Reaction Products of Dimethyl hydrogen phosphite and Alcohols C14-18, Even, Linear

Endpoint summary

Description of key information

The NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 100, 350, 500, and 1000 mg/kg/day groups (OECD 422).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2014 to 22 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Sprague Dawley [Crl:CD(SD)]

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: 66 males and 66 females received in good health from Charles River Laboratories, Inc., Kingston, NY.

- Weight at study initiation: males 321-391g, females 219-268 g. Female body weights ranged from 247-318g on gestation day 0

- Housing: Following receipt and until pairing, all F0 animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study.
During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the males were individually housed in clean, solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in plastic maternity cages with bedding material. The dams and their litters were housed in these cages until euthanasia on lactation day 4.
The 5 rats/sex in the control and high-dose groups that were assigned to the recovery phase were not paired for mating and remained housed in groups of 2-3 in clean solid-bottom cages until euthanasia

- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum throughout the acclimation period and during the study. Exception of all males and recovery phase females which were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

- Water (e.g. ad libitum): Reverse osmosis-purified water, delivered by an automatic watering system

- Acclimation period: 12 days

- Age at study initiation: approximately 11 weeks old for males and females at study day 0.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
-Vehicle was dispensed weekly for administration to the control group (Group 1) and for preparation of the test item formulations.
- Aliquots were prepared for daily dispensation to the control group and stored at room temperature.
- Aliquots were heated in a water bath at 50 °C ± 5 °C on each dosing day prior to dispensation until a homogenous mixture was obtained.
- Aliquots were removed and placed in a second water bath set at 37 °C ± 5 °C for a minimum of 15 minutes prior to dosing.
- The vehicle was mixed throughout the sampling and dose administration procedures.
- Aliquots were maintained at 37 °C ± 5 °C and stirred continuously during dosing.

TEST ITEM
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored at room temperature.
- Aliquots were heated in a water bath at 50 °C ± 5 °C on each dosing day prior to dispensation until a homogenous mixture was obtained.
- Aliquots were removed and placed in a second water bath set at 37 °C ± 5 °C for a minimum of 15 minutes prior to dosing.
- Test item formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.
- Aliquots were maintained at 37 °C ± 5 °C and stirred continuously during dosing.
- The first test item dosing formulations were visually inspected by the study director's designee and were found to be visibly homogenous and acceptable for administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Test item formulations ranging from 1 to 220 mg/mL were previously assessed for homogeneity, resuspension homogeneity, and stability following 5 and 9 days of room temperature storage. Homogeneity, resuspension homogeneity and stability were therefore not assessed in the study.
- Samples for concentration analysis were collected from the middle stratum of the first and last test item dosing formulations (including the control group).
- Formulations that failed to meet acceptance criteria were not used for dose administration.
- One set of samples from each collection was subjected to the appropriate analyses.
- All remaining samples were stored room temperature as back-up.
- All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with charged aerosol absorbance detection.

Duration of treatment / exposure:

- Test duration: 28 days
- Control animals were treated in an identical manner with 5 mL/kg/day of polyethylene glycol.
- Males assigned to the recovery group were maintained for a further 14 days treatment-free period following termination of treatment for males (28 days).
- Females assigned to the recovery group were maintained for a further 15 days treatment-free period following termination of treatment for females (day of euthanasia of the first lactating female).
- The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
- The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-52 doses.
- Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25 or post-cohabitation day 25) for a total of 39-52 doses.
- The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 28 doses for males and 40 doses for females, these animals remained on study for a 14 or 15-day recovery period.

Frequency of treatment:

- The test and control substances were administered once daily, for 28 consecutive days (males) or 39-52 days (females), by gavage.
- The volume of test and control material administered to each animal was based on the most recent bodyweight.
- All animals were dosed at approximately the same time each day.

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

- 15 animals of each sex (m/f) for groups 1 and 5 and 10 animals/sex/group for group 2 to 4. Based on the OECD guidelines 422 which recommends evaluation of at least 8 pregnant females per group.
- In addition, 5 animals of each sex (m/f) were allocated to the control and high-dose group to assess recovery, persistence, or progression of any toxic effects following the cessation of dosing. These animals were not evaluated for reproductive parameters.

Control animals:

yes, concurrent vehicle

other: recovery control (concurrent vehicle)

Details on study design:

- Dose selection rationale: Results of a previous 14-day study in rats.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Regulatory requirement
- Post-exposure recovery period in satellite groups: 15 days, treatment free
- The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
- The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-52 doses.
- The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 28 doses for males and 39 doses for females, these animals remained on study for a 15-day recovery period.

