4-[Difluor(3,4,5-trifluorphenoxy)methyl]-3,5-difluor-4'-propyl-1,1'-biphenyl

Endpoint summary

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:

03 March 2017-10 August 2017

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 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050: Repeated dose 28-day oral toxicity study in rodents,

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: males: approximately 10-12 weeks, females: at pre-test approximately 9-10 weeks, at the start of treatment approximately 10-12 weeks.
- Weight at study initiation: males 247-296 g, females 196-240 g
- Fasting period before study: none
- Housing: general: sterilized sawdust was provided as bedding material and paper was provided as cage-enrichment/nesting material. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cages without cage-enrichment, bedding material, food and water.
Females:
Pre-test and pre-mating: 5 females/cage in Macrolon plastic cages
Mating: cohabitated on a 1:1 basis in Macrolon plastic cages
Post-mating: individually in Macrolon plastic cages
Lactation: in Macrolon plastic cages; pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Males:
Pre-mating: 5 males/cage in Macrolon plastic cages
Mating: cohabitated on a 1:1 basis in Macrolon plastic cages
Post-mating: in their home cases, 5 animals/cage
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except during motor activity measurements when animals were deprived of food and water for max. 2 hours.
- Water: free access to tap water, except during motor activity measurements when animals were deprived of food and water for max. 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 42-60
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 March 2017 (allocation) To: 10 August 2017 (last data collected).

Route of administration:

oral: gavage

Details on route of administration:

This study should provide part of a rational basis for toxicological risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Methocel® K4M; 0.25% (w/v) hydroxypropyl methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Preparation of 1000 mL vehicle
1. Approximately 1/3 of the required volume of Elix water was heated to at least 90°C.
2. 2.5 gram Methocel® K4M powder was added to the heated water with agitation.
3. The mixture was agitated until the particles were thoroughly wetted and evenly dispersed.
4. For complete solubilization, the remainder of the Elix water was added with agitation as cold water to lower the temperature of the dispersion. Once the dispersion reached the temperature at which the Methocel® K4M powder began to hydrate and viscosity increased.
5. Agitation was continued for at least 30 minutes.

Method of formulation:
Formulations (w/w) were prepared daily. The required amount of test item was weighed and the required amount of vehicle added. After homogenization with a blender, the container was wrapped in aluminium foil and the dose preparation was stirred on a magnetic stirrer at room temperature overnight (≥ 16 hours). No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity/composition of the test item.
The formulations were used within 4-6 hours after release by the formulation room.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on information provided by the sponsor and trial formulations performed at Charles River Den Bosch.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted during the treatment phase according to a validated method. Samples of dose preparations were taken after overnight stirring and released on the following morning. Samples of formulations were analyzed for homogeneity and accuracy of preparation. Stability in vehicle over 6 hours and 4 hours at room temperature under normal laboratory light conditions was also determined.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

Males: 29 days (2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy)
Females that delivered healthy offspring (controls only): 50-55 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females that failed to deliver healthy offspring (all females administered the test item) were treated for 39-44 days.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose, if possible

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle controls, Group 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Remarks:

