3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-12-17 to 2021-10-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2019-10-17 to 2020-02-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline required

Principles of method if other than guideline:

The study was non-specific but was expected to meet the requirements of the relevant agencies.

GLP compliance:

no

Remarks:

The study is a non-regulatory study for which a claim of GLP compliance was not made. However, the laboratory procedures used were consistent with International Standards of GLP.

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.: S019341917
- Substance type: Almost clear colorless liquid
- Physical state: liquid
- Molecular weight: 316 g/mol
- Analytical purity: 35.2% in water
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Expiration date of the lot/batch: 28 March 2021
- Purity/weighing factor: none

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed container, 2 to 8°C, protected from light
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Formulations of 0.2 and 100 mg/mL AI were stable for 15 days refrigerated (2 to 8° C), or for 14 days at room temperature (15 to 25°C) in Covance Study 8410552, Covance Laboratories, 2020c).

OTHER:
- Correction factor: 2.85, based on purity

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sixty time-mated female Crl:WI(Han) rats were obtained from Charles River Laboratories, Margate, United Kingdom.
- Age at study initiation: approx. 9 weeks old at the time of mating.
- Weight at study initiation: at least 140 g at time of mating. Animals were delivered to Covance by GD 3 and weighed between 171.6 and 252.2 g at the start of dosing.
- Fasting period before study: not indicated
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes (Home Office, 2014). Females were housed individually during gestation and with their litter during lactation. Bedding was provided on a weekly basis to each cage by use of European Softwood bedding or clean Aspen wood chips (Datesand Ltd; Manchester, United Kingdom). Each batch of bedding was analyzed for specific constituents and contaminants. No contaminants were present in the bedding at levels which might have interfered with achieving the objective of the study. Animals were provided with wooden Aspen chew blocks and nesting materials as forms of environmental enrichment.
- Diet (e.g. ad libitum): Animals had ad libitum access to VRF1 (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels which might have interfered with achieving the objective of the study.
- Acclimation period: Acclimation was limited by mated status, an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2019-10-21 To: 2020-02-11

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulation: formulations were prepared weekly. The test article was formulated as a suspension in purified water.
- Formulations were stored refrigerated (2 to 8 °C) in a sealed container, protected from light.
- Formulations of 0.2 and 100 mg/mL AI were stable for 15 days refrigerated (2 to 8 °C), or for 14 days at room temperature (15 to 25 °C) in Covance study 8410552.
- Formulations were stirred continuously from at least 30 minutes prior to and throughout dosing (excluding the control group). On GD8, group 5, subgroup 1 animals R0802 and R0805 (1000 mg/kg/day AI) were not doses due to significant body weight loss.
- A dose volume of 10 mL/kg was used. Individual dose volumes were based on the most recent individual body weight.

VEHICLE
- Amount of vehicle (if gavage): 10 mL

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: Mating for Crl:WI(Han) rats (overnight at Charles River Laboratories, Margate, United Kingdom) was confirmed by the presence of a vaginal plug in situ, or other evidence of mating, if necessary. The day on which mating was confirmed was designated as Gestation Day (GD) 0.

Duration of treatment / exposure:

- Prenatal development (Subgroup 1) animals were dosed once daily from GD 6 through 20.
- Postnatal development (Subgroup 2) animals were dosed once daily from GD 6 to Lactation Day (LD) 21.

Frequency of treatment:

Once daily

Duration of test:

114 days

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1; Control
Subgroup 1 and 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low)
Subgroup 1 and 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate)
Subgroup 1 and 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

Group 4 (High)
Subgroup 1 and 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 5 (High II)
Subgroup 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 6 (Intermediate II)
Subgroup 1

No. of animals per sex per dose:

- Prenatal Development (Subgroup 1): 6 female animals/dose group (6 groups). Dosing was omitted if an animal was in or near parturition at the time of daily dosing. This included Animals R0501, R0505 and R0506 (Group 2); R0603 (Group 3); and R0703 (Group 4).
- Prenatal Development (Subgroup 2): 6 female animals/dose group (4 groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Species selection rationale: The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics. Historical control data are available at the test facility for this strain of animal.

- Administration route selection rationale: The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.

- Dose selection rationale:
*High II Dose: In the absence of any obvious test article-related effects noted in the lower dose levels to establish a suitable high-dose level for the subsequent OECD test guideline 414 and 443 rat studies, and considering the findings of the previous 28-day study, the limit dose of 1000 mg/kg/day active ingredient (AI) was selected as the high II dose level, to observe for maternal toxicity and effects on prenatal development.
*High Dose: The high-dose level of 125 mg/kg/day AI was expected to elicit some toxicity without causing death.
* Intermediate II Dose: Due to the deaths of two animals noted following administration of 1000 mg/kg/day AI on GD 8, and significant body weight losses, the dose level was considered excessive and the remaining animals were terminated on the same day. The dose level of 500 mg/kg/day AI was considered suitable for exploration in this study.
*Intermediate Dose: An intermediate-dose level of 100 mg/kg/day AI was expected to show minimal test article-related effects.
*Low Dose: A dose level of 75 mg/kg/day AI was selected as the low dose, as it was expected to be well tolerated.
*Background information:
**In a 28-day repeated dose toxicity study in the Sprague-Dawley rat (Safepharm Laboratories Limited Study number 625/051), animals were administered 15, 150, or 1000 mg/kg/day AI by oral gavage. Clinical observations indicative of toxicity; functional incidents, such as hunched posture; sensory reactivity; reduced food consumption; increased water consumption; and reduced body weight gain were observed for animals administered 1000 or 150 mg/kg/day AI. Treatment-related changes in liver and spleen were observed following administration of 1000 or 150 mg/kg/day AI. Microscopic changes were also observed in the kidneys, urinary bladder, and stomach of high-dose animals. Hematological changes (hemolytic anemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time) and elevations in plasma aspartate aminotransferase, alanine aminotransferase, and bilirubin were observed in animals administered 1000 or 150 mg/kg/day AI. Therefore, the no observed effect level (NOEL) for this study was considered 15 mg/kg/day AI.
**In a 90-day repeated oral dose toxicity study in Sprague-Dawley animals were administered 50, 150, or 500 mg/kg/day AI by oral gavage (Covance Study 8280017). At the high dose of 500 mg/kg/day AI, one male was found dead during Week 12; this death was considered treatment-related. Clinical observations at this dose included salivation, staining around the mouth and noisy breathing, in combination with high water consumption values; this indicated irritant test materials and the animals not liking the taste of the formulation. Microscopic examination revealed adverse findings in the (fore) stomach (squamous cell hyperplasia) and urinary bladder (transitional cell hyperplasia), which were considered a result of the irritating properties of the test article. Findings for animals administered 150 mg/kg/day AI included low body weight gains; as no effect on food consumption was observed, this was considered non-specific toxicity. Squamous cell hyperplasia of the forestomach was noted in one male administered 150 mg/kg/day AI and, as this finding matched those recorded for those administered 500 mg/kg/day AI, it was considered adverse. The only treatment-related changes noted in males administered 50 mg/kg/day AI were statistically relevant minor changes in the red blood cell parameters, which were considered treatment-related but not adverse in nature; therefore, the NOEL for this study was considered 50 mg/kg/day AI.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed in the cage twice daily, at the beginning and end of the working day for signs of ill health or overt toxicity. Animals were observed daily immediately after dosing upon return to the home cage, and approximately 1 hour (±30 minutes) postdose. Postdose observations for subgroup 2 animals were not recorded from GD 20 to LD 4, to limit disturbing the litter. Character and timing of reactions to treatment were recorded, together with all observations (including absence of observations but were not reported).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was given a detailed physical examination outside of the cage, daily from the start of dosing until necropsy. An individual record was maintained of the clinical condition of each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded for Subgroup 1 animals on GD 3, 6, 7, 8, 9, 12, 15, 17, 19, 20, and 21. Body weights were recorded for Subgroup 2 animals on GD 3, 6, 7, 8, 9, 12, 15, 17, 19, 20 and 21 and on LD 1, 4, 7, 14, 17, and 22.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Daily food consumptions were recorded for all animals from GD 6 through 21, and from LD 0/1 through 14 for Subgroup 2 animals only. Consumption was calculated as g/animal/day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Terminal procedure - maternal animals:
All animals that died prior to their scheduled sacrifice were examined macroscopically for abnormalities of the thoracic and abdominal viscera.
- Subgroup 1 and 2 females were sacrificed in replicate order. Food was not removed prior to scheduled necropsies.
- Subgroup 1 females were sacrificed on GD21 by cervical dislocation following isoflurane anesthesia; death was confirmed by exsanguination.
- Subgroup 2: Females were sacrificed on LD 22 by isoflurane anesthesia; once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.

