Nitrosodiphenylamine

Endpoint summary

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction/developmental toxicity study (OECD 422) was performed on N-nitrosodiphenylamine. Animals were exposed to the test item at least 28 days. The NOAEL for parental systemic toxicity was considered to be 50 mg/kg/day based on the microscopic findings observed at 150 mg/kg/day in the urinary bladder in males and females.

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:

21 November 2017 - 26 February 2018

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:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

exception: on Days 34 (males) and 51-54 (females), following urobilinogen QC deviations at the beginning of the series on the positive control, the samples were analyzed with an invalid QC (urobilinogen data non GLP): no impact on the study conclusions

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age: on the first day of treatment, the males were approximately 10 weeks old and the females were approximately 11 weeks old
- Mean: on the first day of treatment, the males had a mean body weight of 411 g (range: 389 g to 426 g) and the females had a mean body weight of 274 g (range: 247 g to 312 g)
- Housing: F0 animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the males were acclimated to the study conditions for a period of 7 days before treatment and the females were acclimated to the study conditions for a period of 6 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 03 January 2018 to 26 February 2018.

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a route of administration which is recommended by the Regulatory Authorities for this type of study.
The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time of day.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING FORMULATIONS:
- Suspension in the vehicle (solution in the vehicle at 3 mg/mL)
- Concentration in vehicle: 3, 10 and 30 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV)
Test item concentrations: remained within an acceptable range of variations (-4.1% to +0.6%) when compared to the nominal values (criterion: ± 15% of the nominal concentrations).
Homogeneity/Stability: the dose formulations containing the test item at 3 mg/mL or at 10 and 200 mg/mL in corn oil were found to be homogeneous and stable after 24 hours or 2 days at room temperature and protected from light, respectively.

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- until sacrifice (at least 4 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- during gestation,
- during lactation until Day 13 post-partum inclusive,
- until euthanasia for females with no delivery.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose level selection:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous study performed in the same species, in which the test item was administered daily by gavage to five males and five females at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks.
The dose level of 1000 mg/kg/day was associated, in both sexes, with mortality, premature euthanasia of the surviving animals and signs of poor clinical condition (i.e. thin appearance, hunched posture, piloerection, pallor of extremities, half-closed eyes, hypoactivity, hypotonia, staggering gait, decreased grasping reflex and/or dyspnea), body weight loss leading to a lower body weight, markedly reduced food consumption and macroscopic changes in the stomach (white, black or red discoloration, gas distension), forestomach (white or red discoloration), intestine (distension with feces or with gas and/or thick or liquid brown content), spleen (enlargement), ureters (dilatation), kidneys (enlargement, pelvis dilatation, gelatinous hilus and perirenal adipose tissue and/or yellow discoloration), pancreatic lymph nodes (enlargement) and liver (enlargement and/or yellow discoloration). No microscopic observation was performed.
The dose level of 300 mg/kg/day was associated with poor clinical condition (i.e. and hunched posture and piloerection in one male and one female), body weight loss in males at the beginning of the treatment period, moderate reduced food consumption in both sexes during the first week of the treatment period, organ weight changes in the liver (absolute and relative liver weights moderately increased day in both sexes), the spleen (marked increases in the mean absolute and relative spleen weights in both sexes), and in the kidneys and the heart (mean absolute and relative kidney or heart weights minimally increased in females reaching statistical significance for the absolute weight), macroscopic changes in the spleen (enlargement in both sexes) and ureters (dilatation in males) and microscopic changes in the liver (slight to moderate hepatocellular hypertrophy and minimal hematopoiesis in both sexes), the spleen (increased severity of hematopoiesis and congestion along with increased hemosiderin in both sexes suggestive of hemolysis) and the kidneys (eosinophilic granules in renal tubules in males suggestive of hemolysis).
The dose level of 100 mg/kg/day was associated with slight reduced food consumption in females during the study, macroscopic changes in the spleen (enlargement in both sexes) and microscopic changes in the liver (minimal hepatocellular hypertrophy in males and minimal hematopoiesis in both sexes), and the spleen (increased severity of hematopoiesis in both sexes and congestion along with increased hemosiderin in females suggestive of hemolysis).

As the doses of 300 and 1000 mg/kg/day are considered to be higher than the Maximum Tolerable Dose (MTD), 150 mg/kg/day was selected as the high-dose level. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 15 and 50 mg/kg/day).

- Rationale for animal assignment: stratification procedure

Positive control:

no (not required)

Observations and examinations performed and frequency:

MORTALITY/MORBIDITY:
- Time schedule: Each animal was checked for mortality or signs of morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: detailed clinical examinations were performed on all animals, once before the beginning of the treatment period and then once a week until the end of the study.

CLINICAL SIGNS:
- Time schedule: from arrival, each animal was observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time.

BODY WEIGHT:
- Time schedule: the body weight of each male was recorded on the first day of treatment (Day 1), then once a week until euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated, on Days 0, 7, 14 and 20 p.c. (post-coitum) (and on the day of euthanasia for females which did not deliver), and on Days 1, 4, 8 and 13 p.p.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14 20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
During the mating period, food consumption was not measured for males or females.

WATER CONSUMPTION:
- Time schedule: the quantity of water consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of water consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
During the mating period, water consumption was not measured for males or females.

NEUROBEHAVIOURAL EXAMINATION:
Functional observation battery
The first five males and the first five surviving females euthanized on Day 14 p.p., from each group, were evaluated with a functional observation battery once at the end of the treatment period. For females, this was performed on Day 13 p.p. after euthanasia of the pups.

This included a detailed clinical examination, the assessment of reactivity to manipulation and to different stimuli and motor activity.
All animals were observed in the cage, in the hand and in the standard arena.

Detailed clinical examination
The following parameters were assessed and graded:
- in the cage: “touch escape” or ease of removal from the cage,
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

Reactivity to manipulation and different stimuli
The following measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupillary reflex,
- visual stimulus response,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.

Motor activity
Motor activity was measured once by automated infra-red sensor equipment over a 60-minute period.

HAEMATOLOGY: see tables 1 and 2
The parameters were determined on the day of euthanasia for the first five males and the first five females from each group euthanized on Day 14 p.p.

CLINICAL CHEMISTRY: see table 3
The parameters were determined on the day of euthanasia for the first five males and the first five females from each group euthanized on Day 14 p.p.

URINALYSIS: see table 4
The parameters were determined on the day of euthanasia for the first five males and the first five females from each group euthanized on Day 14 p.p.

THYROIDS HORMONES:
Blood samples were taken, in the first half of the morning (between 7.5 and 10 am), as follows:
- at termination on Day 4 p.p. from at least 2 pups/litter culled,
- at termination on Day 13 p.p. from at least 2 pups/litter,
- at termination on Day 14 p.p. from all F0 females,
- at termination from all F0 males.

The levels of the thyroid hormone (T4) and thyroid stimulating hormone (TSH) were determined respectively by LC-MS/MS or Luminex MAP® technology for samples from pups collected on Day 13 p.p. and from F0 males collected at termination.

Plasma samples obtained on Day 4 p.p. from pups and on Day 14 p.p. from F0 females were put into storage at 80°C pending possible analysis. As there was no indication of an effect on the thyroid glands, no analysis was performed.