Group 3

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a dose range finding study in which male and female Wistar Han rats were treated by oral gavage for 28 days at dose levels of 0, 30, 60 and 100 mg/kg bw/day.
Treatment at 100 mg/kg bw/day caused severe toxicity. Next to mortality (one female on Day 8), clinical signs (piloerection, hunched posture), marked body weight loss together with severely reduced food consumption were noted during the first week of treatment, followed by partial recovery thereafter. No adverse effects were observed at 30 and 60 mg/kg bw/day during in-life. At end of treatment, changes in haematology and clinical biochemistry parameters were noted in all treated groups. These consisted of higher absolute WBC count together with a higher neutrophil count (males at 30, 60 and 100 mg/kg bw/day and females at 100 mg/kg bw/day), lower RBC count together with lower haemoglobin and haematocrit (females at 30, 60 and 100 mg/kg bw/day), a trend towards higher ASAT (males at 30, 60 and 100 mg/kg bw/day) and ALP (both sexes at 30, 60 and 100 mg/kg bw/day), and lower albumin (both sexes at 30, 60 and 100 mg/kg bw/day). At necropsy, all treated males had testes that were flaccid and except for two males at 30 mg/kg bw/day all testes were reduced in size. In addition, the epididymides of all males at 60 mg/kg bw/day were reduced in size. A trend towards higher liver weights (absolute and/or relative to body weight) was noted in males and females at 30, 60 and 100 mg/kg bw/day. Kidney weights were unaffected by treatment up to 100 mg/kg bw/day (both sexes).
- Rationale for animal assignment (if not random): based on the pre-test 40 females with at least two regular estrous cycles were selected at random and further used in the study.
- Section schedule:
Males: following completion of the mating period (a minimum of 28 days of dose administration).
Females that delivered: PND 14-16
Females that failed to deliver or with suspected total litter loss: Post-coitum Days 25-27 (females with evidence of mating) or approximately 24-26 days after the last day of the mating period (females without evidence of mating)
Females with total litter loss observed: within 24 hours from litter loss.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour (± 30 min) after dosing (on the peak period of anticipated effects after treatment). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: selected 5 animals/sex/group
- Parameters examined: WBC, differential leucocyte count, RBC, reticulocytes (as % RBC), RDW, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets, PT, APTT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on the day of scheduled necropsy
- Animals fasted: Yes / No / Not specified
- How many animals: selected 5 animals/sex/group
- Parameters examined: ALAT, ASAT, ALP, total protein, albumin, total billirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: selected males during week 4 of treatment; selected females on PND 7-8 (females with live offspring, 0 mg/kg bw/day treatment group), PND 1 (females with total litter loss; 5 mg/kg bw/day treatment group) or once during Days 24-26 post-coitum (non-pregnant females and females with implantation sites only; 15 and 50 mg/kg bw/day groups). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: selected 5 animals/sex/group from all groups
- Battery of functions tested: sensory activity (heating, pupillary reflex, static righting reflex), grip strength (fore- and hind-limb), motor activity (total movements and ambulations)

IMMUNOLOGY: No

OTHER:
Thyroid hormone analysis:
End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and
all males after at least 4 weeks of treatment (including all males that failed to sire).
Note: Females of the 5, 15 and 50 mg/kg bw/day treatment groups were sacrificed almost 2 weeks earlier than those of the 0 mg/kg bw/day group due to the lack of offspring or early total litter loss of treated females
For males, 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroidstimulating hormone (TSH).
For females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).

Estrous cycle determination:
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of
copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
During pretest, this was done for 48 females.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

For examinations conducted on pups and general reproduction data see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs.
The following organ weights were recorded:
Selected 5 animals/sex/group: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid including parathyroid if detectable, uterus including cervis.
All remaining animals: epididymides, prostate, seminal cesicles including coagulation glands, testes, tyroid including parathyroid if detectable.

HISTOPATHOLOGY: Yes
The following slides were examined by the pathologist:
• The preserved organs and tissues of the selected 5 animals/sex of the 0 and the 50 mg/kg bw/day treatment group.
• Additional slides of the testes of the selected 5 males of the 0 and 50 mg/kg bw/day treatment group and all males that failed to sire to examine staging of spermatogenesis.
• Thymus, thyroid gland, liver, spleen and reproductive organs from all selected 5 animals of the 5 and 15 mg/kg bw/day treatment group, based on (possible) treatment-related changes in these organs in the 50 mg/kg bw/day treatment group.
• All gross lesions of all animals (all dose groups).
• The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups or had a total litter loss. In addition, histopathological examination of the mammary gland was conducted for the females that had total litter loss, except for Group 3.

For examinations of pups see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references)

Other examinations:

See cross-references for general reproduction data and examinations conducted on pups.

Statistics:

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed in males up to 50 mg/kg bw/day.
In females, treatment-related clinical signs of toxicity were noted from 5 mg/kg bw/day onward, generally starting after about five weeks of treatment (i.e. towards the end of the post-coitum period). The main finding was piloerection which was noted in most females at 15 and 50 mg/kg bw/day and in one female at 5 mg/kg bw/day. Hunched posture was noted in one female at 15 mg/kg bw/day and three females at 50 mg/kg bw/day.
No additional clinical signs of toxicity were noted during the arena observations. Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

no mortality observed

Description (incidence):

