerection, skin examination, salivation); open field observation (gait, mobility, arousal, vocalisations, rears, respiration, clonic movement, tonic movement, urination, defecation, sterotypy, bizarre behaviour). For open-field observations, rats were placed (one at a time) in an open arena (size: 42.7 × 28.7 × 19.8 cm) with a flat surface covered with clean absorbent paper and observed for a period of 2 minutes.

Sacrifice and (histo)pathology:

SACRIFICE
- Main male animals: Males of the main groups were treated up to the day 32 and sacrificed on day 33.
- Main female animals: Female rats were sacrificed on LD 15. Females that did not mate were sacrificed by 25 days after the last day of mating period. Females that did not deliver by day 25 post-coitum were sacrificed at that time.
- Recovery animals: Male and female rats belonging to recovery groups were sacrificed 15 days after the first scheduled sacrifice of the main study dams.

GROSS NECROPSY
- Gross necropsy was conducted under the direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out the blood from the rat. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities. Special attention was paid to organs of the reproductive system.

The uteri of all cohabited female rats were examined for the presence and number of implantation sites.

ORGAN WEIGHTS
- The following organs / tissues: testes, epididymides, Levator ani plus bulbocavernosus muscle complex, Cowper’s glands, glans penis, ovaries, thyroid, uterus with oviducts and cervix, adrenals, liver, kidneys, thymus, spleen, brain, heart.

HISTOPATHOLOGY / ORGAN WEIGHTS
- A detailed histopathological examination of 31 preserved organs including gross lesions was performed in high and control dose group rats.

Detailed testicular histopathological examination, paraffin embedding and transverse sections of 4-5 µm thickness) was conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation included identification of treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus and cauda, which was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm. Periodic Acid Schiff (PAS), haematoxylin, and eosin staining was used for examination of the testes, while haematoxylin and eosin were used for epididymides and ovaries.

Histopathological examination of the ovary was carried out to detect treatment-related effects such as qualitative depletion/increase of primordial, secondary, antral, graffian follicles population, persistence and increased/decreased corpus luteum, ovarian degeneration/atrophy and stromal cell proliferation.

In addition, all gross lesion, as well as liver and thyroid were processed from all lower dose groups and all recovery group and examined microscopically.

Other examinations:

UDPG ANALYSIS: Remnant serum samples from the control and high dose animals, stored at -70 +/- 10 degrees C, were analyzed for UDP-glucuronosyltransferase enzyme levels using My BioSource ELISA kits.

Positive control:

No

Statistics:

Non-pregnant female rats were excluded from statistical analysis.
Data such as body weight, body weight gain, food consumption, hind limb foot splay, grip strength, organ weight, organ weight ratio, % pre-natal loss, % post-natal loss, and litter parameters (pups body weight and pups body weight gain) were subjected to Shapiro-Wilk’s test for checking normality wherever applicable, followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data did not meet the normality, data were transformed to check the normality again. When log transfer data did not meet the normality, Kruskal-Wallis test or Mann-Whitney test were performed to calculate significance. When the data did not meet the homogeneity of variance, statistical analysis was extended following the decision tree (Gad, S.C., 2007). AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.
Countable data { viz., litter size, number of implants, pre-coital interval, duration of gestation, number of the oestrous cycles, urination count, defecation count, rearing count, motor activity (total, fine and ambulatory)} were subjected to non-parametric test either Kruskal-Wallis test or Mann-Whitney test.
Non-parametric data such as gestation index, parturition index, pregnancy rate, survival index, mortality index, live birth index and fertility index were analysed using a Chi-Square test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation (mild) was observed approximately 3 to 5 minutes after dosing in male at 450 and in female rats at 150 and 450 mg/kg b. wt./day dose groups and persisted for approximately 40 to 45 minutes. This finding is considered a response to the dose solution and toxicologically non-adverse in nature.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Serum TSH levels of male rats treated at 150 and 450 mg/kg b. wt./day were statistically significantly increased when compared with that of the control group. Statistically significant decrease was noted in serum T3 levels at 150 and 450 mg/kg b. wt./day and T4 at 450 mg/kg b. wt./day when compared with those of the control group.

Serum TSH, T3 and T4 level of male rats at 50 mg/kg b. wt./day were comparable with that of the control group.

TSH, T3 and T4 levels of female rats for all the dose levels were comparable with those of the control group.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases were noted in absolute (18 to 21% increase vs control) and relative (17 to 19% increase vs control) weights of liver in high dose group of both sexes. Effect recovered after recovery period in high dose males, while the effect was continued in high dose recovery females.