Sacrifice and pathology:

ORGAN WEIGHTS: see table 5
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 14 p.p. for females) was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all F0 animals including females that died during the study or were euthanized prematurely. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.
The numbers of corpora lutea and implantation sites were recorded for females euthanized as scheduled on Day 14 p.p. and for females euthanized on Day 25 or 26 p.c. due to no delivery.
For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from the first five euthanized as scheduled males and the first five females euthanized on Day 14 p.p., of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions of all groups,
- reproductive organs and thyroids with parathyroids from from any animal that did not mate or conceive, or from pregnant females that did not deliver, to investigate possible causes: i.e. one male and one female (group 2), one male and one female (group 3) and one male and one female (group 4).

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

In agreement with the Sponsor and based upon the results of the microscopic examination of the high-dose group, other tissues of the low- and intermediate-dose groups were examined in the first five euthanized as scheduled males and the first five females euthanized on Day 14 p.p. as follows:
-heart (females only),
-kidneys (both sexes),
-liver (both sexes),
-sternum with bone marrow (females only),
-spleen (both sexes),
-urinary bladder (both sexes).
In agreement with the Sponsor and based upon the results of the microscopic examination of the high-dose group, microscopic examination of histological slides stained with Perl’s procedures were performed.

Statistics:

Body weight, food consumption
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Hematology, blood biochemistry, urinalysis, hormones, motor activity:
CITOX software was used to perform the statistical analysis.
Organ weight:
PATHDATA software was used to perform the statistical analysis of organ weight data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See table 2.
Non-adverse test item-related clinical signs, namely round back (from Day 29 to Day 33) and piloerection (Day 29) were noted in Week 5 in 1/10 males given 150 mg/kg/day.
Ptyalism was observed with a dose-related incidence in test item-treated males and females during the premating, pregnancy and lactation periods. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.

At 50 mg/kg/day, piloerection or emaciated appearance were noted in 2/8 females for 4 or 2 days, respectively, during the lactation period. As these clinical signs were not dose-related and/or were most probably due to a technical problem of water access (emaciated appearance), a test item relationship was considered to be unlikely.

All the other clinical signs (i.e. areas of hair loss, cutaneous lesions, scabs, chromodacryorrhea, short tail, reflux at dosing and/or reddish vaginal discharge) were considered to be unrelated to the test item as they were present in control animals, were not dose-related, were reported sporadically in only a few animals, are commonly observed findings in this species and strain and/or resulted from the gavage procedure.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

See table 1.
There were no unscheduled deaths in males.

In females:
. At 0 mg/kg/day
One female was found dead on Day 24 p.c. Five dead pups (two males and three females) were found in the litter. No remarkable clinical signs were observed prior to death. At necropsy, there were 11 dead fetuses and 5 placentae without fetuses in the uterus, which suggested that female death was related to parturition issues. The thymus was red, which correlated with agonal congestion/hemorrhage. In the thymus, in addition to agonal congestion/hemorrhage, there was moderately increased apoptosis in the cortex and slightly decreased lymphocyte cellularity in the cortex/medulla, which were both suggestive of stress associated with poor health.

. At 15 mg/kg/day
Two females were euthanized on Days 26 and 25 p.c., respectively, for no delivery. At necropsy, these females were found to be non-pregnant. Ptyalism was transiently noted during the premating and/or gestation period together with chromodacryorrhea and cutaneous lesions on the forelimbs in one female. At necropsy, a vaginal transverse septum was observed in one female, which is associated with the absence of pregnancy.
No necropsy findings were observed in one female. However, follicular cysts and absence of corpora lutea in the ovaries, increased thickness of the squamous epithelium with keratinization in the vagina were suggestive of persistent estrus. This absence of normal estrous cycling explained the absence of pregnancy, and was considered to be incidental.

. At 50 mg/kg/day
One female was euthanized on Day 26 p.c. for no delivery. Ptyalism was transiently noted during the premating and gestation periods. At necropsy, no macroscopic post-mortem findings were observed. This female was found to be non-pregnant and no relevant microscopic changes were observed in the reproductive organs.

One female was prematurely euthanized on Day 13 p.c. due to a subcutaneous mass on the urogenital area (3.0 x 3.0 cm in diameter), which resulted from a nodosity that appeared during the mating period (i.e. on Day 17 of the study). Ptyalism was transiently noted during the premating and gestation periods. At necropsy, there was a large perigenital white mass (3.5x3 cm approximatively), which correlated with an adenocarcinoma of the mammary gland. This isolated finding in a mid-dose individual was considered to be incidental.
This female was found to be pregnant with 10 live concepti and 2 early resorptions.

. At 150 mg/kg/day
Two females were euthanized on Days 26 and 25 p.c., respectively, for no delivery. Ptyalism was transiently noted during the premating period and throughout the gestation period.
One female was found to be pregnant. At necropsy, the spleen was enlarged and 2 implantation sites were observed in uterine horns.
One female was found to be non-pregnant. At necropsy, no macroscopic post-mortem findings were observed and no relevant microscopic changes were noted in the reproductive organs.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See table 3.
There were no effects on mean body weight or mean body weight change at 15 and 50 mg/kg/day in males and females and no effects on mean body weight in females given 150 mg/kg/day.

Lower body weight and body weight gain were observed during the whole exposure period in males given 150 mg/kg/day, reaching statistically significance on some occasions (i.e. body weight gain between Days 1 and 8: +30 g vs. +39 g in controls, p<0.05).
Lower mean body weight gains in females given 150 mg/kg/day were observed between Days 1 and 4 p.p. (+1 g vs. +9 g in controls, not statistically significant).
These findings were considered to be of minor toxicological importance as they were of slight magnitude, isolated and/or with no effects on the mean body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See table 4.
There were no effects on mean food consumption in males at any dose level or in females at 15 mg/kg/day.

Differences between control and test item-treated animals consisted of higher mean food consumption between Days 8 and 15 of the premating period (+13% vs. controls, p<0.05) in females given 50 or 150 mg/kg/day. These differences persisted in females given 150 mg/kg/day over the first week of the gestation period (+16% vs. controls, p<0.01). These test item-related findings were considered to be of minor toxicological importance as they were of slight magnitude, isolated and/or with no effects on the mean body weight.
Other differences from controls were observed during the lactation period (i.e. higher mean food intake in females given 15 mg/kg/day). A relationship with the test item was considered to be unlikely as these variations were not dose-related.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

See table 5.
Differences between control and test item-treated animals consisted of higher mean water consumption
- in males given 15, 50 or 150 mg/kg/day over the second week of the premating period (+19, +16 and +24% vs. controls, respectively, statistically significant),
- in females given 15 mg/kg/day over the first week of the gestation period (+17% vs. controls),
- in females given 50 mg/kg/day or 150 mg/kg/day over the premating period (+13 to +19% and +20 to +22% vs. controls, respectively), throughout the gestation period (+17 to +28% and +31 to +38% vs. controls, respectively) with statistical significance, and on some occasions during the lactation period at all dose levels from Day 4 p.p. without statistically significance and in a poorly dose-related manner. These differences were statistically significant during the premating and gestation periods and more marked between Days 0 and 7 p.c.
These test item-related findings correlated with slightly higher mean urine volumes recorded in females given 15, 50 or 150 mg/kg/day at the end of the study. They were considered to be of minor toxicological importance as they were of slight magnitude and were not associated with other adverse findings.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See table 6.
At 150 mg/kg/day in females, test item-related differences from controls were noted and consisted in low mean red blood cell count (-13%, p<0.05), high mean cell volume (+13%, p<0.01) and mean cell hemoglobin (+14%, p<0.01), and high reticulocyte count (+38%) that was also observed in males (+59%, p<0.01). They correlated with non-adverse histopathological findings (increased extramedullary hematopoiesis and pigment consistent with hemosiderin in the spleen of both sexes and increased erythroid cell numbers in the bone marrow of females). Marginally high mean neutrophil count (+37% vs. controls, p<0.05) was also observed in females. In view of their slight magnitude and as mean values were within or close to the range of the Historical Control Data, hematological findings recorded at 150 mg/kg/day were not considered as adverse.
Low mean platelet count in females from 50 mg/kg/day was considered to be of no toxicological importance as mean values were in the range of the Historical Control Data and were without statistical significance.