There were no premature decedents in both sexes. All test item treated females failed to deliver healthy pups and were sacrificed about 1 to 2 weeks before the control females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: body weight gain was slightly reduced, to about the same extent, at 15 and 50 mg/kg bw/day (statistically significant from Day 1 of the mating period). Mean body weights of these males did not differ statistically significantly from those of controls (relative difference at the end of the treatment period: about 5%).
Females: lower body weight gain occurred at 5 mg/kg bw/day throughout the pre-mating period (statistically significant), and at 15 and 50 mg/kg bw/day throughout the pre-mating and post-coitum periods (statistically significant on Day 1 of the mating period and from Day 14 or Day 17 of the post-coitum period). A finding of note was that all three non-pregnant females in the 15 mg/kg bw/day group and all six non-pregnant females of 50 mg/kg bw/day group lost 4-9% of their body weight from post-coitum Days 11-20. In addition, the two females in the 50 mg/kg bw/day group with implantation sites only lost 10% and 9% of their body weight from Days 14-20 post-coitum and Days 17-20 post-coitum, respectively. This body weight loss resulted in statistically significantly reduced body weights towards the end of the postcoitum period (relative differences from controls at post-coitum Day 20: 16% and 26% at 15 and 50 mg/kg bw/day, respectively).
The relatively low body weight gain in the post-coitum period noted for one female of Group 4 was attributed to the fact that she had 2 implantations only.
Note: Body weight development of lactating treated females could not be evaluated due to the complete lack of healthy offspring at 5, 15 and 50 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: food consumption before or after correction for body weight was not affected by treatment up to 50 mg/kg bw/day.
Females: there was a trend towards lower food consumption before allowance for body weight from Day 7 of the post-coitum period at 15 and 50 mg/kg bw/day. The difference from controls was most marked and statistically significant at 50 mg/kg between Days 17-20. Food consumption after allowance for body weight was similar between treated and control females.
Note: Food consumption of lactating treated females could not be evaluated due to the lack of healthy offspring at 5, 15 and 50 mg/kg bw/day

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):

Males:
• Lower haemoglobin, haematocrit and mean corpuscular volume (MCV) at 50 mg/kg bw/day (11%, 12% and 6%, respectively). The mean number of red blood cells at this dose level was 6% lower compared to controls (not statistically significant).
• Higher percentage of neutrophils from 5 mg/kg bw/day onward in a dose-related manner (50%, 79% and 98% at 5, 15 and 50 mg/kg bw/day, respectively; not statistically significant at 50 mg/kg bw/day). The corresponding absolute numbers of neutrophils were 15%, 54% and 62% higher compared to controls (not statistically significant).
• Higher percentage of monocytes from 5 mg/kg onward in a dose-related manner (70%, 123% and 185% at 5, 15 and 50 mg/kg bw/day, respectively; not statistically significant at 50 mg/kg bw/day). The corresponding absolute numbers of monocytes remained close to the concurrent control values.
• Lower total white blood cell (WBC) count (26%, 17% and 22%) and lower percentage of lymphocytes (8%, 14%, 17% ) at 5, 15 and 50 mg/kg bw/day, respectively. It should be noted that changes in treated males compared to controls did not reach statistical significance, were within normal limits or slightly below (relative lymphocyte count at 50 mg/kg bw/day), and control values were at the upper end of the historical range.

Females:
Note: Treated females had no healthy offspring and were sacrificed at post-coitum Day 25-27 (non-pregnant females and females with implantations only) or shortly after parturition (PND 1-2; females with total litter loss). In contrast, all control females were lactating and sacrificed at PND 14-16. Treated females selected for blood sampling had the following physiological status: five with total litter loss at 5 mg/kg bw/day; one non-pregnant and four with suspected total litter loss at 15 mg/kg bw/day; two non-pregnant, one with implantations only and two with suspected total litter loss at 50 mg/kg bw/day.
Several clinical pathology parameters are known to be influenced by physiological status (lactating versus nulliparous). This was taken into account when assessing the possible relation with treatment of differences in clinical pathology values between treated females and controls. Where useful, values in treated females were compared with historical control data from sub-chronic (90-day) oral toxicity studies with the strain of rats used in this study (rats were about 19 weeks old at the time of blood sampling). Relative changes in mean values compared to the concurrent control group are indicated between parentheses.
• Lower haemoglobin from 5 mg/kg bw/day onward (16%, 17% and 8% at 5, 15 and 50 mg/kg bw/day, respectively; latter difference not statistically significant).
• Lower mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) from 5 mg/kg bw/day onward (9% for MCH, 5-8% for MCHC).
• Lower mean values for the number of red blood cells and haematocrit at 5 and 15 mg/kg bw/day (about 10%, not statistically significant).
• Higher percentage of reticulocytes at 5 and 15 mg/kg bw/day (60% and 130%, respectively). The value in one 15 mg/kg bw/day female was about five-fold higher compared to control values.
• Higher red blood cell distribution width (RDW) at 5 mg/kg bw/day (135%).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Male rats:
• Higher alkaline phosphatase (ALP) at 15 and 50 mg/kg bw/day (74 and 86%, respectively).
• Lower albumin at 15 and 50 mg/kg bw/day (10 and 13%, respectively).
• Lower total protein at 50 mg/kg bw/day (5%).
• Lower bile acids at 15 and 50 mg/kg bw/day (45 and 44%, respectively).
• Lower creatinine from 5 mg/kg bw/day onward (10, 8 and 8% at 5, 15 and 50 mg/kg, respectively; not statistically significant at 50 mg/kg bw/day, but all mean values at the lower end or just below the historical range).