Statistically significant increase was also noted in absolute (17% increase vs control) and relative weight (20% increase vs control) of thyroids in high dose males.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related effects were observed in liver and thyroid.

Microscopic examination of liver revealed centrilobular hypertrophy in two mid-dose male animals and diffuse hypertrophy in high-dose animals of both sexes, as well as in female high-dose recovery animals. The lesions were well correlated with increased liver weight in main and recovery groups. Further these lesions were minimal to mild in nature. These changes might be due to induction (upregulation) of xenobiotic-biotransforming enzymes and transporters, an adaptive process that augments xenobiotic elimination during periods of high xenobiotic exposure (Curtis, 2008).

The hepatocellular hypertrophy ceased completely in recovery male animals, whereas in female animals the effect persisted with lesser incidence, which suggests additional recovery period could have resulted in complete recovery. Moreover, the test item did not result in any treatment related alteration in clinical chemistry parameters related to liver function (ALT, AST, ALP, GGT and total bilirubin). Therefore, this effect could be considered as an adaptive response.

Microscopic examination of thyroid revealed minimal to mild follicular cell hypertrophy in most animals of the mid-dose and high-dose groups, and in two males and two females in the low-dose group. This effect persisted in male and female animals of the high-dose recovery group.

Thyroid gland follicular cell hypertrophy and hepatocellular hypertrophy are often seen concomitantly in rats. An increase in liver weight, associated with liver enzyme induction and seen as centrilobular hypertrophy, is commonly observed along with thyroid follicular cell hypertrophy, and is a physiological response (Catherine and Philip, 2015).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Serum UDPG levels of the high dose group were comparable to the control group levels, based on ELISA kit analysis of remnant serum samples stored at -70 +/- 10 degrees C.

Details on results:

9.11 Neurobehavioral Observations
9.11.1 Home Cage Observations
In the home cage, all rats from treatment, recovery and control groups revealed normal postures asleep
(curled up often asleep), sitting A (sitting but with head hung down), sitting B (sitting normally, feet
tucked in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic movements were
absent in the home cage during NBO.
9.11.2 Handling Observations
NBO performed during handling of rats did not reveal any abnormality related to treatment. All the
rats revealed a normal behaviour during removal (very easy - animals sit quietly) and handling (easy -
alert, limbs put against the body). None of the rats showed lacrimation, salivation or piloerection.
Eyelids were wide open in all rats. Eye and skin examination of rats from all groups did not reveal any
abnormality.
9.11.3 Open Field Observations
In the open field, all rats from treatment and control groups showed normal gait, mobility, arousal and
respiration during the two minutes observation period. Clonic and tonic movements, stereotypy and
bizarre behaviour were absent. No treatment-related significant changes were observed in vocalisation,
rearing, urination, and defecation counts of male and female rats from treatment groups when
compared with that of the control group.
The following are some incidental observations that were not considered to be toxicologically relevant:
There were statistically significant increases in urination count during week 3 in all dose groups (50,
150 and 450 mg/kg b. wt./day dose; G2, G3, G4) and during week 4 in male rats of 450 mg/kg b.
wt./day dose main group (G4).
There were statistically significant increases in rearing count during week 1 in the 150 and 450 mg/kg
b. wt./day dose groups (G3 and G4) and defecation count during week 8 in the in female rats of main
450 mg/kg b. wt./day dose group (G4).
There were statistically significant increases in rearing count during week 1 and week 7 in female rats
of 450 mg/kg b. wt./day dose recovery group (G6).
9.12 Functional Observational Battery
9.12.1 Motor Activity
The motor activity counts of male and female rats from all treatment groups were comparable with
that of the control group except for a statistically significant decrease in ambulatory and total activity
of males in the recovery 450 mg/kg b. wt./day dose group (G6) for duration of 11-20 minutes. This
finding is incidental in nature and not considered to be toxicologically relevant.
9.12.2 Sensory Reactivity Measurements
Sensory reactivity parameters viz., approach response, touch response, click response, pupil response,
tail pinch response and air righting reflex in all treatment and recovery groups were comparable with
that of the respective control group.
9.12.3 Grip Strength
The hindlimb and forelimb grip strength values of rats from treatment and recovery groups were
comparable with that of the respective control group except statistically significant increase in
hindlimb grip strength values of male rats of recovery group (450 mg/kg b. wt./day). This effect was
not observed in male rats of main groups and female rats of main and recovery groups. The other
supportive parameters such as forelimb grip strength and foot splay of male and female rats of main
and recovery groups were also normal as compared to those of the control group. Therefore, this effect
is considered as incidental without any toxicological relevance. This could be considered not related
to treatment effect due to absence of effect in supportive parameter like foot splay in recovery group
and forelimb and hind limb in treatment main group.
9.12.4 Hindlimb Foot Splay
The hindlimb foot splay values of rats from treatment and recovery groups were comparable with that
of the respective control group.