All the other differences from controls, namely high mean red blood cell count and low mean packed cell volume (PCV) in females given 15 mg/kg/day, were of low magnitude and/or not dose-related. Therefore, these differences were considered to be unrelated to the test item treatment.

There were no relevant test item-related effects on hematology parameters at 15 and 50 mg/kg/day in males and females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See table 7.
When compared with controls, statistically significant, dose-related, low mean glucose levels were noted in males at 15, 50 and 150 mg/kg/day (-14 to -20%, p<0.05 to p<0.01). However in females, dose-related high mean glucose levels were observed without statistically significance. As mean values were within the range of the Historical Control Data, as variations were mainly due to higher mean control values in males and were of opposite trend, and/or in absence of any correlates with microscopic observations and in view of their slight magnitude, these differences were not considered as adverse.

From 50 mg/kg/day, higher mean bile acids levels were noted in both sexes without statistically significance. In view of their slight magnitude, as mean values were within the range of the Historical Control Data and in absence of any correlates with microscopic observations, this finding was not considered as adverse.

At 150 mg/kg/day in females and when compared with controls, a statistically significant increase was observed in mean inorganic phosphorus level (+30%, p<0.01). As this difference was not associated with other blood biochemistry or histological changes and as mean values were close to the range of the Historical Control Data, this finding was not considered as adverse.
At the same dose-level, a statistically significant decrease in creatinine level (-9% vs. controls, p<0.05) was observed in males. In view of the slight magnitude and the direction of the change, in absence of this effect in females and as mean values were within the range of the Historical Control Data, this finding was not considered as toxicologically relevant.

All the other differences from controls, namely high mean chloride and sodium levels (males given 15 or 150 mg/kg/day) and higher mean albumin/globulin ratio (males given 15 or 150 mg/kg/day) were slight, within or close to the range of the Historical Control Data, marginal, without any relationship to microscopic findings and/or were not dose-related. Therefore, they were considered to be of no toxicological importance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See table 8.
From 15 mg/kg/day in females and when compared with mean control values, high mean urine volume (+64%) was noted without any impact on the mean specific gravity values. Variations in urine volume correlated with higher mean water intake recorded in females during the study. As there was no dose response in test item-treated females, and the values were not statistically significant and remained within the range of Historical Control Data, these differences were considered to be findings related to the test item but of minor importance.
The same tendency was also observed in males given 15 or 150 mg/kg/day (+67% and +56% vs. controls, respectively). As these differences were not dose-related, they were considered to be of no toxicological importance.

At 15 mg/kg/day in most animals (5/5 males and 3/5 females) and from 50 mg/kg/day in all males and females, presence of nitrites was noted. This finding could be secondary to the presence of metabolites of the test item. In absence of any other correlates, this test item-related finding was considered to be of minor importance.

From 15 mg/kg/day in both sexes and when compared with controls, dose-related higher presence of bilirubin was noted. This effect could be linked to coloration of urine observed in most males given 50 (dark yellow color, 3/5 males) or 150 mg/kg/day (yellow brown, 3/5 males). Presence of bilirubin was associated with histopathological findings suggestive of hemolysis and correlated with variations observed in the red blood cell mass of males and females given 150 mg/kg/day.

At 50 mg/kg/day, proteinuria (=1g/L) was noted in 3/5 males at 50 mg/kg/day. This severity was also recorded in 2/5 males and 1/5 females at 150 mg/kg/day. As proteinuria correlated with renal microscopic changes (female) and was most probably related to urinary bladder microscopic changes (in one male given 150 mg/kg/day), this finding was considered to be test item-related.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Motor activity - see table 12.
There were no differences between test item-treated and control groups in motor activity (horizontal movements and rearing) in males.

In females, low test item-related mean numbers of horizontal and rearing movements were noted at 50 mg/kg/day (-36% and -16% vs. controls, respectively) and at 150 mg/kg/day (-36% and -32% vs. controls, respectively). These findings that correlated with high mean landing foot splay in females at 50 and 150 mg/kg/day were considered to be test item-related but of minor toxicological importance as they were not statistically significant.

Funtional Observation Battery - see table 13.
There were no test item treatment-related neurologic abnormalities.

The presence of urine was noted in 3/5 males given 15 or 150 mg/kg/day vs. 1/5 control males. High mean landing foot splay values were noted in females given 50 mg/kg/day (90 mm vs. 73 mm in controls) and in males and females given 150 mg/kg/day (95 and 96 mm vs. 81 and 73 mm in controls, respectively). This correlated with lower mean numbers of horizontal and rearing movements in females given 50 or 150 mg/kg/day.
Observations recorded in males and females did not correlate with any adverse findings, they were therefore considered to be of minor toxicological importance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See table 10.
Of note, the terminal body weight of males at 150 mg/kg/day was minimally decreased compared with controls.

When compared with controls, the mean absolute and relative liver weights were minimally or slightly increased in males and females at 150 mg/kg/day. These increases correlated with hepatocellular hypertrophy at microscopic examination. In the absence of microscopic correlates, minimal increases in liver weights seen in females at 50 mg/kg/day were considered to be of minor importance.

There were slight increases in the mean absolute and relative spleen weights in males and females at 150 mg/kg/day. These increases correlated with increased extramedullary hematopoiesis and/or congestion microscopically.

The mean heart weight was slightly increased in females at 150 mg/kg/day, which was related to high individual heart weight of one female (absolute weight of 2.253 g versus mean absolute weight of 1.04 g in controls). This increase correlated in this female with macroscopic enlargement of the heart at necropsy and with cardiomyocyte hypertrophy secondary to atrial thrombosis at microscopic examination observed in this female.

The mean kidney weights were minimally increased (up to +18%) in males at 150 mg/kg/day (reaching statistical significance for the relative weight only) and in females at 50 and 150 mg/kg/day (reaching statistical significance for the absolute weight only). In males, the variation was considered to be a reflection of the reduction in body weight and not to be due to test-item-related organ toxicity. In females, in the absence of a dose-relationship, these variations were considered to be most probably incidental and unrelated to the test item administration.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled deaths
In one female (50 mg/kg/day) prematurely sacrificed on Day 31 (i.e. on Day 13 p.c.), there was a large perigenital white mass (3.5x3 cm approximatively) correlated with an adenocarcinoma of the mammary gland. This isolated finding in a mid-dose individual was considered to be incidental.

In one control female, found dead on Day 40 (i.e. on Day 24 p.c.), there were 11 dead fetuses in the uterus, which suggested that death was related to delivery issues. The thymus was red, which correlated with agonal congestion/hemorrhage.