Females:
• Higher alkaline phosphatase (ALP) at 50 mg/kg bw/day (69%).
• Lower albumin at 15 and 50 mg/kg bw/day (7 and 14%, respectively).
• Lower total protein at 15 and 50 mg/kg bw/day (7% at both dose levels).
• Lower creatinine from 5 mg/kg bw/day onward (17% at 5 mg/kg bw/day, 20% at the higher dose levels).
The following statistically significant differences between treated females and concurrent controls were considered to be due to the difference in physiological status: lower values for alanine aminotransferase (ALAT) activity, cholesterol, bile acids, urea and inorganic phosphate, and higher values for sodium and chloride.
The other differences noted in treated females were considered unrelated to treatment due to somewhat high concurrent control values (aspartate aminotransferase (ASAT) activity) or because they occurred in the absence of a dose-related trend (lower ALP and higher fasting glucose at 5 mg/kg bw/day).
Thyroid hormone analyses (males only):
The serum T4 level of F0-males was statistically significantly decreased from 5 mg/kg bw/day onward (relative differences from controls: 24, 41 and 54% at 5, 15 and 50 mg/kg bw/day, respectively). Mean value for the 5 mg/kg bw/day group remained within the historical control range, but was clearly lower for the 15 and 50 mg/kg bw/day group. At the individual level, T4 values in a few 5 mg/kg bw/day males and in most males at the higher dose levels were below the historical control range.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly lower mean values for hind limb grip strength were noted at 15 and 50 mg/kg bw/day in both sexes (relative differences from controls: about 30-40%). Values in treated animals remained in the normal historical range for rats of this strain and age, except for those of two females in Group 3 and two females in Group 4 which were below this range.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
The lower motor activity noted in females treated at 5 mg/kg bw/day, particularly from test interval 4 onwards (i.e. >15 minutes), occurred in the absence of a dose-related trend and was therefore considered to be unrelated to treatment. One female at 15 mg/kg bw/day showed low motor activity throughout the 1-hour test period. This might be related to her health condition (she showed piloerection and had an abnormal pregnancy). It was noted that two other 15 mg/kg bw/day females and several 50 mg/kg bw/day females with piloerection and abnormal pregnancies showed normal motor activity. This female had normal outcomes for other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Therefore, her lower motor activity was considered not to represent a direct effect of the test item on this neuromuscular endpoint.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males: statistically significant, test item-related organ weight changes were present in:
• Liver: Increased relative weight at 15 and 50 mg/kg bw/day
• Testes: Decreased absolute and relative weight at 15 and 50 mg/kg bw/day.
• Epididymides: Decreased absolute weight at 15 mg/kg bw/day and absolute and relative weight at 50 mg/kg bw/day
• Seminal vesicles: Decreased absolute weight at 50 mg/kg bw/day
• Adrenal gland: Increased relative weight at 50 mg/kg bw/day
The statistically significantly lower absolute thymus weight at 15 mg/kg was considered not to be related to treatment due to the lack of a dose-related pattern.

