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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 January 2015 to 06 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
various protocol deviations and one GLP deviation with no impact on outcome of study (see attached document)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tall oil, reaction products with ethanolamine
IUPAC Name:
Tall oil, reaction products with ethanolamine
Test material form:
semi-solid (amorphous): gel
Details on test material:
- Purity: 100%
- Physical state/Appearance: Brown gel
- Storage Conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Crl:CD(SD) rats (66 males and 66 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 06-Jan-2015.
- The animals were approximately 64 days old upon receipt.
- Each animal was examined by a qualified biologist on the day of receipt.
- The day following receipt, all animals were weighed and clinical observations were recorded. - Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period.
- The animals were housed for an acclimation period of 10 days prior to the first day of treatment.
- During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, and the testes were palpated at least once for all males.

ANIMAL 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). The nesting material is periodically analysed by the manufacturer for contaminants. Analyses of the nesting material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research.
- Assignment of individual animals to social groups tool place and, 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 returned to their original social group of 2-3 males until the scheduled necropsy.
- Following positive evidence of mating, the females were individually housed in plastic maternity cages with nesting material, ground corncob bedding (Bed O'Cobs; The Andersons, Cob Products Division, Maumee, OH).
- The dams and their litters were housed in these cages until euthanasia on lactation day 4.
- Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post cohabitation or post-mating day 25.
- 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.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records.
- Feeders were changed and sanitized once per week.
- Municipal water supplying the facility was sampled for contaminants according to WIL Research’s SOPs.
- The results of the diet and water analyses are maintained at WIL Research.
- No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.
- Reverse osmosis-purified (on site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study, with the exception that all males and recovery phase females were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

ENVIRONMENTAL CONDITIONS
- All rats were housed throughout the acclimation period and during the study in an environmentally controlled room.
- The room temperature and relative humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis.
- Actual mean daily temperature ranged from 70.5°F to 71.6°F (21.4°C to 22.0°C) and mean daily relative humidity ranged from 41.0% to 46.3% during the study.
- Fluorescent lighting provided illumination for a 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod.
- The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- Near the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities.
- At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomisation procedure based on body weight stratification in a block design.
- At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated.
- The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the WIL Research colony.
- The experimental design consisted of 4 test item treated groups and 1 control group composed of 10 rats/sex/group.
- An additional 5 rats/sex in the control and high-dose group were selected to be evaluated following a 13- or 14-day recovery period; animals assigned to the recovery period were not evaluated for reproductive toxicity.
- At the initiation of dose administration (study day 0), the males and females were approximately 11 weeks old.
- Male body weights ranged from 320 g to 390 g and female body weights ranged from 220 g to 259 g on study day 0.
- The animals were approximately 13 weeks old.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
arachis oil
Details on exposure:
PREPARATION
- The vehicle was dispensed every 3-5 days 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, protected from light.
- On each day of dose administration, each aliquot of vehicle was placed in a water bath set at 37°C ± 5 °C prior to dosing.
- The vehicle was mixed throughout the preparation, sampling, and dose administration procedures; aliquots were maintained at 37°C ± 5 °C and stirred continuously during dosing.
-Test item formulations were prepared every 3-5 days as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light.
- On each day of dose administration, aliquots were placed in a water bath set at 37°C ± 5 °C prior to dosing.
- The 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 and were found to be visibly homogeneous and acceptable for administration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Test item formulations ranging in concentration from 1 to 250 mg/mL were previously assessed for homogeneity, resuspension homogeneity, and stability following 5 and 10 days of room temperature storage and 5-day stability was established.
- The results of the 10-day stability study did not meet the acceptance criteria and dosing formulations were therefore prepared every 3 5 days.
- Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first test item dosing formulations and from the middle stratum of the first control group dosing formulation.
- The results of the initial assessment of the test item formulations prepared on 14-Jan-2015 failed to meet acceptance criteria. The back up samples were analysed and also failed to meet the acceptance criteria. Therefore, the formulations were re-prepared on 15-Jan-2015, samples were collected, and the results met the acceptance criteria.
- Samples for concentration analysis were also collected from the middle stratum of the last dosing formulations (including the control group) in which all dose levels were prepared.
- One set of samples from each collection was subjected to the appropriate analyses (except as noted above). - All remaining samples were stored at 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 ultraviolet absorbance detection.
Details on mating procedure:
BREEDING PROCEDURES
- The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females.
- A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained.
- Each female was cohabitated with 1 male in a solid-bottom cage.
- Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist.
- Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0.
- If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.
- For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
- Calculation methods for mating, fertility, and copulation/conception indices are attached.
Duration of treatment / exposure:
ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMES
- The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, flexible, Teflon-shafted, stainless steel ball-tipped dosing cannula once daily.
- The males selected for pairing were dosed during study days 0-34 (14 days prior to pairing through the day prior to euthanasia) for a total of 35 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 39-44 doses.
- Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating 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 35 doses for males and 40 doses for females, these animals remained on study for a 13- or 14 day recovery period.
- The dosage volume for all groups was 5 mL/kg.
- Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
Frequency of treatment:
Once daily
Duration of test:
35 to 52 doses (see information provided on duration of treatment/exposure above)
No. of animals per sex per dose:
- Group 1: Vehicle control (15 males and 15 females)
- Group 2: 100 mg/kg bw/day (10 males and 10 females)
- Group 3: 300 mg/kg bw/day (10 males and 10 females)
- Group 4: 500 mg/kg bw/day (10 males and 10 females)
- Group 5: 1000 mg/kg bw/day (15 males and 15 females)
- Five animals in Groups 1 and 5 were necropsied following a 13 or 14 day recovery period.
- Recovery animals were not evaluated for reproductive performance.
Control animals:
yes, concurrent vehicle
Details on study design:
- An overview of study design is given in the attached scheme.
- Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants.
- In addition, WIL Research has reproductive historical control data in the Crl:CD(SD) rat.
- The number of animals selected for this study (15 [Groups 1 and 5] or 10 rats/sex/group [Groups 2-4]) was based on the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 22 Mar 1996, which recommends evaluation of at least 8 pregnant females per group.
- Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, 10 animals/sex/group was an appropriate number of animals to obtain a sample size of 8 at termination.
- In addition, 5 animals/sex in the control and high-dose groups were necessary to assess recovery, persistence, or progression of any toxic effects following the cessation of dosing.