At the end of the treatment period
Test item-related enlargement of the spleen was noted in 1/10 females at 150 mg/kg/day. This correlated with increased congestion and extramedullary hematopoiesis. The heart was enlarged in 1/10 females at 150 mg/kg/day, which correlated with cardiomyocyte hypertrophy secondary to atrial thrombosis.

The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control rats, and were considered to be incidental. Among them was a vaginal septum in one low-dose female (15 mg/kg/day), which explained absence of pregnancy, and unilateral reduced size of the epididymis and testis in two males at 150 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See table 11.
Unscheduled deaths
One female (50 mg/kg/day) prematurely sacrificed on Day 31 (i.e. on Day 13 p.c.), had an adenocarcinoma of the mammary gland in the perigenital region i.e. a malignant tumor. This isolated finding in a mid-dose individual was considered to be incidental.

In one control female, found dead on Day 40 (i.e. on Day 24 p.c.), there were 11 dead fetuses in the uterus, which suggested that death was related to delivery issues. In the thymus, in addition to agonal congestion/hemorrhage, there was moderately increased apoptosis in the cortex and slightly decreased lymphocyte cellularity in the cortex/medulla, which were both suggestive of stress associated with the poor health status.

At the end of the treatment period
In the liver, slight hepatocellular hypertrophy in centrilobular to midzonal location was noted in all males and females at 150 mg/kg/day.

In the urinary bladder, minimal or slight, multifocal or diffuse hyperplasia of the urothelium was noted in males and females at 150 mg/kg/day, associated in some animals with minimal single cell necrosis in the urothelium, and in a single male with slight mixed inflammatory cell infiltrate in the submucosa.

In the spleen, there was an increased severity and/or incidence of extramedullary hematopoiesis (erythropoiesis mainly), golden brown pigment and congestion in males and females at 150 mg/kg/day compared to controls. Pigment correlated with increased severity of Perls positive deposits, which was consistent with hemosiderin.

In the kidneys, minimal brown pigment was noted in tubular cells in all females at 150 mg/kg/day, associated with minimal tubular vacuolation and eosinophilic granules in 2/5 females. This pigment was negative with Perls stain i.e. this pigment was not hemosiderin. In addition in one female, there was increased severity of tubular basophilia suggestive of regeneration along with slight hyaline casts in the tubular lumens and slight glomerulopathy.
Of note, tubular basophilia was observed in test item-treated males and not in any control males. However it is a common background finding in rats, and in the absence of a dose-relationship, this finding in males was considered to be incidental and unrelated to the test item administration.

In the bone marrow, increased erythroid cells was seen in 3/5 females at 150 mg/kg/day. This increased cellularity was associated with decreased adipose tissue in the bone marrow compared to controls.

In the heart, 1/5 females had moderate diffuse cardiomyocyte hypertrophy and left atrial thrombosis (with mineralization) associated with slight multifocal necrosis/fibrosis of the cardiomyocytes.
In males, minimal cardiomyocyte necrosis/fibrosis was noted in two individuals versus no control males, however since this is a more common background observation in males, the difference was considered to be most probably incidental.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

See table 9.
Thyroid hormones
The thyroid hormone levels were considered to be unaffected by the test item treatment in F0 males.

In males, in all treated groups and when compared with controls, there were lower mean T4 concentrations reaching statistical significance at 15 and 150 mg/kg/day but remaining in the range of the Historical Control Data. These variations were considered fortuitous as they were not dose-related, did not correlate with a dose-related response in mean TSH levels and also resulted from higher mean control values (above mean Historical Control Data).

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

bladder

heart

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Mortality

Dose level (mg/kg/day)	0	15	50	150
Humane reasons (mass)	0	0	1(pg)	0
No delivery	0	2(npg)	1(npg)	2(1npg 1pg)
Found dead	1(pg)	0	0	0
Total surviving animals	9/10	8/10	8/10	8/10

pg: pregnant; npg: not pregnant.

Table 2: Clinical signs

 

Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Pre-mating (females) or whole study (males)
Ptyalism	0	4	10	10	0	2	10	10
Round back	0	0	0	1	0	0	0	0
Piloerection	0	0	0	1	0	0	0	0
Total affected animals	0/10	4/10	10/10	10/10	0/10	2/10	10/10	10/10
Gestation
Ptyalism	-	-	-	-	0	3	8	8
Total affected animals	-	-	-	-	0/9	3/8	8/8	8/8
Lactation
Ptyalism	-	-	-	-	0	1	8	8
Piloerection	-	-	-	-	0	0	1	0
Emaciated appearance	-	-	-	-	0	0	1	0
Total affected animals	-	-	-	-	0/9	1/8	8/8	8/8

-: not applicable.

Table 3:  Body weight and body weight change

Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Pre-mating period (females) and whole study (males)
. Day 1	412	410	412	408	276	272	273	276
% from controls	-	0	0	-1	-	-1	-1	0
. Day 8	452	452	450	438*	279	271	277	280
% from controls	-	0	0	-3	-	-3	-1	0
. Day 15	476	481	479	464	284	275	286	288
% from controls	-	+1	+1	-3	-	-3	+1	+1
. Day 29	516	521	522	497*	-	-	-	-
% from controls	-	+1	+1	-4	-	-	-	-
. Days 1 - 8	+39	+41	+38	+30*	+3	-1	+4	+4
. Days 1 - 15	+64	+70	+68	+56	+8	+4	+14	+12
. Days 22 - 29	+24	+26	+28	+18	-	-	-	-
. Days 15 - 29	+40	+41	+43	+33	-	-	-	-
. Days 1 - 29	+104	+111	+111	+89	-	-	-	-
Gestation
. Day 0p.c.	-	-	-	-	285	284	286	293
% from controls	-	-	-	-	-	0	0	+3
. Day 20p.c.	-	-	-	-	424	432	443	446
% from controls	-	-	-	-	-	+2	+4	+5
. Days 0 - 20p.c.	-	-	-	-	+140	+149	+156	+153
Lactation
. Day 1p.p.	-	-	-	-	334	339	340	351
% from controls	-	-	-	-	-	+1	-2	+5
. Day 13p.p.	-	-	-	-	358	367	375	383
% from controls	-	-	-	-	-	+3	+5	+7
. Days 1 - 4p.p.	-	-	-	-	+9	+7	+6	+1
. Days 4 - 8p.p.	-	-	-	-	+10	+18	+12	+17
. Days 8 - 13p.p.					+5	+4	+16	+15
. Days 1 - 13p.p.	-	-	-	-	+24	+28	+34	+32

-: not applicable.

Statistical significance*: p<0.05.

Table 4: Food consumption

 

Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Pre-mating period (females) and whole study (males)
Days 1 - 8	29	30	29	28	17	17	17	18
% from controls	-	+3	0	-3	-	0	0	+6
Days 8 -15	27	28	29	29	16	17	18*	18*
% from controls	-	+4	+7	+7	-	+6	+13	+13
Gestation
Days 0 - 7p.c.	-	-	-	-	19	20	20	22**
% from controls					-	+5	+5	+16
Days 7 - 14p.c.	-	-	-	-	22	23	23	24
% from controls					-	+5	+5	+9
Days 14 - 20p.c.	-	-	-	-	27	27	28	29
% from controls					-	0	+4	+7
Lactation
Days 1- 4p.p.	-	-	-	-	33	37	32	33
% from controls					-	+12	-3	0
Days 4- 8p.p.	-	-	-	-	45	52	48	49
% from controls					-	+16	+7	+9
Days 8- 13p.p.	-	-	-	-	57	63	62	59
% from controls					-	+11	+9	+4

-: not applicable.