POST-MORTEM EXAMINATIONS: Yes
- Subgroup 1: Animals were examined macroscopically. All lesions were recorded and retained in the relevant fixative.
* Organs examined: ovaries and uteri
* The uterus of apparently non-pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.
- Subgroup 2:
*A macroscopic examination was performed, and all lesions were recorded. The following tissues from each animal were preserved in 10% neutral-buffered formalin unless otherwise indicated.
* Organs examined: cervix, thyroid, gross lesions, uterus , ovaries, vagina, pituitary
* The uterus of all mated and littered females and any apparently non-pregnant female was immersed in 10% ammonium sulfide solution, and implantation sites were counted.
The thyroid was dissected free from fat and other contiguous tissue and weighed postfixation and retained in 10% buffered formalin.

OTHER:
- Parturition:
Subgroup 2 animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered, or until a potential GD 25, whichever was earlier. Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation was recorded, where possible, and marked the end of gestation; when not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made. Abnormal observations of nesting, parturition, or nursing were recorded. It was considered important that the dam was disturbed as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).
- Estrous Stage Determination: The stage of the estrous for Subgroup 2 animals was recorded on LD 22 prior to necropsy

Ovaries and uterine content:

The uterus and ovaries were examined for implantations and corpora lutea, respectively. The extent of development of the implantation sites was determined as near as possible.
Maternal Necropsy - Subgroup 1:
- The ovaries and uteri were removed and examined, and the following data were recorded:
* Pregnancy status
* Gravid uterus weight
* Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported)
* Number of corpora lutea
* Number and intrauterine position of implantations subdivided into:
* Live fetuses
* Early intrauterine deaths
* Late intrauterine deaths
* Dead fetuses
- Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or fetal tissue, in addition to placental tissue. Dead fetuses were classified as those which appeared to have died shortly before necropsy.

Blood sampling:

not applicable

Fetal examinations:

Fetal Examinations:
- Litter Size and Sex Determination: On Postnatal Days (PND) 1, 4, 7, 14, 17, and 22, litter size and pup sex were recorded.
- Clinical Observations: Each animal was given a detailed clinical examination on PND 1, 4, 7, 14, 17, and 22.
- Body Weights: On PND 1, 4, 7, 14, 17, and 22, pup body weights were recorded.
- Ano-genital Distance: The Ano-genital distance of all pups was recorded on PND 4.
- Nipple/ Areolae Count: The number of nipples/areolae for male pups was counted on PND 13.
- Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition were given a macroscopic necropsy.

Fetal necropsy:
- Live fetuses were sacrificed by a subcutaneous injection of sodium pentobarbitone followed by confirmation of cessation of circulation.
- Individual fetal and placental weights were recorded, and fetuses were examined externally and externally sexed.
- Fetal abnormalities were classified as malformations (rare and/or potentially lethal defects) or variations (commonly occurring non-lethal abnormalities).

Pup Necropsy:
- Subgroup 2 pups were sacrificed on PND 22 by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. - A macroscopic examination was performed for all pups, including decedents, with particular attention to reproductive organs.
- Thyroids were taken from one pup/sex/litter and dissected free from fat and other contiguous tissue and weighed post fixation and retained in 10% buffered formalin.

Statistics:

See section Any other information on materials and methods incl. tables

Indices:

- Corrected body weight (Carcass Weight) = (Terminal body weight - uterine weight) X 100
- Corrected body weight change = (Carcass weight - GD 6 body weight) X 100
- Total weight change = (GD 21 body weight - GD 6 body weight) X 100
- % male fetuses = (Number of male fetuses/Number of fetuses of determined sex) X 100
- % pre-implantation loss = ((Number of corpora lutea - number of implantations)/Number of corpora lutea) X 100
- % post-implantation loss = ((Number of implantations - number of live embryos)/Number of implantations) X 100
- % male pups = (Number of male pups Day 1/Number of pups of determined sex) X 100
- Gestation index = (Number of females with live pups/Number of pregnant females) X 100
- % post implantation survival index = (Number of pups born/Number of implantation sites) X 100
- % live birth index = (Number of pups alive Day 1/Number of pups born) X 100
- % Day 22 viability index = (Number of pups alive Day 22/Number of pups born alive) X 100

Historical control data:

Historical control data are available at the test facility for this strain of animal.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Test article-related clinical observations of raised hair, hunched posture, irregular respiration, cold to touch body, and/or soft/small/brown feces were noted prior to sacrifice on GD 8 for two animals administered 1000 mg/kg/day AI (Animals R0802 and R0805). These observations in this group were considered adverse. Incidences of raised hair, hunched posture, head shaking, irregular and/or noisy respiration were observed for animals administered 500 mg/kg/day AI. These observations were mostly confined to individual animals, and were considered nonadverse. No test article-related clinical observations were noted for animals administered ≤125 mg/kg/day AI.
Prior to their unscheduled sacrifice, test article-related postdose observations for animals administered 1000 mg/kg/day AI were confined to a single incidence of mouth rubbing upon return to the home cage on GD 8 (Animal R0804). This finding is generally associated with the unpalatable nature or unpleasant taste of the test article formulation, and in isolation was considered not to have represented a toxic effect of the test article. A test article-related increase in the incidence of mouth rubbing (from GD 11 or 12) was observed for animals administered ≤100 mg/kg/day AI, with isolated incidences of paddling behavior or excessive salivation also noted for one or two animals administered 500 mg/kg/day AI. These observations are generally associated with the unpalatable nature or unpleasant taste of test article formulation rather than an indication of test article toxicity. No test article-related postdose observations were noted for animals administered 75 mg/kg/day AI.

Postnatal Development (Subgroup 2): No test article-related clinical observation were noted. The isolated clinical observations recorded were confined to one or two animals within each group, including controls, and were considered low incidence findings occasionally observed in studies of this type and unrelated to the test article. Test article-related postdose observations were confined to a few incidences of mouth rubbing upon return to home cage during gestation (100 or 125 mg/kg/day AI) and lactation (125 mg/kg/day AI), with two animals from the latter dose group (Animals R0701 and R0702) also observed with isolated incidences of excessive salivation. Postdose observations of mouth rubbing and salivation are common following gavage administration and were considered associated with the unpalatable nature of unpleasant taste of the test article formulation, rather than an indication of test article toxicity.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Prenatal Development (Subgroup 1): Test article-related mortality occurred following administration of 1000 mg/kg/day AI. One female administered 1000 mg/kg/day AI (Animal R0806) was found dead on GD 8. The remaining animals from this group were also sacrificed on that same day because this dose level was considered excessive due to marked body weight losses and reduced food consumption. No unscheduled deaths occurred for the remaining groups.

Postnatal Development (Subgroup 2): No unscheduled deaths occurred as a consequence of test article toxicity. One female administered 75 mg/kg/day AI (Animal R0501) was sacrificed on LD 0 due to total litter loss. Only three live pups were noted in the litter, and in the absence of similar observations at the higher dose levels, this finding was considered incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Adverse, test article-related marked mean body weight loss was observed, between GD 7 and 8 following administration of 1000 mg/kg/day AI (mean body weight loss - 16.9 g; P < 0.01), and, as such, this dose level was considered excessive for further dosing.
Initial test article-related body weight losses were observed between GD 6 and 8 for females administered 500 mg/kg/day AI, and lower mean body weight gain was noted between GD 8 and 9 for these animals, with mean overall body weight gain approximately -19%, compared with controls over the dosing duration (GD 6 to 21). Overall mean body weights were not overtly affected, compared with controls; as such, the initial body weight losses were considered nonadverse. No test article-related effect on body weights was noted following administration of ≤125 mg/kg/day AI.

Postnatal Development (Subgroup 2): A test article-related reduction in mean body weight gain was noted during lactation (LD 4 to 14) for animals administered 125 mg/kg/day AI, compared with controls. Lower mean body weight gains were observed for animals administered 125 mg/kg/day AI compared with controls, between LD 4 and 7 (-56%), and the effect was still evident between LD 7 and 14 (-30%, compared with controls). This resulted in a lower overall mean body weight gain for these animals during lactation (approximately -35%). No test article-related effect on body weights was observed during gestation following administration of 125 mg/kg/day AI, compared with controls. No effect on body weights was noted following 75 or 100 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Adverse, test article-related reductions in food consumption were observed following administration of 1000 mg/kg/day AI. Mean food consumption between GD 6 and 8 was statistically significantly lower than controls (up to -83%; P < 0.05 or P < 0.001) prior to the unscheduled sacrifice of this dose group. Test article-related reductions in food consumption were also observed following administration of 500 mg/kg/day AI, compared with controls, with an up to 40% reduction in mean food consumption between GD 6 and 9, and statistically significant reductions between GD 7 and 9 (P < 0.05 or P < 0.01). Some improvement in food consumption was noted thereafter; however, mean food consumption values remained lower than controls throughout the remainder of the in-life phase. Overall food consumption (GD 6 to 21) was approximately -16% lower than controls. In the absence of any associated adverse changes, these small reductions in food consumption were considered nonadverse at this dose level. No test article-related effect on food consumption was noted following administration of ≤125 mg/kg/day AI.