Females:
There were statistically significant organ weight changes at 15 and 50 mg/kg bw/day in liver (decreased absolute and relative weight) and thymus (increased relative weight). These organ weight changes were regarded to be related to the different physiologic state of the test item-treated females compared to the controls: lactating females show a decreased thymus weight and increased liver weight compared to non-lactating females. In the current study all females of the control group were lactating, compared to none of the test item-treated groups. There were no microscopic correlates to these weight changes. Therefore these weight changes in liver and thymus of females were regarded to be unrelated to the treatment with the test item.
Other organ weight changes of note were present in females at 5 mg/kg bw/day and consisted of an increased relative spleen weight (microscopic correlate: increased hematopoiesis) and increased absolute and relative ovary and uterus weight (considered correlating to the time of necropsy: shortly after delivery of the pups). To a lesser extent, the weights of the ovaries and uterus were also increased in females at 15 and 50 mg/kg bw/day, which was considered to be related to their physiologic state (not pregnant, implantations only or suspected total litter loss).
The remaining statistically significant organ weight changes in females were in line with the lower terminal body weight (kidneys, brain, adrenals) or unrelated to treatment due to the lack of a dose-related pattern (thyroid gland).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic findings were noted in males at all dose levels as listed below.
• Testes: flaccid and/or reduced in size in 1/10 males at 5 mg/kg bw/day and all 10 males at 15 and 50 mg/kg bw/day.
• Epididymides: reduced in size in 1/10 males at 15 and 50 mg/kg bw/day.
• Prostate gland: reduced in size in 1/10 males at 5 mg/kg bw/day and 3/10 males at 50 mg/kg bw/day.
There were no test item-related macroscopic findings in female rats.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were observed in the thymus, thyroid gland, liver, spleen, and male and female reproductive organs, as described below.
• Thymus: Increased apoptosis of lymphocytes was recorded in males starting at 15 mg/kg bw/day and in females starting at 5 mg/kg bw/day.
• Thyroid gland: An increased incidence and severity of hypertrophy of follicular cells was recorded in males starting at 15 mg/kg bw/day and in females at 50 mg/kg bw/day.
• Liver: An increased incidence and/or severity of centrilobular hepatocellular hypertrophy was recorded in males starting at 15 mg/kg bw/day.
• Spleen: An increased incidence and severity of extramedullary hematopoiesis was recorded in males and females at 50 mg/kg bw/day.
The high incidence and severity of extramedullary hematopoiesis in the spleen (and liver) of females at 5 and 15 mg/kg bw/day was considered to be mainly related to blood loss, due to abnormal pregnancies.
• Testes: Tubular degeneration/atrophy was recorded in a few males at 5 mg/kg bw/day and in all males at 15 and 50 mg/kg bw/day. In most males at 5 mg/kg bw/day and one male at 15 mg/kg bw/day sperm retention and/or tubular vacuolation, which are considered precursor findings of tubular degeneration/atrophy, were recorded. The PAS-stain showed no normal stages or all stages degenerative in all males at 15 and 50 mg/kg bw/day. In males of the control group and 5 mg/kg bw/day group all spermatogenic stages were present.
• Epididymides: Cell debris was recorded in a few males at 5 mg/kg bw/day and in all males at 15 and 50 mg/kg bw/day. Reduced sperm was recorded in all males at 15 and 50 mg/kg bw/day. These findings were regarded to be secondary to the degenerative changes in the testes.
• Uterus: Vascular necrosis at the implantation sites was recorded in six females at 5 mg/kg bw/day and one female at 15 mg/kg bw/day.
Hemorrhage in the uterine lumen and/or implantation sites was recorded in seven females at 5 mg/kg bw/day and three females at 15 mg/kg bw/day. The hemorrhage was considered to be related to the vascular necrosis and/or recent parturition.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

None of the treated animals had healthy offspring. Treatment at 50 mg/kg bw/day resulted in abnormal estrous cycles in 7/10 females: one female (not pregnant) had an irregular cycle (persistent di-estrus); six females (not pregnant, implantations only or suspected total litter loss) had an acyclic cycle (at least 10 days without estrus). The remaining three females (all not pregnant) had regular cycles of 4 days.
At 15 mg/kg bw/day, one female (suspected total litter loss) had an irregular cycle (persistent di-estrus). An irregular cycle occasionally occurs at low incidence in untreated controls. However, taken in the context of the cycling abnormalities at 50 mg/kg bw/day, it cannot be excluded that the irregular cycle in this female was related to treatment.
Length and regularity of the estrous cycle were not affected at 5 mg/kg bw/day. All 5 mg/kg bw/day females had regular cycles of 4 days.
For further details on reproductive and developmental toxicity see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).