Statistical significance*: p<0.05 and **: p<0.01.

Table 5: Water consuption

 

Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Pre-mating period (females) and whole study (males)
Days 1 - 8	38	42	40	42	27	27	32**	33^#
% from controls	-	+11	+5	+11	-	0	+19	+22
Days 8 -15	37	44**	43*	46#	30	31	34*	36**
% from controls	-	+19	+16	+24	-	+3	+13	+20
Gestation
Days 0 - 7p.c.	-	-	-	-	29	34	37*	40^#
% from controls					-	+17	+28	+38
Days 7 - 14p.c.	-	-	-	-	38	42	45*	51^#
% from controls					-	+11	+18	+34
Days 14 - 20p.c.	-	-	-	-	52	55	61*	68^#
% from controls					-	+6	+17	+31
Lactation
Days 1- 4p.p.	-	-	-	-	57	60	58	60
% from controls					-	+5	+2	+5
Days 4- 8p.p.	-	-	-	-	69	77	75	80
% from controls					-	+12	+9	+16
Days 8- 13p.p.	-	-	-	-	84	92	97	94
% from controls					-	+10	+15	+12

-: not applicable.

Statistical significance*: p<0.05, **: p<0.01 and ^#: p<0.001.

Table 6: Haematology

 

	Hematology
Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Red blood cell count (T/L)	9.07	9.25	9.11	8.79	8.55	7.85*	8.33	7.42*
HCD	9.06 (7.93-9.57)				7.99 (6.58-9.97)
Mean cell volume (fl)	53.3	53.7	53.6	55.6	58.4	59.1	58.5	66.2**
HCD	54.0 (50.9-57.2)				57.3 (53.0-63.4)
Mean cell hemoglobin (pg)	17.4	17.4	17.5	18.0	19.0	19.7	19.5	21.7**
HCD	17.5 (16.4-18.6)				18.8 (17.1-21.1)
Reticulocyte count (T/L)	0.22	0.25	0.27	0.35**	0.32	0.31	0.30	0.44
HCD	Not available				Not Available
Reticulocyte (%)	2.44	2.76	2.93	4.01**	3.76	3.89	3.63	5.94**
HCD	2.43 (1.85-3.19)				4.10 (0.78-8.60)
Platelet count (G/L)	857	735	862	856	876	846	781	717
HCD	744 (344-1062)				966 (229-1583)
Neutrophil count (G/L)	3.02	2.50	2.66	3.77	4.82	4.38	4.33	6.61*
HCD	2.74 (1.66-4.40)				3.84 (1.42-6.74)

Statistically significant: *: p<0.05 and **: p<0.01.

HCD: mean (minimum-maximum) Historical Control Data values

Table 7: Blood biochemistry

 

	Blood biochemistry
Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Inorganic phosphorus (mmol/L)	2.19	2.25	2.18	2.33	2.64	2.52	2.53	3.43**
HCD	2.37 (2.15-2.61)				2.55 (2.01-3.23)
Glucose (mmol/L)	8.31	7.17*	7.16*	6.64**	5.59	6.84	6.78	7.22
HCD	6.67 (5.80-7.78)				6.44 (4.65-8.54)
Creatinine (µmol/L)	34.33	35.25	34.27	31.09*	39.32	41.52	40.04	39.97
HCD	32.70 (25.03-38.43)				37.29 (28.87-46.07)
Bile acids (µmol/L)	13.51	14.63	22.55	24.71	53.52	59.42	123.14	126.02
HCD	71.86 (22.11-209.83)				65.00 (23.27-209.78)

Statistically significant: *: p<0.05 and **: p<0.01.

HCD: mean (minimum-maximum) Historical Control Data values.

Table 8: Urinalysis

 

	Urinary parameters
Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Volume (mL)	9	15	8	14	11	18	18	18
HCD	9 (5-13)				15 (5-29)
Presence of proteins
. negative	0	1	0	2	3	5	4	4
. 0.3 g/L	4	2	2	1	2	0	1	0
. 1 g/L	1	2	3	1	0	0	0	1
.=3 g/L	0	0	0	1	0	0	0	0
Presence of bilirubin
. negative	3	0	1	0	2	0	0	0
. low	2	3	1	0	3	3	1	0
. moderate	0	2	2	0	0	2	4	2
. high	0	0	1	5	0	0	0	3
Color
. pale yellow	1	4	0	0	2	4	4	1
. yellow	4	1	2	2	3	1	1	4
. dark yellow	0	0	3	0	0	0	0	0
. yellow/brown	0	0	0	3	0	0	0	0
Presence of nitrites
. negative	5	0	0	0	5	2	0	0
. positive	0	5	5	5	0	3	5	5

HCD: mean (minimum-maximum) Historical Control Data values.

Table 9: Thyroid hormones

 

	Thyroid hormones
Sex	F0 males
Dose level (mg/kg/day)	0	15	50	150
T4 (ng/mL)	39.32	34.65*	36.10	32.53**
HCD	36.6 Minimum: 26.9 Maximum: 46.9
Standard deviation	6.183	2.746	2.668	3.664
n	10	10	10	10
% from controls	/	-12	-8	-17
TSH (pg/mL)	1435	1974	2193	1622
HCD	1830 Minimum: 424 Maximum: 6935
Standard deviation	699.8	1292.0	1032.1	476.3
n	10	10	10	10
% from controls	/	+38	+53	+13

Statistically significant: *: p<0.05 and **: p<0.01.

HCD: Historical Control Data.

n: number of animals with available values; /: not applicable.

Table 10: Organ weights

 

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	15	50	150	15	50	150
- Final body weight	+1	+1	-4*	+1	0	+1
- Liver
. absolute	0	+8	+16*	+11	+15	+29**
.       relative to body weight	+1	+8	+22**	+8	+10*	+23**
- Spleen
. absolute	+2	+8	+23*	-3	+7	+21
.       relative to body weight	+3	+8	+29**	-5	+3	+16
- Heart
. absolute	-3	-3	-8	-3	+6	+27
. relative to body weight	-1	-3	-3	-5	+2	+21
- Kidneys
.absolute	0	+3	+13	+13	+15*	+14*
.relative	0	+3	+18*	+11	+11	+9

Statistically significant from controls: *: p<0.05, **: p<0.01.

The significance concerned the organ weights values and not the percentages.