Postnatal Development (Subgroup 2): No test article-related effect on food consumption was observed at any dose level investigated. Any intergroup differences observed during gestation or lactation were considered to have arisen incidentally and not as a consequence of test article administration.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Postnatal Development (Subgroup 2): No test article-related effect on maternal thyroid weights was noted. Any minor intergroup differences were not dose related and considered incidental.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Test article-related macroscopic changes were noted in the intestinal tract for animals administered 1000 mg/kg/day AI. Following the unscheduled sacrifice of this group, abnormal contents in the jejunum, ileum, cecum, and colon were observed, with one female (Animals R0804) noted with slight to moderate distension of the jejunum, ileum, and colon. No test article-related macroscopic observations were noted in the remaining animals at scheduled sacrifice.

Postnatal Development (Subgroup 2): No test article-related macroscopic observations were noted for the maternal animal at scheduled sacrifice.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Postnatal Development (Subgroup 2):
Estrous Stage Determination: No test article-related effect on the stage of estrus was observed at scheduled sacrifice. Most females across all dose groups were in di-estrus.

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Prenatal Development (Subgroup 1): All uterine parameters, including the mean number of implantations, live fetuses, and post-implantation losses, for test article-treated animals were comparable with controls. Any minor differences were not dose-related and considered incidental.

Postnatal Development (Subgroup 2): The mean number of live pups born following test article administration at all dose levels was slightly lower than controls, and post-implantation loss was slightly higher than controls. However, no dose-relationship was evident; taking into consideration the small group sizes, this was considered incidental. One female administered 75 mg/kg/day AI (Animal R0501) was sent to necropsy on LD 0 due to the death of all three live born pups. In the absence of similar findings in groups administered 100 or 125 mg/kg/day, this finding was considered incidental.

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

Prenatal Development (Subgroup 1): At the terminal necropsy, all remaining animals (five controls, six animals administered 75 mg/kg/day, five animals administered 100 mg/kg/day AI, six animals administered 125 mg/kg/day AI, and six animals administered 500 mg/kg/day AI) had live fetuses.

Postnatal Development (Subgroup 2): The mean number of live pups born following test article administration at all dose levels was slightly lower than controls, and post-implantation loss was slightly higher than controls. However, no dose-relationship was evident; taking into consideration the small group sizes, this was considered incidental. One female administered 75 mg/kg/day AI (Animal R0501) was sent to necropsy on LD 0 due to the death of all three live born pups. In the absence of similar findings in groups administered 100 or 125 mg/kg/day, this finding was considered incidental.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Prenatal Development (Subgroup 1): Pregnancy was unaffected by administration of up 500 mg/kg/day AI. All animals administered 1000 mg/kg/day AI and prematurely terminated on GD 8 were pregnant.
One animal administered 100 mg/kg/day AI (Animal R0202) littered prior to necropsy on GD 21. This is a low incidence finding occasionally observed in studies of this type, was considered related to the time of mating of animals at the animal supplier, and did not represent an effect of the test article.

Postnatal Development (Subgroup 2): No test article-related effect on parturition or litter data was observed up to 125 mg/kg/day AI. Four controls and all six females from each test article-treated group completed parturition with live offspring. No impact on the gestation length, gestation index, number of still births or live births, and live birth or Day 22 viability indices was observed for test article-treated females compared with controls

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Prenatal Development (Subgroup 1): Pregnancy was unaffected by administration of up 500 mg/kg/day AI. All animals administered 1000 mg/kg/day AI and prematurely terminated on GD 8 were pregnant.
Of the surviving dose groups, all animals were pregnant, except one control animal (Animal R0004) and one animal administered 100 mg/kg/day AI (Animal R0204); these females were supplied non- pregnant from the animal supplier, which was not test article related. One animal administered 100 mg/kg/day AI (Animal R0202) littered prior to necropsy on GD 21. This is a low incidence finding occasionally observed in studies of this type, was considered related to the time of mating of animals at the animal supplier, and did not represent an effect of the test article.

Postnatal Development (Subgroup 2): No test article-related effects on pregnancy were observed up to a dose level of 125 mg/kg/day AI. Of the six females in each group, five controls and all six females from each group administered 75, 100, or 125 mg/kg/day AI were pregnant. One control female (Animal R0402) was noted as non-pregnant, while one pregnant control female (Animal R0405) did not litter and was observed with no viable fetuses on GD 26. These findings were confined to the control group and considered a low incidence finding, with no relationship to test article administration.

Other effects:

not specified

Key result

Dose descriptor:

dose level: 500 mg/kg/day AI

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: suitable high dose in OECD414 study

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal Observations: A possible test article-related effect on fetal weights was observed following 500 mg/kg/day, compared with controls. Mean fetal weights, adjusted for litter size, following maternal exposure of 500 mg/kg/day AI was -8% lower than controls, although statistical significance was not attained. The etiology of this finding was unclear, and a relationship to test article administration could not be ruled out. Nevertheless, this was considered nonadverse. Mean corrected body weight, mean corrected body weight change, and mean total weight change for dams administered 500 mg/kg/day AI were -6, -48, and -13% lower than controls, respectively. In view of the marginally lower mean body weight gains observed in-life, this was considered a nonadverse test article-related effect.

Pup Observations: No test article-related effect on offspring body weight was observed throughout lactation.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Prenatal Development (Subgroup 1): All uterine parameters, including the mean number of implantations, live fetuses, and post-implantation losses, for test article-treated animals were comparable with controls. Any minor differences were not dose-related and considered incidental.

Postnatal Development (Subgroup 2): The mean number of live pups born following test article administration at all dose levels was slightly lower than controls, and post-implantation loss was slightly higher than controls. However, no dose-relationship was evident; taking into consideration the small group sizes, this was considered incidental. One female administered 75 mg/kg/day AI (Animal R0501) was sent to necropsy on LD 0 due to the death of all three live born pups. In the absence of similar findings in groups administered 100 or 125 mg/kg/day, this finding was considered incidental.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Fetal observations: Fetal sex was unaffected by test article administration.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal observations: Mean gravid uterine weight in the groups administered 500 mg/kg/day AI was slightly higher than controls; no dose-relationship was evident, and this was considered likely due to slightly lower litter size in the control group, and unrelated to the test article.

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal observations: No test article-related effect on fetal ano-genital distance was noted. Minor decreases in ano-genital distance, noted for the female fetuses maternally administered the test article up to a dose level of 500 mg/kg/day AI were not statistically significant and considered to have arisen incidentally.

Pup observations: No test article-related effect on pup ano-genital distance was observed. Statistical analysis of the data did not show any statistically significant intergroup differences.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Pup observations: No test article-related mortality or clinical observations were noted for any offspring. The mortality or clinical observations recorded were low incidence findings occasionally observed in rat offspring of this age and were considered not related to maternal administration of the test article.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal observations: No test article-related fetal malformations or variations were noted on GD 21. One fetus from the litter of a single animal administered 125 mg/kg/day AI (Animal R0302-L3 Male) had a number of external malformations and variations, including omphalocele, short trunk, malrotated right hind limb at the ankle joint, micromelia of the left forelimb and right hind limb, small entire genital tubercle, edema of the head/neck, and subcutaneous hematoma of the abdomen. These observations were confined to a single fetus from one litter and were not recorded for the litters maternally exposed to 500 mg/kg/day; as such, they were considered unrelated to the test article.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Fetal observations: No test article-related fetal malformations or variations were noted on GD 21.

Visceral malformations:

no effects observed

Description (incidence and severity):

Fetal observations: No test article-related fetal malformations or variations were noted on GD 21.

Other effects:

no effects observed

Description (incidence and severity):

Pup Nipple/ Areolae Count: Maternal administration of the test article had no effect on mean nipples/areolae count in male pups.

Pup macroscopy: No test article-related macroscopic observations were noted for offspring at scheduled sacrifice. Any macroscopic observations were low incidence findings occasionally observed in rat offspring of this age and were considered not related to maternal administration of the test article.

Pup Organ Weights: No test article-related effect on pup thyroid weights was noted. An increase in mean absolute and body weight-related thyroid/parathyroid weights was noted for male pups maternally exposed to the test article at all dose levels, compared with controls. No dose- relationship was observed, and the corresponding values in females were similar to controls. Taking into consideration the small groups size, this was considered to have arisen incidentally.