Examinations

Maternal examinations:
PARTURITION
- All females selected for pairing were allowed to deliver naturally and rear their young to PND 4.
- During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia.
- On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.
Ovaries and uterine content:
- See details of macroscopic examination (below)
Fetal examinations:
LITTER VIABILITY AND DEATHS
- Each litter was examined daily for survival, and all deaths were recorded.
- All pups were individually identified by application of tattoo markings on the digits following completion of parturition.
- A daily record of litter size was maintained.
- Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).
- Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings.
- The carcass of each pup was then discarded.

CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behavior.
- Each pup received a detailed physical examination on PND 1 and 4.
- Any abnormalities in nesting and nursing behavior were recorded.

BODY WEIGHTS
- Pups were individually weighed on PND 1 and 4.
- Mean pup weights were presented by sex for each litter and by dose group.
- When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data.
- The time periods a given pup was not weighed were left blank or designated as “NA” on the individual report tables.

SEX DETERMINATION
- Pups were individually sexed on PND 0 and 4.

EUTHANASIA
- On PND 4, surviving F1 rats were euthanised via an intraperitoneal injection of sodium pentobarbital and discarded.
Statistics:
See below
Indices:
- Litter parameter definitions are attached.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL OBSERVATIONS AND SURVIVAL
- All males and females in the control, 100, 300, 500, and 1000 mg/kg/day groups survived to the scheduled necropsies.
- Test item-related clinical findings of clear and/or red material around the mouth and/or red material around the nose were noted for F0 males and females in all test item-treated groups approximately 1 hour following dose administration, generally throughout the dosing period. These findings generally did not persist to the daily examinations on the following days and in the absence of any other signs of toxicity for F0 animals, the aforementioned material findings were not considered to be adverse.
- Other clinical findings, including red material around the eyes and hair loss on the forelimbs, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related and therefore were not attributed to the test item. Clinical findings noted during the recovery period in the 1000 mg/kg/day group were limited to hair loss on the facial area for the 1000 mg/kg/day group F0 females.

GESTATION
- Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day groups 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 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test item administration during lactation.
- None of the differences from the control group were statistically significant.

REPRODUCTIVE PERFORMANCE
- No test item-related effects on reproductive performance were observed at any dosage level. - No statistically significant differences were noted between the control and test item treated groups and all values were within the WIL Research historical control ranges.
- Males that did not sire a litter numbered 0, 1, 1, 1, and 2 in the control, 100, 300, 500, and 1000 mg/kg/day groups, respectively.
- Females that had evidence of mating but did not deliver numbered 0, 0, 1, 1, and 2 in the same respective groups.
- The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
- Mean gestation lengths in the 100, 300, 500, and 1000 mg/kg/day groups were similar to those in the control group.
- No statistically significant differences were noted. No signs of dystocia were noted in these groups.