Positive control:

Not used

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT: Yes
- Time schedule for examinations: For males, recorded weekly throughout the study and prior to the scheduled euthanasia. For females, recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase).
Once evidence of mating observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period).
-Food consumption was measured on a per cage basis for the corresponding body weight intervals.
- Food consumption was normalized to the number of animals/cage and was reported in g/animal/day.
- Food intake was not recorded during the breeding period for animals selected for pairing. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4; food consumption was reported as g/animal/day and g/kg/day during gestation and lactation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected from 5 animals/sex/group at the primary necropsy (study day 28 for males and lactation day 4 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 14 or 15-day recovery period; study day 42 for males and study day 55 for females).
- Blood for serum chemistry and hematology was collected from the retro-orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy.
- Animals fasted: Yes. Overnight prior to blood collection for all males and recovery phase females.
- Parameters examined: Total leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, activated partial thromboplastin time, reticulocyte count, mean platelet volume, differential leukocyte count (neutrophil, lymphocyte, monocyte, eosinophil, basophil, large unstained cell), red cell distribution width, hemoglobin distribution width, platelet estimate, red cell morphology.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected from 5 animals/sex/group at the primary necropsy (study day 28 for males and lactation day 4 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 14 or 15-day recovery period; study day 42 for males and study day 55 for females).
- Animals fasted: Yes. Overnight prior to blood collection for all males and recovery phase females.
- Parameters examined: Albumin, total protein, globulin, albumin/globulin ratio, total bilirubin, urea nitrogen, creatinine, AP, ALAT, ASAT, gamma GT, glucose, total cholesterol, calcium, chloride, phosphorus, potassium, sodium, triglycerides, bile acids, appearance

NEUROBEHAVIOURAL EXAMINATION AND CAGE SIDE OBSERVATIONS: Yes
FOB Assessments
- Functional Observational battery were recorded for 5 animals/sex/group following approximately 28 days of dose administration (study week 4 for males selected for pairing, and on lactation day 4 for females).
- The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB.
- Battery of functions tested:
Home Cage Observations: Posture, convulsions/tremors, feces consistency, biting, palpebral (eyelid) closure)
Handling Observations: Ease of removal from cage, lacrimation/Chromodacryorrhea, piloerection, palpebral closure, eye prominence, red/crusty deposits, ease of handling animal in hand, salivation, fur appearance, respiratory rate/character, mucous membranes/eye/skin color, muscle tone
Open Filed Observations: mobility, rearing, convulsions/tremors, grooming, bizarre/stereotypic behavior, time to first step, grait, arousal, urination/defecation, grait score, backing
Sensory Observations: Approach response, startle response, pupil response, forelimb extension, air righting reflex, touch response, tail pinche response, eyeblink response, hindlimb extension, olfactory orientation
Neuromuscular Observations: Hindlimb extensor strenght, hindlimb foot splay, grip strenghts-hind and forelimb, rotarod performance
Physiological Observations: Catalepsy, Body weight, Body temperature

Motor Activity
- Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration (study week 4 for males selected for pairing, and on lactation day 4 for females).
- Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes a series of
infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity.
- 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 (print intervals) and the test session duration was 60 minutes.
- These data were compiled as six, 10-minute subintervals for tabulation.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period or following the 14-day recovery period. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in a 10 % ammonium sulphide solution for detection of early implantation. Females not selected for pairing were euthanized following the 15-day recovery period. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.
HISTOPATHOLOGY: Yes
- At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):
Adrenal glands, aorta, bone with marrow, brain, coagulating glands, eyes with optic nerve, gastroinstestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), heart, kidneys, liver, lymph node (axillary, mandibular, mesenteric), lungs, ovaries and oviduct, pancreas, peripheral nerve (sciatic), pituitary gland, prostate gland, salivary gland, seminal vesicles, skeletal muscle (rectus femoris), skin with mammary gland, spinal cord (cervical), spleen, testes with epididymides and vas deferens, thymus gland, thyroids, trachea, urinary bladder, uterus with cervix and vagina, all gross lesions
- Microscopic examination was performed on all tissues listed above from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and on gross lesions from all animals in all groups at the scheduled necropsies.
The following organs were weighed from all F0 animals at the scheduled necropsies: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, spleen, testes, thymus gland, thyroids with parathyroids.