Details on results:

There were no premature decedents in males up to 50 mg/kg bw/day and control females. All test item treated females failed to deliver healthy pups and were sacrificed about 1 to 2 weeks before the control females.
Clinical signs of toxicity occurred in treated females towards the end of the post-coitum period. These included piloerection in one female at 5 mg/kg bw/day and most females at 15 and 50 mg/kg bw/day and hunched posture in a few females at the higher dose levels. It cannot be excluded that these clinical signs, at least in part, were secondary to pregnancy difficulties.
Functional observations showed lower mean values for hind limb grip strength at 15 and 50 mg/kg bw/day in both sexes. As there were no corroborative clinical signs or changes in other measures in the neuromuscular domain (including forelimb grip strength, gait, air righting reflex and motor activity), these differences in hind limb grip strength were considered not to be adverse.
Body weight gain was reduced at 15 and 50 mg/kg bw/day in both sexes throughout the treatment period, and at 5 mg/kg bw/day in females during the first two weeks of treatment. In males, the resulting decreases in body weight were slight (about 5% compared to concurrent control at the end of treatment) and considered non-adverse. Females at 15 and 50 mg/kg bw/day initially had only slightly lower mean body weights (up to about 5%), but between Days 11-20 of the post-coitum period, 4-9% body weight loss occurred in all non-pregnant females in the 15 mg/kg bw/day (n=3) and 50 mg/kg bw/day (n=6) groups. In addition, the two females in the 50 mg/kg bw/day group with implantation sites only lost 10% and 9% of their body weight from Days 14-20 post-coitum and Days 17-20 post-coitum, respectively. This weight loss was considered adverse. Females at 15 and 50 mg/kg bw/day consumed less food than controls during the post-coitum period. A possible relation with the observed aborted pregnancies and/or pup mortality cannot be excluded.
Changes in haematology parameters were noted in males at 50 mg/kg bw/day and females at all dose levels. The decreases in haemoglobin and haematocrit, accompanied by extramedullary hematopoiesis in the spleen, in 50 mg/kg bw/day males were considered adverse as the differences from controls (slightly) exceeded 10%. Increases in neutrophils (percentage and absolute number) and monocytes (percentage only) together with decreases in lymphocytes (percentage and absolute number) were recorded in males from 5 mg/kg bw/day onward. Total white blood cell count was lower in all treated groups. In the absence of morphological correlates, these changes were likely to be non-adverse.
The red blood cell changes in females at 5 and 15 mg/kg bw/day (lower haemoglobin, haematocrit, red blood cell count, MCH and MCHC, higher percentage of reticulocytes and red blood cell distribution width), accompanied by extramedullary hematopoiesis in spleen and liver, were considered to be mainly related to blood loss due to abnormal pregnancies. The decreases in MCH and MCHC in females at 50 mg/kg bw/day were considered non-adverse as they occurred in the absence of decreases in haemoglobin, red blood cell count and haematocrit.
Clinical biochemistry changes consisted of higher alkaline phosphatase (ALP) activity and lower albumin and total protein starting at 15 mg/kg bw/day in both sexes, lower creatinine starting at 5 mg/kg bw/day in both sexes and lower bile acids in males at 15 and 50 mg/kg bw/day. Based on their modest magnitude (ALP less than two-fold, total protein less than 10%, creatinine approximately 10% in males and 20% in females, and bile acids approximately 45% in males) and/or absence of morphologic correlates, these changes were likely to be nonadverse.
Serum levels of thyroid hormone T4 (measured only in males) were decreased from 5 mg/kg bw/day onward in a dose-related manner. At 15 and 50 mg/kg bw/day most values were below the historical control range, and the incidence and severity of follicular cell hypertrophy in the thyroid were increased in males from 15 mg/kg bw/day and females at 50 mg/kg bw/day (thyroid weight was unchanged). Therefore, the decreases in T4 at 15 and 50 mg/kg bw/day were regarded as adverse. The decrease in T4 at 5 mg/kg bw/day was regarded as non-adverse based on its slight magnitude (in the normal range) and the absence of accompanying thyroid changes.
Non-adverse, treatment-related microscopic changes were observed in the thymus (increased apoptosis of lymphocytes in males from 15 mg/kg bw/day and females from 5 mg/kg bw/day), thyroid (see above), spleen (increased extramedullary haematopoiesis in both sexes at 50 mg/kg bw/day), and liver (hepatocellular hypertrophy, correlating to increased liver weight, in males from 15 mg/kg bw/day). Based on their low severity and the absence of accompanying degenerative or inflammatory changes, these findings were regarded as non-adverse. No microscopic correlate was observed for the decreased absolute weight of seminal vesicles and increased relative weight of adrenal glands in males at 50 mg/kg bw/day. Therefore, these changes were considered to be treatment-related, but not adverse.
Adverse morphological changes in parental reproductive organs were observed from 5 mg/kg bw/day onward (in males at all dose levels, in females at 5 and 15 mg/kg bw/day):
Microscopic testicular changes consisted of tubular degeneration/atrophy (in a few males at 5 mg/kg bw/day and all males at 15 and 50 mg/kg bw/day), sperm retention and/or tubular vacuolation (precursor lesions of tubular degeneration/atrophy, at 5 and 15 mg/kg bw/day), and absence of normal spermatogenic stages/all stages degenerative (at 15 and 50 mg/kg bw/day). The changes in the
epididymides (cellular debris from 5 mg/kg bw/day, reduced sperm from 15 mg/kg bw/day) were considered to be secondary to the degenerative changes in the testes. These microscopic changes correlated with a flaccid appearance of the testes and reduced testicular and epididymidal size and weight.
Microscopic changes in the uterus consisted of vascular necrosis of arteries at the implantation sites and hemorrhage in the uterine lumen and/or implantation sites in most females at 5 mg/kg bw/day and a few females at 15 mg/kg bw/day. These morphological changes may at least in part be the underlying cause of the aborted pregnancies and pup mortality observed in this study.
Other treatment-related adverse changes in reproductive parameters included abnormalities in estrous cyclicity (1/10 female at 15 mg/kg bw/day and 7/10 females at 50 mg/kg bw/day), and decreases in the number of implantation sites and fertility index at 15 and 50 mg/kg bw/day (respectively, 7/10 and 4/10 females with evidence of mating were pregnant).
Both, the adverse morphological changes in testes, epididymides and uterus and abnormalities in estrous cyclicity were regarded as the cause of the lack of (healthy) offspring in the test item-treated dose groups.
For further details on reproductive and developmental toxicity see sections 7.8.1 and 7.8.2 of this IUCLID (cross-references).