MACROSCOPIC EXAMINATIONS
- There were no test item-related gross changes.
- The mean numbers of unaccounted-for sites and implantation sites in the 100, 300, 500, and 1000 mg/kg/day groups were similar to the control group values.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEC
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
PND0 LITTER DATA AND POSTNATAL SURVIVAL
- The mean number of pups born, live litter size and the percentage of males at birth in the 100, 300, 500, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test item administration.

GENERAL PHYSICAL CONDITION OF F1 PUPS
- The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of the test item.
- Pups that were found dead numbered 14(4), 3(2), 3(3), 2(2), and 6(3) in the control, 100, 300, 500, and 1000 mg/kg/day groups, respectively. Two, 2, 2, and 3 pups each in the 100, 300, 500 and 1000 mg/kg/day groups, respectively, was missing and presumed to have been cannibalised.

OFFSPRING BODY WEIGHTS
- Mean male and female pup body weights and body weight changes during PND 1-4 in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by parental administration of the test item.
- No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead during PND 0-4 numbered 14(4), 3(2), 3(3), 2(2), and 6(3) in the control, 100, 300, 500, and 1000 mg/kg/day groups, respectively.
- Aside from the presence or absence of milk in the stomach, no other internal findings were noted in the test item-treated groups.
- Pup no. 5920-08 in the control group had visceral developmental variations that included a pale liver and an accessory lobule in the liver.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item-related effects were observed on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

ANALYSIS OF DOSING FORMULATIONS

- The analysed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%) and were homogeneous with noted exceptions. Summary tables detailing exceptions are attached.

- The mean concentration results of the initial assessment of the 20, 60, 100, and 200 mg/mL formulations prepared and processed on 14-Jan-2015 failed to meet acceptance critera. The back-up samples were processed and analysed on 15-Jan-2015.

- The mean concentrations of the 20 and 60 mg/mL formulations were 81.4% and 82.4% of the target concentrations, respectively, and failed to meet acceptance criteria for concentration. In addition, the 100 and 200 mg/mL back-up samples had 15% RSD and failed to meet acceptance criteria for homogeneity. 

- These formulations were not used for dose administration; therefore, there was no impact on the study. 

- Furthermore, all formulations were re-prepared and analyzed on 15-Jan-2015 with results that met the WIL Research SOP acceptance criteria for homogeneity and concentration. 

- The test item was not detected in the vehicle formulation that was administered to the control group (Group 1).

BODY WEIGHTS

- Mean body weights and body weight gains in the 100, 300, 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.

- Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating or entire dosing and recovery periods. Differences from the control group were slight and not statistically significant, with the following exceptions. 

- A significantly (p<0.05) higher mean body weight gain was noted for the 500 mg/kg/day group compared to the control group during study days 7-13 which resulted in a significantly (p<0.05) higher mean body weight gain for this group when the overall pre-mating period (study days 0-13) was evaluated. In the absence of a dose response, these differences were not attributed to the test item.

- In addition, a significantly (p<0.01) higher mean body weight gain was noted for the 1000 mg/kg/day group when compared to the control group during study days 20-27. This transient increase in mean body weight gain did not affect absolute mean body weights or the overall dosing period interval (study days 0-39) and therefore was not considered test item-related. 

- During the recovery period, a significant (p<0.05) mean body weight loss was noted for the 1000 mg/kg/day group females when the entire recovery period (study days 39-52) was evaluated. 

- In the absence of body weight effects during the dosing period and effects on absolute body weights, this difference was not attributed to the test item.

FOOD CONSUMPTION

- Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group males was similar to that in the control group throughout the study. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05 or p<0.01) lower mean food consumption was noted for the 300 mg/kg/day group during study days 0-7 and the 300, 500, and 1000 mg/kg/day groups during study days 27-34. In the absence of a dose response and/or lack of corresponding effects on body weights and body weight gains, these differences were not attributed to test item administration.

- Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test item administration during the pre mating or entire dosing and recovery periods. None of the differences from the control group were statistically significant.

- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, 500, and 1000 mg/kg/day groups was unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.

- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 300, 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.