Other examinations:

None

Statistics:

ANALYSIS CONDUCTED BY WIL RESEARCH
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex.
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, pre-coital intervals, gestation length, numbers of former implantation sites, corpora lutea, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, and FOB data values were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. FOB parameters that yield scalar or descriptive data in the test item-treated groups were compared to the control group using Fisher’s Exact test (Steel and Torrie, 1980). Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test item-treated groups to the control group.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

not adverse and/or not dose-related (see below)

Mortality:

mortality observed, treatment-related

Description (incidence):

not adverse and/or not dose-related (see below)

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

transient (see below)

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

not dose related (see below)

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

not dose related (see below)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

not dose related (see below)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

not dose related (see below)

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg bw/day

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 100, 350, 500, and 1000 mg/kg/day groups

Critical effects observed:

not specified

ANALYSES OF DOSING FORMULATIONS

With noted exceptions, the analysed dosing formulations were within the WIL Research SOP range for suspensions (85 % to 115 %). Formulations that did not meet acceptance criteria were not used for dosing.

 

CLINICAL SIGNS AND MORTALITY

All F0 animals survived to the scheduled necropsies. Test item-related clinical findings

included red material around the mouth for females in the 1000 mg/kg/day group and

clear material around the mouth for females in the 500 mg/kg/day group and males and females in the 1000 mg/kg/day group at approximately 1 hour following dose administration sporadically throughout the treatment period. Due to the sporadic nature of these findings, they were not considered adverse. Other clinical findings noted in the test item-treated groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

 

BODY WEIGHT AND WEIGHT GAIN

Males

Mean body weights and body weight gains for the 50, 100, 500, and 1000 mg/kg/day group males were unaffected by test item administration throughout the study. None of the differences from the control group were statistically significant.

Females pre-mating

Mean body weights and body weight gains in the 50, 100, 500, and 1000 mg/kg/day

group females were unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant.

Gestation

Mean body weights and body weight gains in the 50, 100, 500, and 1000 mg/kg/day group females were unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

Lactation

Mean body weights and body weight gains in the 50, 100, 500, and 1000 mg/kg/day group females were unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

 

FOOD CONSUMPTION AND COMPOUND INTAKE

Males

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 100, 500, and 1000 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.

Females pre-mating

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 100, 500, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating period. None of the differences from the control group were statistically significant, with the exception of significantly (p<0.05) higher mean food consumption in the 50 and 1000 mg/kg/day groups during study days 0-7. However, this was transient and did not affect mean body weight gains.

Gestation

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 100, 500, and 1000 mg/kg/day group females was unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

Lactation

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 100, 500, and 1000 mg/kg/day groups was unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

 

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)

Home cage observations

Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

Handling observations

Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

Open field observations

Open field parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

Sensory observations

Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

Neuromuscular observations

Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

Physiological observations

Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

Motor activity

Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all dosage levels when evaluated during study week 4 (males) or on lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL historical control data ranges and/or did not occur in a dose -related manner. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups.

 

CLINICAL PATHOLOGY

For the presentation of clinical pathology results in this report, the terms “higher” or “lower” refer to comparisons between test item-treated group means versus the concurrent control group mean.

Hematology and coagulation

There were no test item-related effects on hematology or coagulation parameters. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Hemoglobin distribution width was higher in the 1000 mg/kg/day recovery group at necropsy, but was within the range of mean values in the WIL Research historical control database. Additionally, there was no alteration in this parameter in the primary test-item groups; thus, this alteration was considered to be due to biologic variability. Prothrombin time was higher for females in the 50 mg/kg/day group at the primary necropsy, but this alteration was not seen in the other 3 treatment groups, and therefore lacked a dose-related response.

Serum chemistry

There were no test item-related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test item-treated groups were compared. A lower mean cholesterol level was observed for males in the 1000 mg/kg/day group at the primary necropsy; however, there were no histologic lesions or other clinical chemistry alterations to substantiate this change and the level was within the range of mean values in the WIL Research historical control database; therefore, the apparent alteration in cholesterol level was considered due to biologic variability. Females in the 1000 mg/kg/day recovery necropsy group had higher albumin and total protein. These alterations were not present in the primary test item-treated groups and these values were within the range of mean values in the WIL Research historical control database; therefore, they were considered to be due to biologic variability. A mild elevation in triglyceride level was observed in the female 1000 mg/kg/day recovery animals. This alteration was both statistically significant and slightly outside of the WIL Research historical control database range; however, the lack of elevation in the primary necropsy for any dosage group suggests that this was not test item-related and due to biologic variation.