Key result

Dose descriptor:

dose level: 500 mg/kg/day AI

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: suitable high dose in OECD414

Conclusions:

Once daily oral (gavage) administration of Amides, C12-C18 (even numbered), N-[3- (dimethylamino) propyl], N’-oxides at a dose level of 75, 100, 125, 500, or 1000 mg/kg/day AI to pregnant female rat to assess prenatal development, resulted in mortality following administration of 1000 mg/kg/day AI. The nature and severity of these observations demonstrated that this dose level exceeded the maximum-tolerated dose, and this dose level was considered excessive. A dose level of 500 mg/kg/day AI was better tolerated, with nonadverse postdose observations, minor reduction in body weight gain and food consumption, reduced fetal weight, and reduced corrected body weight and corrected body weight change. These were considered nonadverse, and a dose of 500 mg/kg/day AI was considered a suitable high-dose level for further exploration in a subsequent prenatal development study in the rat (Covance Study 8410555, [Covance Laboratories Ltd, 2020a]).
For assessment of postnatal development, once daily oral (gavage) administration of Amides, C12-C18 (even numbered), N-[3-(dimethylamino) propyl], N’-oxides at dose levels of 75, 100 or 125 mg/kg/day AI to the female rat from Gestation Day 6 through to Lactation Day 21 was well tolerated with only minor test article-related effects noted. The effects were confined to postdose observations which were considered attributable to the nature of the test article formulations and not a direct toxic effect of the test article, and lower mean body weight gains observed following 125 mg/kg/day AI during lactation. These effects were considered nonadverse. Furthermore, no effect on the subsequent offspring was noted. In view of the overall findings noted in this study, and taking into consideration the findings from previous studies performed on this test article over a much longer duration (Covance Study 8280017; Covance Laboratories Ltd, 2014), a dose level of 150 mg/kg/day AI was selected as the high-dose level for the subsequent OECD 443 study (Covance Study 8410556). This dose was anticipated to induce minimal parental toxicity (i.e., changes in red blood cell parameters and local irritancy of the stomach) without causing death, or affecting parturition, pup growth, or mating of the first generation.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-07-24 - 2009-12-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Physical state: pale amber liquid
- Analytical purity: 31.8% (99.9% w/w active ingredient), 35.46% (based on solid substance)
- Lot/batch No.: PA19100368
- Storage condition of test material: room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK
- Age at study initiation: 12 weeks old
- Weight at study initiation: males: 305-358 g; females: 189-219 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages.
Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): ad libitum; pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: From: September 15, 2009 To: November 8, 2009

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in Distilled water. The stability and homogeneity of the test material formulations were determined. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.

Details on mating procedure:

- M/F ratio per cage: on Day 15, all animals were paired on a 1 male:1 female basis within each dose group
- Length of cohabitation: Maximum of 14 days
- Proof of pregnancy: The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
- After successful mating each pregnant female was caged (how): Females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each test material formulation were taken and analysed for concentration. The test material formulations were diluted with water to give a final, theoretical test material concentration of approximately 3 mg/ml. Standard solutions of test material were prepared in water at a nominal concentration of 3 mg/ml.

The concentration was determined by high performance liquid chromatography (HPLC) using an external standard technique.
HPLC : Agilent Technologies 1100 or 1200, incorporating autosampler and workstation
Column : Gemini 3µ C18 (100 x 4.6 mm id)
Column temp : 40 °C
Mobile phase : Eluent A:acetonitrile with 0.1% trifluoroacetic acid; Eluent B:water with 0.1% trifluoroacetic acid
Flow-rate : 1 ml/min
UV detector wavelength: 205 nm
Injection volume : 25 µl
Retention time : Profile of peaks from ~ 1.4 to 10.5 mins

The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery. The results indicate that the prepared formulations were within ± 2% of the nominal concentration.

Duration of treatment / exposure:

54 days

Frequency of treatment:

Daily

Details on study schedule:

i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
iii) Following evidence of mating (designated as Day 0 post coitum), the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Dose / conc.:

0 mg/kg bw/day

Remarks:

actual ingested

Dose / conc.:

15 mg/kg bw/day

Remarks:

actual ingested, incorporating a correction factor for 31.8% purity, converted to 16.7 mg/kg bw/day considering correction for 35.46% purity

Dose / conc.:

40 mg/kg bw/day

Remarks:

actual ingested, incorporating a correction factor for 31.8% purity, converted to 44.5 mg/kg bw/day considering correction for 35.46% purity

Dose / conc.:

100 mg/kg bw/day

Remarks:

actual ingested, incorporating a correction factor for 31.8% purity, converted to 111.35 mg/kg bw/day considering correction for 35.46% purity

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination. Females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1 - 4).

WATER CONSUMPTION AND COMPOUND INTAKE:
Water intake was observed daily by visual inspection of water bottles for any overt changes. Inter-group differences did not indicate any need for more formal gravimetric measurements.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded and offspring were individually identified within each litter by a tattoo on Day 1 post partum.
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Days 1 and 4 post partum

All live offspring were assessed for surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females that failed to achieve pregnancy were killed after Day 26 post coitum.

GROSS NECROPSY
All adult animals including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).

HISTOPATHOLOGY
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where indicated:
Coagulating gland, Prostate, Epididymides, Seminal vesicles, Ovaries, Testes, Mammary tissue, Uterus/Cervix, Pituitary, Vagina.
The tissues from control and 100 mg/kg/day dose group animals, and any animals which failed to achieve a pregnancy, were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from control and 100 mg/kg/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

GROSS NECROPSY
Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis:
Bodyweight and bodyweight change, Food consumption for females during gestation and lactation, Litter data, Sex ratio, Implantation losses and viability indices, Offspring bodyweight and bodyweight change, Offspring surface righting, Adult absolute and bodyweight relative organ weights.

Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnetts test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U test. The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.

In addition, histopathological findings were analysed (excluding non-mated females and females not producing a pregnancy/litter) using the following methods: Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.

Reproductive indices:

- Pre-coital Interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices: mating index (%) and pregnancy index (%)
- Gestation length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition index

Offspring viability indices:

- Implantation losses (%)
- Live birth and viability indices
- Sex ratio (% males)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant findings were observed throughout the study. Sporadic episodes of increased salivation were evident in animals of either sex treated with 100 mg/kg/day, one male from this treatment group also had red/brown staining around the mouth. One 40 mg/kg/day female had noisy respiration. Generalised fur loss was detected in one 15 mg/kg/day and one control female. Increased salivation is often recorded following the oral administration of an unpleasant tasting test material formulation and is considered not to be indicative of systemic toxicity. The remaining observations are considered incidental and not to represent an adverse effect of treatment. No such effects were detected in the 40 or 15 mg/kg/day males.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically significant effect on bodyweight development was detected for treated animals, in comparison to controls. A slight but statistically significant increase in bodyweight gain (P<0.05) was evident in the 40 and 100 mg/kg/day males during Weeks 5 and 6. This increase was also seen in the 15 mg/kg/day males during Week 6 only. There was no adverse effect on overall bodyweight gain between the treated and control animals. In the absence of an effect on dietary intake or supporting histopathological correlates this finding is considered to be of no toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effect on dietary intake was detected for treated animals, in comparison to controls.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Water consumption was monitored daily by visual inspection of the water bottles. There was no adverse effect on water consumption.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No adverse effect on mating performance was detected. All animals from the control and treated groups mated within four days of pairing with the exception of one 15 mg/kg/day female which mated after thirteen days of pairing.

Key result

Dose descriptor:

NOEL

Remarks:

for reproductive toxicity

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

NOEL is 111.35 mg/kg bw/day based on 35.46% purity

Key result

Dose descriptor:

NOEL

Remarks:

for systemic toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

NOEL is 111.35 mg/kg bw/day based on 35.46% purity

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs were detected

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No adverse effect on total litter weights, offspring bodyweight development or surface righting reflex was detected in treated animals, in comparison to controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At terminal kill, one female treated with 15 mg/kg/day and one treated with 100 mg/kg/day produced a litter with one pup which showed a dark left testis. One 40 mg/kg/day female produced a litter with one pup which showed a dark right testis. There were no abnormalities detected in the remaining animals. The incidental signs detected were considered to be of no toxicological importance.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

VIABILITY (OFFSPRING)
There were no obvious differences in the number of corpora lutea or implantation sites from treated females when compared to controls; and pre-implantation and postimplantation losses from treated animals were comparable to controls. Furthermore, no obvious effect on litter size, sex ratio and offspring viability were detected for treated animals, in comparison to controls.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

NOEL is 111.35 mg/kg bw/day based on 35.46% purity

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The oral administration of the test material to rats for a period of up to fifty-four consecutive days at dose levels of up to 100 mg/kg/day, did not result in any treatment-related effects. The No Observed Effect Level (NOEL) for systemic toxicity was therefore considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). The No Observed Effect Level (NOEL) for reproductive toxicity was considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). However, taking into account the purity based on solid substance (35.46%) is recommended and based on this purity and expert judgement by the registrant, and thus factor 2.82, the NOEL for systemic and reproductive toxicity can be considered to be 111.35 mg/kg.