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 concentrations when evaluated during study week 4 (males) and 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 Research historical control data. 

- Differences from the control group were slight, not statistically significant when analysed by a repeated measures analysis, within the WIL Research 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 treated groups when the F0 animals were evaluated during study week 4 (males) or on lactation day 4 (females).

HEMATOLOGY AND COAGULATION

- There were no test item-related alterations in hematology and coagulation parameters. However, a significant difference (p<0.05) was observed when the control and test item treated groups were compared. 

- Hematocrit was lower in the 1000 mg/kg/day group males at the recovery necropsy. Values were within the WIL Research historical control range and this change was not observed at the primary necropsy. Therefore, this alteration was considered due to biologic variation and not administration of the test item. 

- There were no statistically significant alterations in hematology and coagulation parameters in females.

- Statistically significant findings that involved percentage leukocyte differential counts were not itemised above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

SERUM CHEMISTRY

- There were no test item-related effects on serum chemistry parameters. 

- Some statistically significant differences were observed when the control and test item treated groups were compared. 

- Alanine aminotransferase (ALT) was higher in the 1000 mg/kg/day group males (p<0.05) at the primary necropsy. Although the group mean was slightly outside the WIL Research historical control mean value, individual animal values were within the WIL Research historical control range and were generally within the range of the concurrent control group. Additionally, this change did not correlate to any histologic findings. Therefore, the higher group mean ALT in the 1000 mg/kg/day group males was considered due to biologic variation and not administration of the test item.

 - Potassium was lower in the 500 mg/kg/day group females only at the primary necropsy. This change lacked a dose-response, and was therefore considered spurious. 

- At the recovery necropsy, globulin was higher in the 1000 mg/kg/day group females (p<0.05) and lower in the 1000 mg/kg/day group males (p<0.05), triglyceride was higher in the 1000 mg/kg/day group females (p<0.05), and total bilirubin was lower in the 1000 mg/kg/day group males (p<0.01). Changes in these parameters were not observed at the primary necropsy, and all values were within the WIL Research historical control ranges. Therefore, all were considered due to biologic variation and not administration of the test item. 

- Higher alkaline phosphatase (ALP) was also observed in the 1000 mg/kg/day group males (p<0.05). Although the group mean was slightly outside the WIL Research historical control mean values, individual animal values were within the WIL Research historical control range. Additionally, this alteration was not observed at the primary necropsy, and was therefore considered not due to administration of the test item.

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.

- Lower group mean absolute and relative (to body and brain weights) thymus weights were observed for males at ≥300 mg/kg/day (p<0.05 or p<0.01) at the primary necropsy. No statistically significant effects on thymus weights were observed in the 1000 mg/kg/day group males at the recovery necropsy. The lower thymus weights correlated with the gross change of small thymus in 1 male and the histopathologic finding of minimal lymphocyte depletion of the thymus in 2 males from the 1000 mg/kg/day primary necropsy group. These organ weight changes were not associated with a dose response, group means were within the WIL Research historical control database ranges, and there were no correlating clinical pathology changes. Additionally, in females, there were no thymus weight changes at the primary necropsy; while at the recovery necropsy, higher absolute and relative (to brain weight) thymus weights were observed at 1000 mg/kg/day. Therefore, the decrease in absolute and relative thymus weights was considered likely due to biologic variation and not the test item.

- Higher thyroid/parathyroid absolute and relative to body and brain weights were noted in the 500 and 1000 mg/kg/day primary group males (p<0.05 or p<0.01). This alteration was not associated with any gross or histopathologic changes. Additionally, in the 500 mg/kg/day group males, all group mean and individual animal values were within the WIL Research historical control ranges. In the 1000 mg/kg/day group, all group mean values and the majority of individual animal values were also within the WIL Research historical control ranges. Therefore, the changes were considered within the range of normal biologic variability, and not due to the test item. Also at the primary necropsy, absolute and relative (to body or brain weights) kidney weights were lower in the 100, 300, and 500 mg/kg/day group males (p<0.05). These changes lacked a dose response and were considered spurious and not related to test item administration. At the recovery necropsy, several other statistically significant changes were noted in organ weights, but these changes were within the WIL Research historical control ranges and were considered due to biologic variation and not due to administration of the test item. In the 1000 mg/kg/day group males, these changes included lower absolute left testis weight and left testis relative to brain weight (p<0.01). In the 1000 mg/kg/day group females these changes included higher absolute adrenal weight and adrenal relative to body and brain weights (p<0.01 or p<0.05); higher absolute heart and heart relative to brain weights (p<0.01 or p<0.05); and higher absolute thymus and thymus relative to brain weights (p<0.05).