 

ORGAN WEIGHTS

There were no test item-related effects on organ weights. However, some statistically significant differences were observed when the control and test item-treated groups were compared. Both the left and right testis to brain weights were statistically significantly higher in the 1000 mg/kg/day recovery necropsy groups, but this difference was not seen in the primary test item-treated groups, the weights were not outside the range of the WIL Research historical control database, and there were no test item-related lesions in these organs, therefore, this alteration was considered to be due to biologic variation. The mean heart weight for females in the 1000 mg/kg/day recovery group was statistically significantly higher than the control group, but this difference was not seen in the primary test item-treated groups, the weights were within the range of the WIL Research historical control database, and there were no histologic lesions in the treated animals; therefore, this apparent weight variation was considered to be due to biologic variation. All female groups, including control group animals, had liver weights that were above the study means in the WIL Research historical control database. This variation is likely due to the reproductive status of these animals (4 days post-lactation) and lack of fasting at the time of euthanasia. Mean absolute liver weight was statistically significantly higher for females in the 1000 mg/kg/day group at the primary necropsy compared to the control group; however, the difference in weight was minor (14.4% increase in absolute weight) and the relative liver to body weight and liver to brain weight ratios did not show significance, suggesting this alteration was related to increased body weight (8.0% increase in total body weight).

 

MACROSCOPIC EXAMINATIONS

No test item-related internal findings were observed at any dosage level in females that failed to deliver, the female with total litter loss or males and females at the scheduled necropsy. Macroscopic findings observed in the test item-treated groups occurred infrequently and/or in a manner that was not dose-related. The mean numbers of unaccounted-for sites and implantation sites in the 50, 100, 500, and 1000 mg/kg/day groups were similar to the control group values.

 

MICROSCOPIC EXAMINATIONS

Due to the effects on lactation in prohibiting normal estrus cycling in rats and changes in the vaginal epithelium secondary to recent parturition, estrus staging was not performed in the female rats that were 4 days post-lactation at the time of euthanasia. Nongravid animals did not have histologic sections of uterus for evaluation due to ammonium sulphite staining for identification of implantation sites, and were therefore not staged for estrus as well. Test item-related microscopic findings were noted in the glandular and nonglandular portions of the stomach of the 1000 mg/kg/day groups. These changes included dilatation of the glands of the glandular stomach and submucosal edema of the nonglandular stomach. Although these lesions were present in the test item-treated animals and not the control group, both lesions were minimal to mild severity and in low numbers of animals. Additionally, there were no significant changes found in final body weight between control and test item-treated groups. Therefore, the gastric changes were considered to be non-adverse. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test item. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Conclusions:

Under the conditions of this screening study, based on the lack of adverse effects at any dosage level, the highest dose evaluated was considered to be the No Observed Adverse Effect Level (NOAEL) for F0 reproductive and systemic toxicity when test item was administered orally by gavage to Crl:CD(SD) rats. The NOAEL for F1 neonatal toxicity was considered to be 1000 mg/kg/day based on the lack of test item-related effects.

Executive summary:

OBJECTIVE

This study was designed to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development.

STUDY DESIGN

The test item in the vehicle (arachis [peanut] oil) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 50, 100, 500, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. Males and females were approximately 11 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39 -52 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-cohabitation day 25 or post-mating day 25 respectively) for a total of 39 -52 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for females, these animals were assigned to a 15-day non-dosing recovery period.

All animals were observed twice daily for mortality and morbidity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on five F0 animals/sex/group at the primary and recovery necropsies. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day or post-cohabitation day 25 for females that failed to deliver, or following the 15-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

 

RESULTS

All F0 animals survived to the scheduled necropsies. Test item-related clinical findings included red material around the mouth for females in the 1000 mg/kg/day group and clear material around the mouth for females in the 500 mg/kg/day group and males/females in the 1000 mg/kg/day group at approximately 1 hour following dose administration throughout the dosing period. Due to the sporadic nature of these findings, they were not considered adverse. No other test item-related clinical findings were noted.

Mean F0 male and female body weights, body weight gains and food consumption in the test item-treated groups were unaffected by test item administration throughout the study. There were no test item-related effects on functional observational battery parameters or motor activity for F0 animals at any dosage level.

Mean male and female mating, fertility, copulation, and conception indices in the 50, 100, 500 and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration. There were no test item-related gross necropsy observations noted for males/females at any dosage level. In addition, clinical pathology parameters and organ weights in the test item-treated groups were unaffected by test item administration.

Test item-related microscopic finding were noted in the glandular and nonglandular portions of the stomach of the 1000 mg/kg/day groups. These changes included dilation of the glands of the glandular stomach and submucosal edema of the nonglandular stomach. Although these lesions were present in the test item-treated animals and not the control group, both lesions were minimal to mild severity and in low numbers of animals. Additionally, there were no significant changes found in the final body weight between control and test item-treated groups. Therefore, the gastric changes were considered to be non-adverse.