Executive summary:

The study was performed to screen for potential adverse effects of the test material on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study complies with the recommendations of the OECD Guideline No. 421 (Reproduction/Developmental Toxicity Screening Test). The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han:HsdRccHan:WIST strain rats, for up to fifty-four consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 15, 40 and 100 mg/kg/day (incorporating a correction factor for 31.8% purity). A control group of ten males and ten females was dosed with vehicle alone (Distilled water). Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Adult males were terminated on Day 43, and all surviving females and offspring on Day 5 post partum. Any females that failed to achieve pregnancy were killed after Day 26 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed. The oral administration of the test material to rats for a period of up to fifty-four consecutive days at dose levels of up to 100 mg/kg/day, did not result in any treatment-related effects.

The 'No Observed Effect Level' (NOEL) for systemic toxicity was therefore considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). The 'No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 100 mg/kg/day (incorporating a correction factor for 31.8% purity). However, taking into account the purity based on solid substance (35.46%) is recommended and based on this purity and thus factor 2.82, the NOEL for systemic and reproductive toxicity can be considered to be 111.35 mg/kg. 

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-04 - 2014-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl], N’-oxides or Rewominox B 204
- Physical state: clear liquid
- Analytical purity: 35.6%
- Purity test date: 18 January 2013
- Lot/batch No.: ST03102288
- Expiration date of the lot/batch: 31 January 2015
- Stability under test conditions: 14 days at 2 to 8°C
- Storage condition of test material: 2-8°C in the dark

Species:

rat

Strain:

other: CR:CD (Sprague-Dawley)

Details on species / strain selection:

The rat was selected because it is a rodent species known to be accepted by various regulatory authorities and because of the availability of background data within these laboratories.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: between 202.1 and 263.3g. Females: between 150.8 to 205.1
- Fasting period before study: None (but one fasting night during week 13 for blood sampling)
- Housing: The animals were housed in a single, exclusive room, and in groups up to 4 rats of the same sex and dose level par cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45 to 65 %
- Air changes (per hr): 15-20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours nominal

IN-LIFE DATES: From: 22 March 2013 to 24 June 2013

Route of administration:

oral: gavage

Details on route of administration:

The oral (gavage) route of administration was chosen because it is a possible human exposure route.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was formulated as a solution in purified water following dispensary standard operation procedures and a formulation method develpoped in the laboratory for this study. Formulations were prepared weekly and stored refrigerated (2 to 8°C) in a sealed container, protected
from the light. The formulations were stirred on arrival at the animal room and were stirred continuously before and throughout dosing.

The dose levels of 50, 150 and 500 mg/kg/day were expressed as the active ingredient. The test article was supplied as a 356 mg/mL solution and final concentrations of 5, 15 and 50 mg/mL were formulated by serial dilution.

OTHER:
After the dose formulation was drawn into the syringe, the catheter was washed in potable water, wiped clean and then washed again before dosing the animal. he test article was administered orally by gavage, as it is possible human exposure route. A dose volume of 10 mL/kg was used. Individual dose volumes were based on individual body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- SAMPLE COLLECTION:
Samples (3 x 1 mL aliquots from test formulations; 2 x 1 mL aliquots from control formulations) prepared for use on Week 1 and Week 13 were taken for analysis of achieved concentration. Samples were stored at ambient until and during analysis that occurred within 2 hours of the formulations being prepared.

- SAMPLE ANALYSIS:
A set of standard solutions over the approximate concentration range 100 to 600 μg/mL was analysed by HPLC. A six point calibration graph was constructed for the standard solutions. Injections of the lowest calibration standard diluted by factors of 100 and 50 were used to determine a limit of detection (LOD). Formulations were diluted to within the range of the calibration line (approximately100.0 to 600.0 μg/mL) using water.

Duration of treatment / exposure:

The animals were dosed once for at least 13 weeks excluding the day of necropsy.

Frequency of treatment:

The test material was administered daily.

Dose / conc.:

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

Remarks:

active ingredient, actual ingested

Dose / conc.:

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

Remarks:

active ingredient, actual ingested

Dose / conc.:

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

Remarks:

active ingredient, actual ingested

No. of animals per sex per dose:

10 animals per sex per dose
5 spare males and females were available to replace any animals due to extremes of body weight, ophthalmoscopy changes or ill health.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
- High dose: The high dose of 500 mg/kg/day was chosen as, based on the background information (28 day toxicity study), it was not expected to reveal overt toxicity and should set a No-Observed-Adverse-Effect Level (NOAEL).
- Intermediate dose: The intermediate dose of 150 mg/kg/day was chosen to set an NOAEL if effects were seen at 500 mg/kg/day.
- Low dose: The low dose of 50 mg/kg/day was chosen to set a No-Observed-Effect Level (NOEL).
- Background information:
In a previous 28 day toxicity study conducted by the Sponsor (Study number 625/051) a dose of 1000 mg/kg/day resulted in the death of a single female on Day 12. Noisy respiration, increased salivation, red/brown staining of the fur and wet fur, and hunched posture were recorded in animals at this level with diuresis, tiptoe gait, gasping respiration, pallor of the extremities and staining around the ano-genital region also recorded sporadically. No overt signs were recorded in animals at 150 or 15 mg/kg/day. Body weight gain and food consumption were initially affected but improved as the study progressed. Haemolytic anaemia was noted in animals at 1000 or 150 mg/kg/day. Histopathology identified target organs as liver, spleen, kidneys, urinary bladder and stomach with adverse findings recorded at 150 or 1000 mg/kg/day. Findings included centrilobular hepatocyte enlargement in the liver, splenic extramedullary haemopoiesis in the liver, splenic extramedullary haemopoiesis, haemosiderin pigment deposition/accumulation in the liver, spleen and kidneys, hyperplasia in the urinary bladder and acanthosis, hyperkeratosis occasionally associated with subepithelial inflammatory cell infiltrates in the (fore)stomach.

Positive control:

No positive control.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at the beginning and the end of the working day.
- Cage side observations : animals were observed for signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
Each animal was given a detailed physical examination at daily intervals, one hour after dosing only. An individual record was maintained of the clinical condition of each animal. Post dose observations were also performed in relation to the time of dosing to coincide with clinical examinations one hour post dose daily from Day 1 to termination.

BODY WEIGHT: Yes
Individual body weights were recorded twice weekly, before dose, from Dosing Day 1 to Week 4, and at weekly intervals from Week 5 to Week 13 and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The amount of food consumed by each cage of animals was determined twice weekly.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
The amount of water consumed by each cage of animals was determined daily for a three day period (Friday to Monday) in Week 1, 6 and 12.

OPHTHALMOSCOPIC EXAMINATION: Yes
Investigations were performed on all animals pre-treatment and on Groups 1 and 4 in Week 12.

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Once at Day 90.
- Animals fasted: Yes, samples were collected after an overnight period without food.
- How many animals: Investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90)
- Parameters examined: Haematology Tests: haemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte count, red cell distribution width, haemoglobin distribution width, total and differential white cell count, platelet count (includes platelet clump assessment - clump count below 100 considered as none detected; clump count over 100 considered as plateletclumps present and confirmed by review of Advia cytogram or blood film examination), platelet crit, mean platelet volume, platelet distribution width. Coagulation Tests: prothrombin time activated partial thromboplastin time. Clinical Chemistry Tests: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, creatinine, urea, glucose.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and at once weekly intervals thereafter. In Week 13, an detailed assessment was done.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity and some open filed test were performed weekly. Functional observationanl battery, open field examinations and locomotor activity were assessed in Week 13.

Sacrifice and pathology:

NECROPSY AND GROSS PATHOLOGY: All animals including the decedent animal were subjected to necropsy. The scheduled necropsies were performed in Week 14 (Day 95), after an overnight period without food. Where possible, they were carried out in replicate order. Each animal was given an overdose of isoflurane anaesthesia. Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal. A full macroscopic examination was performed under the general supervision of a pathologist and all lesions were recorded.

ORGAN WEIGHTS: Animals were weighed immediately before being killed for necropsy. The organs denoted by ‘W’ in the tissue list were dissected free from fat and other contiguous tissue and weighed before fixation (see table below). Left and right organs were weighed together. For 2F 54, the heart was weighed with adhered pericardium.