Table 11: Microscopic examination

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	15	50	150	0	15	50	150
No. animals	5	5^$$	5	5	5	5	5	5^$
Liver
- Hypertrophy; hepatocyte
Slight (grade 2)	-	-	-	5	-	-	-	5
Urinary bladder
- Single cell necrosis; urothelium
Minimal (grade 1)	-	-	-	4	-	-	-	1
- Hyperplasia; urothelium
Minimal (grade 1)	-	-	-	2	-	-	-	3
Slight (grade 2)	-	-	-	2	-	-	-	-
Spleen
- Hematopoiesis
Minimal (grade 1)	5	6	5	2	4	5	5	1
Slight (grade 2)	-	-	-	3	1	-	-	5
- Pigment
Minimal (grade 1)	-	-	-	4	3	5	3	1
Slight (grade 2)	-	-	-	-	2	-	2	5
- Perls positive deposit
Minimal (grade 1)	4	NA	NA	-	3	NA	NA	1
Slight (grade 2)	1	NA	NA	1	2	NA	NA	2
Moderate (grade 3)	-	NA	NA	4	-	NA	NA	2
- Congestion
Minimal (grade 1)	1	-	-	4	3	-	-	3
Slight (grade 2)	-	-	-	-	-	-	1	-
Moderate (grade 3)	-	-	-	-	-	-	-	1
Kidneys
- Pigment; tubule
Minimal (grade 1)	-	-	-	-	-	-	-	5
- Vacuolation; tubule
Minimal (grade 1)	-	-	-	-	-	-	-	2
- Eosinophilic granules; tubule
Minimal (grade 1)	-	-	-	-	-	-	-	2
- Basophilia; tubule
Minimal (grade 1)	-	3	2	3	1	1	2	1
Slight (grade 2)	-	-	-	-	-	-	-	1
- Cast; hyaline
Slight (grade 2)	-	-	-	-	-	-	-	1
- Glomerulopathy
Slight (grade 2)	-	-	-	-	-	-	-	1
Bone marrow (sternum)
- Increased erythroid cells
Minimal (grade 1)	-	-	-	-	-	-	-	3

-: finding not present. NA: not applicable.

^$: n=5 except for spleen examined in 6 high-dose females because of a macroscopic finding in an additional individual.

^$$: n=5 except for liver, spleen and kidneys examined in 6 low-dose males because of a macroscopic finding in an additional individual.

Table 12: Motor activity

 

Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Horizontal movements	692	689	569	652	529	460	341	339
SD	204.5	113.6	109.6	279.0	250.0	150.8	153.1	163.1
% from controls	-	0	-18	-6	-	-13	-36	-36
Rearing	166	172	178	190	128	135	107	87
SD	26.1	21.8	50.1	48.1	64.2	37.1	50.6	43.7
% from controls	-	+4	+7	+14	-	+5	-16	-32

-: not applicable.

Statistical analysis: no significance.

Table 13: Functional Observation Battery

 

Sex	Male				Female
Dose level (mg/kg/day)	0	15	50	150	0	15	50	150
Urination
. absence	4	2	3	2	4	3	5	4
. presence	1	3	2	3	1	2	0	1
Landing foot splay (mm)	81	77	82	95	73	82	90	96
SD	23	38	29	25	19	22	19	28
% from controls	-	-5	+1	+17	-	+12	+17	+32

-: not applicable.

Conclusions:

The test item was administered daily by oral gavage to male and female Sprague Dawley rats for 2 weeks before mating, during mating and, for females, throughout gestation and until Day 13 p.p. at the dose levels of 15, 50 or 150 mg/kg/day.

Based on the experimental conditions of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 50 mg/kg/day based on the microscopic findings observed at 150 mg/kg/day in the urinary bladder (i.e. hyperplasia and single cell necrosis of the urothelium) of some males and females and in heart (i.e. cardiac atrial thrombosis and hypertrophy) of one female,
- the No Observed Adverse Effect Level (NOAEL) for reproductive performance (mating, fertility and delivery) was considered to be 150 mg/kg/day in the absence of adverse findings at this high-dose level,
- the No Observed Adverse Effect Level (NOAEL) for toxic effects on progeny was considered to be 15 mg/kg/day (based on clinical signs and live birth and viability indexes at 50 and 150 mg/kg/day).

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for the females, throughout gestation and until Day 13 post-partum (p.p.).

This study provides information on:

. the possible health hazards likely to arise from repeated exposure over a relatively limited period of time,

. male and female reproductive performance, such as gonadal function, mating behavior, conception, conceptus development, and parturition.

The study was performed according to OECD guideline No. 422 (29 July 2016) and in compliance with GLP regulations.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item.

The test item was administered daily by the oral route (gavage) at dose levels of 15, 50 or 150 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of 4 weeks: two weeks before mating, during the mating period (up to 1 week) and until the day before sacrifice. Females were treated for an overall period of 7 to 8 weeks: two weeks before mating, throughout mating (up to one week) and gestation (3 weeks) until Day 13 post-partum (p.p.) inclusive.  A control group of ten males and ten females received the vehicle only (corn oil) under the same experimental conditions.

The actual test item concentrations in the dose formulations were determined in Weeks 1 and 6 using a validated HPLC/UV detection analytical method.

The F0 animals were checked daily during the treatment period for mortality, morbidity and clinical signs. Detailed clinical observations were conducted weekly. Body weight, and food and water consumption were recorded weekly during the premating, mating (with the exception of food and water consumption), gestation and lactation periods. Estrous cycle stage was determined each morning from 2 weeks before the treatment period until the females had mated and on Day 14 p.p. before euthanasia.

The F0 animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups not selected on Day 4 p.p. were euthanized and discarded without further examination.

A Functional Observation Battery (FOB) was performed on five F0 animals per sex and group at the end of the treatment period.

Laboratory investigations (hematology, blood biochemistry and urinalysis) were carried out on at least five F0 males and five F0 females from each group at the end of the study. Plasma thyroid hormone levels (TSH and T4) were determined at terminationin all F0 males and in pups euthanized on Day 13 p.p.

The F0 males were euthanized after completion of the mating period and F0 females were euthanized on Day 14 p.p.

A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups, on all macroscopic lesions (all groups) and on the heart (females only), kidneys, liver, sternum with bone marrow (females only), spleen and urinary bladder from five males and lactating females at the low- and intermediate dose levels. Reproductive organs and thyroids with parathyroids from one pair at each test item dose level were also microscopically observed as the females were found to be non-pregnant. Perl’s staining was performed on the spleen and kidneys of the five males and/or five lactating females.

 

Results

 

Chemical analysis:

The actual test item concentrations in the analyzed dose formulations (i.e. in Weeks 1 and 6) remained within an acceptable range of variation (-4.1% to +0.6% when compared with the nominal values; nominal concentration ± 15% required).

 

F0 animals:

Mortality: there were no test item-related unscheduled deaths.

 

Clinical signs: round back and piloerection were transiently noted in 1/10 males given 150 mg/kg/day. These findings were considered to be related to the test item but of minor toxicological importance. Ptyalism occurred in both sexes and in all test item treated groups with dose-related increased incidences. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect.

 

Functional Observation Battery and motor activity: slightly lower incidences of horizontal and rearing movements were recorded in females given 50 or 150 mg/kg/day, along with higher landing foot splay values at the same dose levels in females. These effects were considered to be test item-related but of minor toxicological importance.

 

Body weight and body weight change: at 150 mg/kg/day and when compared with controls, a few variations of body weight gain were noted in males during the whole treatment period and in females on the first 4 days of the lactation period. These differences resulted in slightly transient lower mean body weights in males. They were considered to be related to the test item but of minor toxicological importance in view of their magnitude and/or the absence of effects on the body weight.

 

Food consumption: at 50 and 150 mg/kg/day and when compared with controls, a few episodes of slightly higher food consumption were noted in females in the premating period and/or at the beginning of the gestation period. These findings were considered to be related to the test item but of minor toxicological importance in view of their magnitude and the absence of effects on the body weight.

There were no effects on food consumption at any dose level in males.

Water consumption: at 15, 50 and 150 mg/kg/day and when compared with controls, higher water consumption was recorded in males and/or females over the premating period. High water intake was also observed in all dose females throughout the gestation period and then on a few occasions during the lactation. These effects were more marked at the beginning of the pregnancy period. These findings were considered to be related to the test item but of minor toxicological importance in view of their magnitude and in absence of any correlates with adverse findings.