Viability, growth and clinical condition of F1 pups was unaffected by parental test item administration at all dosage levels.

 

CONCLUSION

Under the conditions of this screening study, based on the lack of adverse effects at any dosage level, the highest dose evaluated was considered to be the No Observed Adverse Effect Level (NOAEL) for F0 reproductive and systemic toxicity when test item was administered orally by gavage to Crl:CD(SD) rats. The NOAEL for F1 neonatal toxicity was considered to be 1000 mg/kg/day based on the lack of test item-related effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch grade 1, combined 28-day repeated dose oral (gavage) toxicity study with reproduction/developmental toxicity screeening test in rats, with recovery.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

The key study was performed in accordance with OECD 422 to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development.

The test item in the vehicle (arachis [peanut] oil) was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 50, 100, 500, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. Males and females were approximately 11 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39 -52 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-cohabitation day 25 or post-mating day 25 respectively) for a total of 39 -52 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for females, these animals were assigned to a 15-day non-dosing recovery period.

All animals were observed twice daily for mortality and morbidity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (hematology and serum chemistry) were performed on five F0 animals/sex/group at the primary and recovery necropsies. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day or post-cohabitation day 25 for females that failed to deliver, or following the 15-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

All F0 animals survived to the scheduled necropsies. Test item-related clinical findings included red material around the mouth for females in the 1000 mg/kg/day group and clear material around the mouth for females in the 500 mg/kg/day group and males/females in the 1000 mg/kg/day group at approximately 1 hour following dose administration throughout the dosing period. Due to the sporadic nature of these findings, they were not considered adverse. No other test item-related clinical findings were noted.

Mean F0 male and female body weights, body weight gains and food consumption in the test item-treated groups were unaffected by test item administration throughout the study. There were no test item-related effects on functional observational battery parameters or motor activity for F0 animals at any dosage level.

Mean male and female mating, fertility, copulation, and conception indices in the 50, 100, 500 and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration. There were no test item-related gross necropsy observations noted for males/females at any dosage level. In addition, clinical pathology parameters and organ weights in the test item-treated groups were unaffected by test item administration.

Test item-related microscopic finding were noted in the glandular and nonglandular portions of the stomach of the 1000 mg/kg/day groups. These changes included dilation of the glands of the glandular stomach and submucosal edema of the nonglandular stomach. Although these lesions were present in the test item-treated animals and not the control group, both lesions were minimal to mild severity and in low numbers of animals. Additionally, there were no significant changes found in the final body weight between control and test item-treated groups. Therefore, the gastric changes were considered to be non-adverse.

Viability, growth and clinical condition of F1 pups was unaffected by parental test item administration at all dosage levels.

Under the conditions of this screening study, based on the lack of adverse effects at any dosage level, the highest dose evaluated was considered to be the No Observed Adverse Effect Level (NOAEL) for F0 reproductive and systemic toxicity when test item was administered orally by gavage to Crl:CD(SD) rats. The NOAEL for F1 neonatal toxicity was considered to be 1000 mg/kg/day based on the lack of test item-related effects.

Inhalation

The test substance is a waxy solid with a melting point of 26 to 42 °C. The substance has been shown to have a low vapour pressure (0.323 Pa at 25 °C) and high onset boiling point range (volatilised with decomposition from approximately 300 °C at 101 kPa). As a result, the potential for generation of inhalable forms of the substance is low and exposure of humans via the respiratory route is predicted to be negligible under normal use conditions. Furthermore, the Log Pow value of >10.0 does not favour absorption directly across the respiratory tract epithelium by passive diffusion (Log10 Pow > 4) and the substance will not be readily soluble in blood because it is poorly water soluble (8.18 mg/L at 30 °C). Thus experimental evidence is in agreement with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7c: Endpoint specific guidance (Version 2.0; November 2014), and investigation of repeated dose toxicity via the inhalation route is scientifically invalid.

 

Dermal

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. However, no evidence of systemic toxicity was demonstrated during an acute study via the dermal route and, in order to minimise the number of vertebrate animals tested, it was considered most appropriate to dose rats orally by gavage as part of the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP guideline study

Justification for classification or non-classification

No classification is required for repeat dose toxicity under the terms of Regulation (EC) No 1272/2008 because there were no adverse effects at up to and including the maximum dose level of 1000 mg/kg bw/day.