HISTOPATHOLOGY: The tissues from each animal presented in the following table were preserved in 10% neutral-buffered formalin unless otherwise indicated.
The following tissues were embedded in paraffin wax BP (block stage), sectioned at anominal 5 μm and stained with haematoxylin and eosin:
Group 1 and Group 4: All tissues denoted by ‘E’ in the tissue list above
Group 2 and Group 3: Lungs, liver, spleen, kidney, urinary bladder, (fore)stomach and gross lesions
Decedent: All tissues denoted by ‘E’ in the tissue listed below

MICROSCOPIC OBSERVATIONS
The following tissues were examined microscopically by the Principal Investigator for Histopathology:
Group 1 and Group 4: All tissues denoted by ‘E’ in the tissue list above
Group 2 and Group 3: Lungs, liver, spleen, kidney, urinary bladder, (fore)stomach and gross lesions
Decedent: All tissues denoted by ‘E’ in the tissue listed below

Other: Bone Marrow Smear Evaluation
Bone marrow smears were prepared at necropsy. Tissues were taken into bovine serum albumin. The smears were prepared, air dried and then were fixed in methanol, but not examined.

Statistics:

Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
The control group (Group 1) was taken as the baseline group with which the treated groups (Groups 2, 3, 4) were compared.
Body weight gains, terminal body weights, functional observational battery, motor activity, haematology and clinical chemistry variables were analysed using one-way analysis of variance (ANOVA), separately for each sex. Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P>0.05), and the group effect from the ANOVA was significant (P≤0.05), pairwise comparisons with the control group were made using Dunnett's test.
Where Levene’s test was significant (P≤0.05), a rank-transformation was applied to the data prior to analysis. No analysis was performed for variables with values above or below the limit of the assay. Some variables were not analysed due to there being too few values for meaningful analysis.
Organ weights were analysed using Analysis of Covariance (ANCOVA), for each sex separately, using the terminal body weight as the covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this assumption was tested. Where there was no evidence of heterogeneity among the slopes (P>0.01), and the group effect from the ANCOVA was significant (P≤0.05), pairwise comparisons with the control group were made using Dunnett's test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Post dosing observations of salivation and staining around the mouth are indicative of the treated animals not liking the taste of the test article and/or being dosed with an irritant material and are considered a secondary action of the test article. Noisy breathing was generally recorded for animals given 500 mg/kg/day but also in a single male at 150 mg/kg/day and a single female at 50 mg/kg/day; this finding being commonly associated with oral administration of irritant materials.

Mortality:

mortality observed, treatment-related

Description (incidence):

A single male dosed at 500 mg/kg/day was found dead during Week 12; although a cause of death could not be established, as the macroscopic findings (large caecum and liver, dark lungs and thymus, a thickened stomach and red fluid in the thoracic cavity) along with the microscopic findings (congestion of the liver, aggregates of foamy alveolar macrophages within the lung (graded as slight, multifocal), squamous cell hyperplasia with hyperkeratosis (both graded as moderate), together with erosion/ulcer (slight, focal) in the (fore)stomach, and minimal transitional cell hyperplasia in the urinary bladder) match the findings in the high dose animals, it is considered that this death was related to the treatment of the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When compared with control values, body weight gains for males during the first 8 weeks of treatment did not reveal an effect of treatment with the test substance. Over the second half of the study gains were low for males given 150 or 500 mg/kg/day, when compared with control, resulting in an overall slightly low gain in males at 500 mg/kg/day. There was no effect on gains for males given 50 mg/kg/day. Body weight gains for female at 500 mg/kg/day were variable, but high, over the first 5 weeks of treatment resulting in slightly high overall gains. There was no effect on body weight gains for females at 50 or 150 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect on food consumption in any test substance-treated male group or for females given 50 or 150 mg/kg/day. Food consumption values were high for females given 500 mg/kg/day.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption values were high in males and females given 500 mg/kg/day. There was no effect seen in groups given 50 or 150 mg/kg/day. The increase in the water consumed could be associated with the oral dosing of an irritant material resulting in the behavioural response to drink. There was no histopathological evidence of any effect on the kidney although transitional cell hyperplasia in the urinary bladder was recorded, suggesting urinary excretion of an irritant material.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular effects after treatment with the test substance in any group and sex.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant haematological changes (decreased haemoglobin, red blood cell counts, mean cell haemoglobin concentration, haemoglobin distribution width and red cell distribution width and increased reticulocyte counts and percentage, mean cell volume and mean cell haemoglobin) consistent with a decreased red blood cell mass associated with regeneration were seen in all treated males and female groups with the severity of the finding being dose dependant. The haemopoietic response in the spleen is considered to account for the increased weight of this organ.
For haemoglobin levels and red blood cell numbers, females given 500 mg/kg/day showed a change of between 7 and 17% from control values while males showed a change of between10 and 19% meaning the findings are on the border of non adverse and adverse. Males given 150 mg/kg/day showed a change of between 7 and 11% compared to controls with females showing a change between 1 and 5% with these findings being considered non-adverse due to the smaller magnitude of change. The low dose (50 mg/kg/day) percentages were between 0 and 2% for females and 4 and 6% for males and also not considered adverse. A reduced red blood cell mass of more than 30% is considered adverse and a change of less than 10% is considered non-adverse for rodents (Principles of Methods of Toxicology 5th Ed. (A Wallace Hays) 2003; In Chapter 26 (Robert L Hall and Nancy E Everds) p1331-1332.) This finding was not considered to be anaemia as it wasn’t associated with any additional findings such as an increase plasma and urinary bilirubin.
Compound-related changes in the haematology parameters consisted of generally dose-dependent and statistically significant decreases in a number of erythroid parameters (erythrocyte count and haemoglobin concentrations) in compound-treated rats of both genders. These changes were accompanied by evidence for a robust regenerative response, strongly suggesting an appropriate response by the bone marrow. This regenerative response was indicated by one or more of the following in the compound-treated groups in both genders: 1) robust and dose-dependent reticulocyte response; 2) generally dose-dependent increase in the mean corpuscular volume and decreases in the mean corpuscular haemoglobin concentration which is consistent with increased number of reticulocytes. With this convincing evidence of appropriate bone marrow regeneration (increased reticulocyte response) an investigation of the bone marrow smears was not required (References: Provencher Bollinger, A. Cytologic evaluation of bone marrow in rats: Indications, methods, and normal morphology. Veterinary Clinical Pathology 33(2):58-67, 2004; Reagan, W.J., Irizarry-Rovira, A., Poitout-Belissent, F., Provencher Bollinger, A., Ramaiah, S.K., Travlos, G., Walker, D., Bounous, D., Walter, G. (Bone Marrow Working Groups of the ASVCP/STP). Best practice for evaluation of bone marrow in nonclinical toxicity studies. Toxicologic Pathology 39: 435-448, 2011).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical chemistry changes revealed low cholesterol and high urea levels in males and female at 500 mg/k/g/day and high inorganic phosphate in males at 500 mg/kg/day. However, the reason for these findings are not clear and in the absence of any histopathological correlate in the liver and kidney, they were considered slight and not adverse they were not taken into consideration when setting the NOAEL.

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no effect on functional observational battery tests or locomotor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment related organ weight changes included a statistical decrease in organ weight was seen in the thymus of males at 500 mg/kg/day. Liver weights were high in females at 500 mg/kg/day, with spleen weights high in females at 150 mg/kg/day and males and females at 500 mg/kg/day. Adrenal gland weights were high in males given 500 mg/kg/day compared to concurrent controls. Increased adrenal and decreased thymus weights are considered to be an effect of the low terminal body weight recorded in the males and the high liver weight likely to be a consequence of the high body weight gain seen in the females and as there is no histopathological correlate, these changes are considered not to be of toxicological significance.
There was no effect on organ weights for animals given 50 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

On macroscopic examination, findings related to treatment with the test substance were present in the stomach and caecum of animals treated with 500 mg/kg/day: the (fore)stomach of 5/10 males and 2/10 females of the 500 mg/kg/day treated group showed macroscopic abnormalities consisting of thickening and in some of those 7 animals also a change in colour (recorded as abnormal and pale), and a distention was noted in the caecum of 5/10 males treated with this dose level. These findings comprised: thickening and/or an abnormal/pale colour of the (fore)stomach, and a large or distended caecum.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