 

Hematology: at 150 mg/kg/day and when compared with controls, there was a decrease in red blood cell count in females (-13%, p<0.05) along with increased mean cell volume and mean cell hemoglobin (+13% and +14%, respectively, p<0.01). These changes were associated with increased reticulocyte count (+38%), which was also observed in males (+59%, p<0.01).These hematological variations correlated with histopathological findings suggestive of hemolysis (increased extramedullary hematopoiesis and pigment consistent with hemosiderin in the spleen of both sexes and increased erythroid cell numbers in the bone marrow of females). Marginally higher mean neutrophil count (+37%vs. controls, p<0.05) was also observed in females. In view of their magnitude and as mean values remained within or close to the range of the Historical Control Data, hematological changes were considered to be non-adverse.

 

Blood biochemistry: when compared with controls, blood biochemistry changes includeda dose-related decreased glucose level in males at all dose levels (-14 to -20%, p<0.05 to p<0.01) and the opposite trend in females (+21% to +29%, not statistically significant) in females, an increase in the inorganic phosphorus level in females (+30%, p<0.01) and an increase in the bile acids level in males and females from 50 mg/kg/day. In view of their magnitude, as mean values remained within the range of the Historical Control Data and in absence of histopathological findings, these changes were considered to be non-adverse.

 

Urinalysis: urinalysis changes consisted of higher volume in females at all dose levels (+64% vs. controls) corresponding to non-adverse higher mean water consumption, increased bilirubin levels in both sexes from 50 mg/kg/day, colored urine in males from 50 mg/kg/day, presence of nitrites in all males at all dose levels and in all females from 50 mg/kg/day, and of moderate to marked proteinuria in 3/5 males at 50 mg/kg/day and in 2/5 males and 1/5 females at 150 mg/kg/day.Bilirubinuria correlated with variations observed in the red blood cell mass of males and females given 150 mg/kg/day and with histopathological findings suggestive of increased red blood cell turnover and proteinuria could be related to adverse microscopic changes observed in the kidneys and/or in the urinary bladder at 150 mg/kg/day. As mean values remained within the range of the Historical Control Data, were poorly dose-related and without statistically significance, in absence of any other correlates and/or as findings could be consecutive from histopathological findings, these changes were considered to be non-adverse.

 

Thyroid hormones: there were no test item-related effects on the T4 and TSH levels in F0 males or pups at any dose level.

 

Pathology: At 150 mg/kg/day, at microscopic examination, adverse hyperplasia and single cell necrosis of the urothelium was observed in the urinary bladder in both sexes. Non-adverse microscopic findings were noted in the spleen (increased congestion and pigment consistent with hemosiderin) and kidneys (brown pigment), with compensatory hematopoiesis (erythropoiesis) in the spleen, correlated with increased spleen weights (both sexes), and bone marrow (females). Minimal and non-adverse tubular vacuolation was noted in two females in the kidneys at 150 mg/kg/day. In addition in one female at this dose-level, there were adverse cardiac atrial thrombosis and hypertrophy, glomerulopathy and renal tubular hyaline casts, potentially related to the test item administration. Non-adverse slight hepatocellular hypertrophy correlated with increased liver weights was noted in the liver at 150 mg/kg/day (both sexes).

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats for 2 weeks before mating, during mating and, for females, throughout gestation and until Day 13 p.p. at the dose levels of 15, 50 or 150 mg/kg/day.

 

Based on the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 50 mg/kg/day based on the microscopic findings observed at 150 mg/kg/day in the urinary bladder (i.e. hyperplasia and single cell necrosis of the urothelium) of some males and females and in heart (i.e. cardiac atrial thrombosis and hypertrophy) of one female.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is considered as reliable, as conducted with GLD and OECD guidelines.

System:

urinary

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

14 -day repeated toxicity study (Michel 2018):

The objective of this preliminary study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 2 weeks in order to assist the selection of dose levels for a further OECD 422 study to be performed in this species.

Three groups of five male and five female Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item at dose levels of 100, 300 or 1000 mg/kg/day. Another group of five males and five females received the vehicle alone (corn oil) and acted as a control group. The test item was administered for 2 weeks at 0, 100 or 300 mg/kg/day and for 2 days at 1000 mg/kg/day due to severe toxic effects. The test item was administered as a suspension in the vehicle under a constant dosage volume of 5 mL/kg/day.

The animals were observed daily for mortality and clinical signs. Body weight was recorded once pre-test, on the first day of treatment and then at least twice a week. Food consumption was recorded twice a week.

On completion of the treatment period, the surviving animals were euthanized and a full macroscopicpost-mortemexamination was performed. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from control, low and intermediate-dose animals euthanized at the end of the treatment period.

Four males and one female given 1000 mg/kg/day were found dead on Day 3 or 4. Prior to death, signs of poor clinical condition were observed with body weight loss. Test item-related necropsy changes consisted of black/red foci in the forestomach and stomach in all four males and in the female, and kidney and liver enlargement and intestinal distension in two males. Signs of poor clinical condition such as hunched posture, hypoactivity, piloerection, half-closed eyes, dyspnea, thin appearance, lacrimation and staggering gait were also noted in almost all surviving animals given 1000 mg/kg/day. These surviving animals were prematurely euthanized for ethical reasons on Day 6 and were not treated from Day 3.

Lower body weight gain was noted during the whole treatment period in males given 300 mg/kg/day. Body weight losses were noted at 1000 mg/kg/d in both sexes until Day 4.

Lower food consumption was noted during the first four days at 1000 mg/kg/day and eight days at 300 mg/kg/day, in both sexes.

Test item-related macroscopic changes in surviving animals treated at 1000 mg/kg/day and euthanized on Day 6 were seen in the spleen (enlargement in all females), ureters (dilatation in the male and three females), kidneys (enlargement and dilated pelvis in the male and one female, with gelatinous hilus and yellow focus in this female), stomach (white discoloration in the male and one female), forestomach (white discoloration in the male), intestine (distension in the male and three females), pancreatic lymph nodes (enlargement in the male and one female) and liver (enlargement and yellow foci in one female).

At the end of the treatment period, the test item administration at 300 mg/kg/day induced microscopic changes in the spleen, liver, and kidneys suggestive of a hemolytic anemia. In the spleen, there was an increased severity of hematopoiesis and congestion, correlated with macroscopic enlargement and markedly increased spleen weights, and increased pigment suggestive of hemosiderin. In the liver, there was hepatocellular hypertrophy, correlated with slight or moderate increases in liver weights in both sexes. In the kidneys, there was tubular vacuolation, regeneration and eosinophilic granules (suggestive of hemoglobin) in males, and minimal increases in kidney weights in females. Additional changes consisted of minimal increases in heart weights in females, and macroscopic dilatation of the ureters in one male.
The test item administration at 100 mg/kg/day induced changes in the spleen suggestive of hemolysis (increased hematopoiesis in both sexes, increased congestion and pigment in females), and hepatocellular hypertrophy correlated with minimal increases in liver weights in males.

In summary :

-At 1000 mg/kg/day, the test item administration induced mortality in four males and one female on Day 3 or 4, and the surviving animals were prematurely sacrificed on Day 6 due to signs of poor clinical condition, as well as body weight loss and reduced food consumption. Adverse macroscopic changes were seen in kidneys.