MICROSCOPIC EXAMINATIONS

- All female rats that were 4 days post-lactation at the time of euthanasia exhibited lactational anestrus. Nongravid animals were not staged for estrus as uteri from these females were discarded at necropsy following ammonium sulfide staining for identification of implantation sites.

- There were no test item-related histologic changes. At the primary necropsy, minimal lymphocyte depletion of the thymus was observed in 2 males from the 1000 mg/kg/day primary necropsy group. This change correlated with the gross change of small thymus in 1 male at 1000 mg/kg/day and lower absolute and relative thymus weights. The thymus was, therefore, identified as a potential target organ and examined microscopically in the 100, 300, and 500 mg/kg/day primary group males. Lymphocyte depletion was not observed in males dosed at or below 500 mg/kg/day. As discussed under Organ Weights (Section 6.2.9.2.), the thymus weight changes were not associated with a dose response, group means were within the WIL Research historical control database ranges, and there were no correlating clinical pathology changes. Additionally, in females, there were no thymus weight changes at the primary necropsy or histopathologic findings of lymphocyte depletion; while at the recovery necropsy, higher absolute and relative (to brain weight) thymus weights were observed at 1000 mg/kg/day. As such, the histologic and organ weight changes observed in the thymus of males at the primary necropsy were considered due to biologic variation and not the test item. 

- 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.

Applicant's summary and conclusion

Conclusions:
The developmental toxicity potential of the test item was assessed in accordance with OECD Guideline 422. Under the conditions of this screening study, no test item-related effects on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no‑observed-adverse-effect level (NOAEL) for reproductive toxicity of test material when administered orally by gavage to Crl:CD(SD) rats. 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, 300, 500, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.
Executive summary:

GUIDELINE

The study, conducted in accordance with OECD 422, 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.

 

METHODS

 

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 1000 mg/kg/day group (Group 5) consisted of 15 rat/sex and the 100, 300, and 500 mg/kg/day groups (Groups 2, 3, and 4, respectively) consisted of 10 rats/sex. The dosage volume was 5 mL/kg. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 11 weeks of age at the beginning of test item administration. Ten males/group received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 35 doses. Ten females/group received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-44 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 postmating day 25, respectively) for a total of 39-52 doses. The extra 5 males and 5 females in the control and 1000 mg/kg/day groups that were not used for mating were treated beginning on study day 0; following 35 doses for males and 40 doses for female, these animals were assigned to a 14- or 13-day nondosing recovery period, respectively. 

 

All animals were observed twice daily for mortality and moribundity. 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 coagulation and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy following the dosing period and from 5 F0 animals/sex in the control and 1000 mg/kg/day groups at the recovery necropsy. 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, on postcohabitation or post-mating day 25 for females that failed to deliver, or following the 13-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 at the primary and recovery necropsies.

 

RESULTS

 

All F0 males and females in the control, 100, 300, 500, and 1000 mg/kg/day groups survived to the scheduled necropsies. Test item-related clinical findings noted for males and females in all test item-treated groups consisted of clear and/or red material around the mouth and/or red material around the nose approximately 1 hour following dose administration. These findings did not persist to the daily examinations on the following days and therefore were not considered adverse. In addition, clinical findings noted during the recovery period were limited to hair loss on the facial area for the 1000 mg/kg/day group F0 females.

 

No test item-related effects on F0body weight or food consumption parameters were noted for males and females at any dosage level. There were no test item-related effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level. 

 

Mean male and female mating, fertility, copulation, and conception indices in the 100, 300, 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, microscopic findings, or effects on organ weights noted for F0 males and females at any dosage level during the dosing and recovery periods. No test item-related alterations in clinical pathology parameters (hematology, coagulation, and serum chemistry) were noted for F0 males and females in the 100, 300, 500, and 1000 mg/kg/day groups during the dosing and recovery evaluations. 

 

Mean numbers of former implantation sites and F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, and pup body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test item administration. Necropsy findings for F1 pups that died were not suggestive of any association with maternal administration of the test item.

 

CONCLUSION

 

Under the conditions of this screening study, no test item-related effects on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the noobserved-adverse-effect level (NOAEL) for reproductive toxicity of test material when administered orally by gavage to Crl:CD(SD) rats. 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, 300, 500, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.