On microscopic examination, findings related to treatment with the test substance were observed in the (fore)stomach, lung, spleen, liver and urinary bladder.
In the stomach, changes observed comprised squamous cell hyperplasia, erosions/ulcers within the affected epithelium, some acute inflammatory changes and submucosal oedema. These changes were noted in a single male treated with 150 mg/kg/day and in males and females treated with 500 mg/kg/day.
In the spleen an increase in severity of (brownish) pigment was observed in males and females from the 50 mg/kg/day treated group onwards: The pigment is believed to be haemosiderin. The incidence of the pigment in the spleen at 50 mg/kg bw/day was 7/10 (slight) and 3/10 (moderate) in males and 1/10 (slight), 7/10 (moderate) and 2/10 (severe) in males. At 150 mg/kg bw/day, 1/10 (minimal), 6/10 (slight) and 3/10 (moderate) in males and, in females, 8/10 (moderate) and 2/10 (severe). At 500 mg/kg bw/day, 5/10 (slight) and 5/10 (moderate) in males and, in females, 9/10 (moderate) and 1/10 (severe). This pigment appeared even in the controls at a moderate grade in females (7/10) and slight in males (8/10).
The changes observed in the urinary bladder were all mild and comprised diffuse transitional cell hyperplasia and/or increased epithelial folding. The transitional epithelium of the urinary bladder showed an increased epithelial folding in a 2/10 males treated with 150 mg/kg/day and in 5/10 males and 7/10 females of the 500 mg/kg/day treated group, while a low grade diffuse transitional cell hyperplasia was observed in few males and one female treated with 500 mg/kg/day. These findings are considered to be due to the irritating properties of the test substance. The epithelium of the urinary bladder is closely related to the squamous epithelium in the (fore)stomach and it is considered that the changes in both organs are related.
An increase in the presence of aggregates of (slight foamy) alveolar macrophages was noted in the lung of the 500 mg/kg/day treated animals, with the significance of these findings unclear.
A treatment-related increase in the incidence of pigmented Kupffer’s cells was observed in the liver of females of the 500 mg/kg/day group only. No microscopic findings were observed in the liver and in the caecum to support the liver weight changes observed in females dosed at 500 mg/kg/day, and the gross changes observed in the caecum of animals dosed at 500 mg/kg/day.

The kidney was not considered target tissue in this 13-week study.

No treatment-related histopathological changes were noted in any of the reproductive organs investigated in this study.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

The analytical results indicate that the prepared formulations were within ±10 % of the nominal concentration. Test article was
not detected in the control samples.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Toxicologically significant changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg bw/day and in the (fore)stomach at 150 mg/kg bw/day.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: gastrointestinal, urinary, and hematopoietic system

Organ:

bladder

blood

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Daily oral administration of the test substance to Sprague-Dawley rats for 13 weeks (test performed according to OECD 408) at dose levels of 50, 150 and 500 mg/kg/day, resulted in toxicologically significant effects at dose levels of 500 mg/kg/day and 150 mg/kg/day. The changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg/day and the changes in the (fore)stomach at 150 mg/kg/day were considered adverse and toxicologically significant. No adverse effect was observed at 50 mg/kg/day. Therefore the No Observed Adverse Effect Level is set at 50 mg/kg/day. With the LOAEL set at 150 mg/kg/day, the substance is not to be classified as STOT RE toxicant according to CLP Regulation.

Cross-reference 4

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1999-09-17 - 2000-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 27 July 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name: NINOX HCDO
- Purity: approximately 81%
- Lot/batch No.: 876 TK, sample IX
- Physical state: off white paste
- Storage condition of test material: room temperature, in the dark

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK)
- Age at study initiation: approximately five to seven weeks old.
- Weight at study initiation: the males weighed 139 to 173 g, and the females weighed 127 to 155 g
- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: nine days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared as a solution in distilled water. The analytical results indicate that the prepared formulations were within ±10 % of the nominal concentration. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

VEHICLE
- Concentration in vehicle: 3.68, 36.8, and 245 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance was determined by gas chromatography (GC) using an external standard technique. Samples were taken from the top, middle and bottom of the container. Sampling was performed in triplicate. The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.

Duration of treatment / exposure:

Twenty-eight consecutive days.

Frequency of treatment:

The test material was administered daily.

Dose / conc.:

18.4 CFU/kg bw/day (actual dose received)

Remarks:

15 mg/kg bw/day of active ingredient, actual ingested

Dose / conc.:

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

Remarks:

150 mg/kg bw/day of active ingredient, actual ingested

Dose / conc.:

1 227 mg/kg bw/day (actual dose received)

Remarks:

1000 mg/kg bw/day of active ingredient, actual ingested

No. of animals per sex per dose:

5 females and 5 males per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A range-finding toxicity study in Sprague-Dawley rats was performed. Six animals, 3 males and 3 females, were included in the study. The test material was administered daily, for fourteen consecutive days, by gavage. Control animals were treated in an identical manner with 5 ml/kg/day of distilled water. The studied dose was 1250 mg/kg/day. The level doses were defined based on the results of necropsy data, bodyweights and clinical observations.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study
- Animals fasted: No
- How many animals: investigations were performed on all surviving animals from each test and control group at the end of the study (Day 28)
- Parameters checked: see tables below.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity, grip strength and motor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS: Yes. The following organs removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, kidneys, testes, brain, liver, thymus, epididymides, ovaries, heart, spleen.

HISTOPATHOLOGY: Yes. Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: adrenals, aorta (thoracic), bone and bone marrow (femur including stifle joint), bone and bone marrow ( sternum), (including cerebrum, cerebelum and pons), caecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.

From all control animals and all the animals at the 1000 mg/kg/day dose group, the following tissues were preapred as paraffin blocks, sectioned and stained with haematoxylin and eosin for subsequent microscopic examination: adrenals, bone & bone marrow (sternum), brain (including cerebrum, cerebelum and pons), caecum, colon, duodenum, epididymides, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.
The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed.

Statistics:

Data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous pairwise comparisons were conducted using Dunnetts's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals treated with 1000 mg/kg/day began to show clinically observable signs of toxicity from Day 3. Symptoms gradually worsened and became more prevalent during weeks 1 and 2 before stabilising during the second half the study. Signs seen in most animals included fur loss, noisy respiration, increased salivation, red/brown staining of the fur, wet fur and hunched posture. The only observations seen in animals from the 150 or 15 mg/kg/day dose groups were incidents of fur loss, which is a common finding in group housed rats and in isolation is not toxicologically significant and a single incident of increased salivation entirely associated with the dosing procedure and not indicative of toxicity.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female treated with 1000 mg/kg/day was found dead on Day 12 of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males and females treated with 1000 mg/kg/day showed a substantially reduced bodyweight gain during week 1 of the study only. In subsequent weeks bodyweight gain in these animals was similar to controls but a full recovery was not apparent in males with terminal bodyweight remaining notably lower than controls. No adverse effect on bodyweight development was detected for animals of either sex from the 150 or 15 mg/kg/day dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males and females treated with 1000 mg/kg/day showed reduced food consumption and reduced food efficiency during the first week of the study only. No adverse effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption measurement, initiated on Day 15, revealed a substantial increase for animals of either sex treated with 1000 mg/kg/day, which persisted until the end of the study. There was no convincing effect on water consumption in animals from the 150 or 15 mg/kg/day dose groups.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological changes consistent with haemolytic anaemia were detected in animals of either sex treated with 1000 or 150 mg/kg/day. The condition was dose related in severity and involved statistically significant reductions in erythrocyte count, haemoglobin, haematocrit and mean corpuscular haemoglobin and increases in mean corpuscular volume, mean corpuscular haemoglobin and reticulocyte count. The majority of 1000 mg/kg/day reticulocyte values were outside the normally expected range for rats of the strain and age used. Males and females from the 1000 mg/kg/day dose group also showed a statistically significant increase in neutrophil numbers, but not in the total leucocyte count, together with a slightly elevated prothrombin time. Animals of either sex treated with 15 mg/kg/day showed no toxicologically or statistically significant haematological changes.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females treated with 1000 mg/kg/day showed statistically significant elevations in plasma aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) including individual values outside the normally expected range for rats of the strain and age used, particularly for ALAT. The group mean ALAT concentration for males from the 150 mg/kg/day dose group was also significantly elevated and there was a slight, but not statistically significant increase in this and ASAT levels for animals of either sex at this dose level. Plasma bilirubin levels were increased for animals of either sex from the 1000 mg/kg/day dose group but statistical significance was confined to the males.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Weekly behavioural assessments supported the clinical observations recorded during the study with incidents of tiptoe gait, hunched posture and noisy respiration seen at each observation time for animals treated with 1000 mg/kg/day. There were no treatment-related changes in the functional performance parameters measured. Males treated with 1000 mg/kg/day showed an enhanced starle reflex compared with controls. The percentage peak response was statistically significantly elevated and the percentage average response and RMS response were also higher than controls, although the differences did not achieve statistical significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females treated with 1000 mg/kg/day showed a statistically significant increase in spleen weight, both absolute and relative to bodyweight, compared with controls. Many of the individual values were outside the normally expected range for rats of the strain and age used and the effect extended to either sex from the 150 mg/kg/day dose group, although statistical significance was not always achieved at this dose level. A statistically significantly higher relative testes weight for 1000 mg/kg/day males was considered to result from the lower terminal bodyweight for these animals and was not indicative of a target organ effect. Reductions in absolute liver and kidney weight for 1000 mg/kg/day males were not reflected in the relative weights and were considered to be of no toxicological importance. There were no toxicologically significant effects on organ weight for animals of either sex treated with 15 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related changes were observed:
- Liver: centrilobular hepatocyte enlargement was observed for three males and scattered deposits of pigment were seen in a few animals of either sex dosed at 1000 mg/kg/day. One female dosed at 150 mg/kg/day similarly demonstrated hepatic pigment accumulation. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.
- Spleen: increased severities of extramedullary haemopoiesis and pigment accumulation were observed in relation to treatment for rats of either sex dosed at 1000 or 150 mg/kg/day. Female rats receiving 15 mg/kg/day demonstrated increased severities of splenic pigment accumulation but no alteration in extramedullary haemopoiesis compared with control animals. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.
- Kidneys: an increased severity of pigment accumulation was observed in the renal tubular epithelium of animals of either sex dosed at 1000 mg/kg/day. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.
- Urinary bladder: hyperplasia of the transitional cell epithelial lining of the urinary bladder was seen for males and females dosed at 1000 mg/kg/day.
- Stomach: acanthosis and hyperkeratosis, occasionally with associated subepithelial inflammatory cell infiltrates, were observed in relation to treatment in the forestomach of animals of either sex receiving 1000 mg/kg/day of the test material.
- No treatment-related changes were reported in the ovaries, protate, seminal vesicles, testes and uterus.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