-At 300 mg/kg/day, some test item-related clinical signs were observed in one male and one female associated with lower body weight gain and food consumption only in males. Splenic changes, liver extramedullary hematopoiesis and hepatocellular hypertrophy were seen in both sexes, and adverse renal tubular lesions in males only.

-At 100 mg/kg/day, the test item was clinically well tolerated. Splenic changes and liver extramedullary hematopoiesis in both sexes and hepatocellular hypertrophy in males were noted at microscopic examination.

Consequently, under the experimental conditions of the study, based on adverse effect observed at 300 and 1000 mg/kg/day, the Maximal Tolerated Dose (MTD) level after 2-week treatment period was established at 300 mg/kg/day in both sexes.

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity study (Papineau 2018):

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, through mating and, for the females, throughout gestation and until Day 13 post-partum (p.p.).

This study provides information on:

. the possible health hazards likely to arise from repeated exposure over a relatively limited period of time,

. male and female reproductive performance, such as gonadal function, mating behavior, conception, conceptus development, and parturition.

The study was performed according to OECD guideline No. 422 (29 July 2016) and in compliance with GLP regulations.

 

Three groups of ten male and ten female Sprague-Dawley rats received the test item.

The test item was administered daily by the oral route (gavage) at dose levels of 15, 50 or 150 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of 4 weeks: two weeks before mating, during the mating period (up to 1 week) and until the day before sacrifice. Females were treated for an overall period of 7 to 8 weeks: two weeks before mating, throughout mating (up to one week) and gestation (3 weeks) until Day 13post-partum(p.p.) inclusive.  A control group of ten males and ten females received the vehicle only (corn oil) under the same experimental conditions.

Mortality: there were no test item-related unscheduled deaths.

 

Clinical signs: round back and piloerection were transiently noted in 1/10 males given 150 mg/kg/day. These findings were considered to be related to the test item but of minor toxicological importance. Ptyalism occurred in both sexes and in all test item treated groups with dose-related increased incidences. This sign, commonly observed when a test item is administered by gavage, was not considered as an adverse effect.

 

Functional Observation Battery and motor activity: slightly lower incidences of horizontal and rearing movements were recorded in females given 50 or 150 mg/kg/day, along with higher landing foot splay values at the same dose levels in females. These effects were considered to be test item-related but of minor toxicological importance.

 

Body weight and body weight change: at 150 mg/kg/day and when compared with controls, a few variations of body weight gain were noted in males during the whole treatment period and in females on the first 4 days of the lactation period. These differences resulted in slightly transient lower mean body weights in males. They were considered to be related to the test item but of minor toxicological importance in view of their magnitude and/or the absence of effects on the body weight.

 

Food consumption: at 50 and 150 mg/kg/day and when compared with controls, a few episodes of slightly higher food consumption were noted in females in the premating period and/or at the beginning of the gestation period. These findings were considered to be related to the test item but of minor toxicological importance in view of their magnitude and the absence of effects on the body weight.

There were no effects on food consumption at any dose level in males.

Water consumption: at 15, 50 and 150 mg/kg/day and when compared with controls, higher water consumption was recorded in males and/or females over the premating period. High water intake was also observed in all dose females throughout the gestation period and then on a few occasions during the lactation. These effects were more marked at the beginning of the pregnancy period. These findings were considered to be related to the test item but of minor toxicological importance in view of their magnitude and in absence of any correlates with adverse findings.

 

Hematology:at 150 mg/kg/day and when compared with controls, there was a decrease in red blood cell count in females (-13%, p<0.05) along with increased mean cell volume and mean cell hemoglobin (+13% and +14%, respectively, p<0.01). These changes were associated with increased reticulocyte count (+38%), which was also observed in males (+59%, p<0.01).These hematological variations correlated with histopathological findings suggestive of hemolysis (increased extramedullary hematopoiesis and pigment consistent with hemosiderin in the spleen of both sexes and increased erythroid cell numbers in the bone marrow of females). Marginally higher mean neutrophil count (+37%vs. controls, p<0.05) was also observed in females. In view of their magnitude and as mean values remained within or close to the range of the Historical Control Data, hematological changes were considered to be non-adverse.

 

Blood biochemistry:when compared with controls, blood biochemistry changes includeda dose-related decreased glucose level in males at all dose levels (-14 to -20%, p<0.05 to p<0.01) and the opposite trend in females (+21% to +29%, not statistically significant) in females, an increase in the inorganic phosphorus level in females (+30%, p<0.01) and an increase in the bile acids level in males and females from 50 mg/kg/day. In view of their magnitude, as mean values remained within the range of the Historical Control Data and in absence of histopathological findings, these changes were considered to be non-adverse.

 

Urinalysis: urinalysis changes consisted of higher volume in females at all dose levels (+64%vs.controls) corresponding to non-adverse higher mean water consumption, increased bilirubin levels in both sexes from 50 mg/kg/day, colored urine in males from 50 mg/kg/day, presence of nitrites in all males at all dose levels and in all females from 50 mg/kg/day, and of moderate to marked proteinuria in 3/5 males at 50 mg/kg/day and in 2/5 males and 1/5 females at 150 mg/kg/day.Bilirubinuria correlated with variations observed in the red blood cell mass of males and females given 150 mg/kg/day and with histopathological findings suggestive of increased red blood cell turnover and proteinuria could be related to adverse microscopic changes observed in the kidneys and/or in the urinary bladder at 150 mg/kg/day. As mean values remained within the range of the Historical Control Data, were poorly dose-related and without statistically significance, in absence of any other correlates and/or as findings could be consecutive from histopathological findings, these changes were considered to be non-adverse.

 

Thyroid hormones: there were no test item-related effects on the T4 and TSH levels in F0 males or pups at any dose level.

 

Pathology: At 150 mg/kg/day, at microscopic examination, adverse hyperplasia and single cell necrosis of the urothelium was observed in the urinary bladder in both sexes. Non-adverse microscopic findings were noted in the spleen (increased congestion and pigment consistent with hemosiderin) and kidneys (brown pigment), with compensatory hematopoiesis (erythropoiesis) in the spleen, correlated with increased spleen weights (both sexes), and bone marrow (females). Minimal and non-adverse tubular vacuolation was noted in two females in the kidneys at 150 mg/kg/day. In addition in one female at this dose-level, there were adverse cardiac atrial thrombosis and hypertrophy, glomerulopathy and renal tubular hyaline casts, potentially related to the test item administration. Non-adverse slight hepatocellular hypertrophy correlated with increased liver weights was noted in the liver at 150 mg/kg/day (both sexes).

 

Based on the experimental conditions of this study:

. the No Observed Adverse Effect Level (NOAEL) for parental systemic toxicity was considered to be 50 mg/kg/day based on the microscopic findings observed at 150 mg/kg/day in the urinary bladder (i.e. hyperplasia and single cell necrosis of the urothelium) of some males and females and in heart (i.e.cardiac atrial thrombosis and hypertrophy) of one female.

Justification for classification or non-classification

Based on the available data, a classification for repeated toxicity is required for nitrodiphenylamine according to the Regulation EC 1272/2008 : STOT RE 2, H373, target organ = urinary bladder.

Justification : The threshold-dose for classification into Category 2 is 300 mg/kg/d based on an oral 28 -day repeated toxicity study. Adverse effects on urinary bladder were observed at 150 mg/kg/day.