Dose descriptor:

NOAEL

Remarks:

Neurotoxicity

Effect level:

>= 450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No neurotoxicity effects observed at the highest dose tested.

Critical effects observed:

no

Conclusions:

No neurotoxic effects were observed in this study. Based on these results the NOAEL for neurotoxicity of tris 2-propylheptyl phosphite in Wistar rats is considered to be greater than or equal to the highest dose tested, 450 mg/kg bw/day.

Executive summary:

An OECD 422 guideline GLP study was conducted of tris 2-propylheptyl phosphite.  Groups of 15 male and 15 female Wistar rats were administered the test substance by oral gavage at doses of 0 (corn oil vehicle only), 50, 150 or 450 mg/kg/day.  Doses were administered daily for two weeks prior to mating, and during a two-week mating period. Following mating, males continued on treatment until sacrifice on study day 33.  Females continued on treatment through gestation, parturition, and until sacrifice on lactation day 14.  Two separate groups of 5 male and 5 female animals were treated at the high dose or with corn oil for seven weeks (without mating) and then held for an additional 2 weeks to evalute the reversibility of any effects. 

Parental animals were evaluated for systemic effects (including neurotoxicity), fertility, and reproduction. Litters were culled on Day 4 of lactation and development of the remaining pups evaluated until study termination on lactation day 14.

Evaluation of neurotoxicity included neurobehavioral observations once before treatment and weekly throughout the treatment and recovery periods.  In addition, functional battery observations including motor activity were conducted on 5 rats/sex/group near the end of the treatment and recovery periods.

Treatment-related systemic effects in parental animals were limited to the liver and thyroid. The Lowest Effect Level (LOEL) in parental animals was considered to be 150 mg/kg b. wt./day based on minimal to mild thyroid follicular cell hypertrophy (males and females) and thyroid hormone changes (males only).  Details of these findings are provided in IUCLID Section 7.5.1.

No neurotoxic effects were observed in this study.  Based on these results the NOAEL for neurotoxicity of tris 2-propylheptyl phosphite in Wistar rats is considered to be greater than or equal to the highest dose tested, 450 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

other: rat

Quality of whole database:

The available data are considered suitable and reliable for REACH requirements.

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An OECD 422 guideline GLP study was conducted of tris 2-propylheptyl phosphite.  Groups of 15 male and 15 female Wistar rats were administered the test substance by oral gavage at doses of 0 (corn oil vehicle only), 50, 150 or 450 mg/kg/day.  Doses were administered daily for two weeks prior to mating, and during a two-week mating period. Following mating, males continued on treatment until sacrifice on study day 33.  Females continued on treatment through gestation, parturition, and until sacrifice on lactation day 14.  Two separate groups of 5 male and 5 female animals were treated at the high dose or with corn oil for seven weeks (without mating) and then held for an additional 2 weeks to evalute the reversibility of any effects. 

Parental animals were evaluated for systemic effects (including neurotoxicity), fertility, and reproduction. Litters were culled on Day 4 of lactation and development of the remaining pups evaluated until study termination on lactation day 14.

Evaluation of neurotoxicity included neurobehavioral observations once before treatment and weekly throughout the treatment and recovery periods.  In addition, functional battery observations including motor activity were conducted on 5 rats/sex/group near the end of the treatment and recovery periods.

Treatment-related systemic effects in parental animals were limited to the liver and thyroid. The Lowest Effect Level (LOEL) in parental animals was considered to be 150 mg/kg b. wt./day based on minimal to mild thyroid follicular cell hypertrophy (males and females) and thyroid hormone changes (males only).  Details of these findings are provided in IUCLID Section 7.5.1.

No neurotoxic effects were observed in this study.  Based on these results the NOAEL for neurotoxicity of tris 2-propylheptyl phosphite in Wistar rats is considered to be greater than or equal to the highest dose tested, 450 mg/kg bw/day.

Justification for classification or non-classification

No neurotoxic effects were observed in an OECD 422 guideline GLP rat study of tris 2-propylheptyl phosphite. These results are considered suitable and reliable for REACH requirements.

Based on the available data, tris 2-propylheptyl phosphite is not classified for neurotoxicity under Regulation (EC) 1272/2008.