Key result

Dose descriptor:

LOAEL

Effect level:

5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

cauda epididymis

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/kg bw/day (actual dose received)

System:

female reproductive system

Organ:

uterus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

15 mg/kg bw/day (actual dose received)

System:

endocrine system

Organ:

thyroid gland

The concentrations analyzed in the formulations of the 5 -50 mg/kg bw treatment groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%; actual range: 97-102%, n = 6 for the 5 mg/kg bw group and the 50 mg/kg bw group, n=2 for the 15 mg/kg bw group) and prepared homogenously (i.e. coefficient of variation ≤ 10%; actual range: 1.2-6.1%), except for those of the 5 mg/kg bw treatment group prepared and analyzed on treatment day 2 (mean accuracy of 83%). This lower accuracy was attributed to inhomogeneity of the formulation (coefficient of variation was 30%). To facilitate the homogenization process, it was decided to keep all formulations for a longer period on the magnetic stirrer. Formulations prepared according to this revised protocol were used first from treatment Day 4 onward and both accuracy and homogeneity were confirmed (coefficient of variation 6.1%, mean accuracy 97% (n = 6)).

The formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours (mean accuracy of 86% and 101% for groups 5 mg/kg bw and 50 mg/kg bw, respectively; n = 2 for both groups).

Conclusions:

In a GLP-compliant OECD guideline 422 study with rats treated by gavage, adverse test item-related effects were observed at the lowest dose level of 5 mg/kg bw/day. They were observed in reproductive organs, including testes (reduced weight, degeneration and atrophy together with tubular sperm retention), epididymides (luminal cell debris) and uterus (vascular necrosis of arteries at the implantation sites and hemorrhage in the uterine lumen and/or implantation sites). Remaining findings (at higher dose levels) considered related to the treatment with the test item were present in thymus (increased apoptosis, both sexes), thyroid glands (follicular cell hypertrophy, both sexes), spleen (increased extramedullary hematopoiesis, both sexes), male liver (hepatocellular hypertrophy, correlating to increased liver weight), and male adrenal gland (increased weight without microscopic correlates). Based on these findings, the lowest dose level of 5 mg/kg bw/day is considered to be a LOAEL for parental toxicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

other: histopathological findings

Justification for classification or non-classification

A classification and labelling of this substance as Repr Cat 1B is warranted.