water consumption and compound intake

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: hematopoietic, urinary, gastrointestinal and hepatobiliary system

Organ:

bladder

kidney

liver

spleen

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The "No Observed Effect Level" (NOEL) was considered to be 15 mg/kg/day.

Executive summary:

The Repeated dose 28-Day Oral toxicity Study in Rodents for the test substance was performed in Sprague-Dawley rats.The test material was administered by gavage to three groups, each of five male and five female, for up to twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day of active ingredient (incorporating a correction factor for test substance purity). A control group of five males and five females was dosed with vehicle alone (distilled water). Clinical signs, functional observations, bodyweight gain and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to a gross necropsy examination. Histopathological evaluation of selected tissues was performed in all control animals (0 mg/kg/day) and animals at the highest dose group (1000 mg/kg/day). The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed.

Clinical signs of toxicity, functional incidents as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption and reduced bodyweight gain were observed at dose of 1000 mg/kg/day.

In the blood chemistry, elevations in plasma aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and bilirubin was noted at the dose of 1000 mg/kg/day; ASAT and ALAT were also elevated for 150 mg/kg/day animals. Haematological changes as haemolytic anaemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time were observed at doses of 1000 and 150 mg/kg/day.

A statistically significant increase in spleen weight was observed in males and females treated with 1000 mg/kg/day. The effect extended to the 150 mg/kg/day dose group although statistical significance was not always achieved.

Treatment-related changes in liver and spleen were detected at 1000 mg/kg/day and 150 mg/kg/day. Histopathological changes were also observed in kidneys, urinary bladder and stomach of animals treated with 1000 mg/kg/day. No treatment-related changes were reported in the ovaries, prostate, seminal vesicles, testes and uterus of control animals or animals at 1000 mg/kg/day.

Oral administration of the test substance to rats for a period of up to twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in toxicologically significant effects at dose levels of 1000 and 150 mg/kg/day. No such effects were observed in animals of either sex treated with 15 mg/kg/day. The "No Observed Effect Level" (NOEL) was, therefore, considered to be 15 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3700

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The study was performed following design described in final decision number CCH-D-2114453649-36-01/F.
An extended one-generation reproductive toxicity study (annex X, according to OECD 443) in rats, oral route with the registered substance was performed including: ten weeks premating exposure duration for the parental (P0) generation; dose level setting shall aim to induce systemic toxicity at the highest dose level; cohort 1A; cohort 1B without extension to mate the cohort 1B animals to produce the F2 generation

Test material

Test material information

Constituent 1

Reference substance name:

3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide

EC Number:

939-581-9

Cas Number:

1471314-81-4

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Amides, C12-C18 (even numbered), N-[3-(dimethylamino)propyl], N'-oxides
- Substance type: Clear liquid
- Lot/batch No.: BLBX017090
- Storage condition of test material: Room temperature in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): Amides, C12-C18 (even numbered), N [3 (dimethylamino) propyl], N’-oxides
- Substance type: Almost clear colorless liquid
- Physical state: liquid
- Analytical purity: 35.2% in water
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: S019341917
- Expiration date of the lot/batch: 28 March 2021
- Action of test substance: no data
- purity/weighing factor: none
- total correction factor: 2.85, based on purity

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed container, 2 to 8°C, protected from light
- Stability under storage conditions: not specified
- Stability under test conditions: not specified
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Formulations of 0.2 and 100 mg/mL AI were stable for 15 days when stored refrigerated (2 to 8°C) or for 14 days when stored at room temperature (15 to 25°C) and formulations of 0.1 mg/mL AI were stable for 1 day when stored at room temperature (15 to 25°C) in Covance Study 8410552.
Formulations of 0.1 and 100 mg/mL AI were confirmed to be homogenous in Covance Study 8410552.

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Residual samples were taken, retained for up to 15 days and stored refrigerated (2 to 8°C), and then discarded.

Test animals

Species:

rat

Strain:

other: Crl:WI(Han) rats

Details on species / strain selection:

The Crl:WI(Han) rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics. The rat is normally the preferred species because of the extent of background data and the comparability to general toxicity tests. The strain used is not known for low fecundity or high incidence of spontaneous developmental defects.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: One hundred male and one hundred female Crl:WI(Han) rats from Charles River Laboratories, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at animal arrival: 28 or 31 days old
- Weight at study initiation: (P) Males: 0,1362 - 0,2004 kg; Females: 0,1069 - 0,153 kg
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes (Home Office, 2014).
- The F0 generation animals were housed in groups of up to four by sex and dose group (pre-pairing phase for both sexes and post-pairing phase for males). During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed, or until the end of the pairing phase. Following mating, females were housed individually during gestation or post-pairing and with their litter during the lactation phase.
- During the maturation phase, the F1 generation animals were housed in groups of up to four by sex and dose group.
- Bedding was provided at least weekly to each cage by use of clean Aspen wood chips or European Softwood bedding during gestation and lactation phases (Datesand Ltd; Manchester, United Kingdom). Each batch of bedding was analyzed for specific constituents and contaminants. No contaminants were present in the bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
- Diet (e.g. ad libitum): ad libitum, VRF1 (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants.
No contaminants were present in the diet at levels that might have interfered with achieving the objective of the study. Results are retained on file at Covance.
A 50-g sample of each batch of diet used in the study was collected and stored at ambient temperature until study finalization.
- Water (e.g. ad libitum): ad libitum, Water from the main tap supply was provided via water bottles. The water is periodically analyzed for specific contaminants.
No contaminants were present in the water at levels that might have interfered with achieving the objective of the study. Results are retained on file at Covance.

- Acclimation period: Animals were acclimated for 13 or 11 days, and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 40% to 70%
- Air changes (per hr): The rooms were air conditioned to provide a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark.

IN-LIFE DATES: From: 2019-12-30 To: 2020-08-07

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: The test article was formulated as a suspension in purified water following dispensary SOPs and the formulation method (Method 8410555_O_01D), as maintained in the study data.
- Frequency of preparation: weekly
- Storage of formulation: Refrigerated (2 to 8°C) in a sealed container protected from light.
- Formulations prepared for use on the first dosing occasion of the F0 generation, the start of dosing for the F1 generation, and the last day of the F1 generation were analyzed to determine the achieved concentration. Triplicate samples were taken from the middle of the test article formulations and were analyzed. A single sample was taken from the middle of the control formulations and was analyzed.
- Formulations (excluding vehicle control article) were stirred continuously for at least 30 minutes before and throughout dosing.

- Test item was prepared at the appropriate concentration as solution in purified water.

VEHICLE
- Justification for use and choice of vehicle: not applicable (vehicle is water)
- Concentration in vehicle: 0, 5, 10, 15 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed, or until the end of the pairing phase.
- Proof of pregnancy: Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. The day on which mating was confirmed was designated as GD 0.
- After successful mating each pregnant female was caged (how): individual
- Any other deviations from standard protocol: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations prepared for use on the first dosing occasion of F0 generation, the start of dosing for the F1 generation and the last day of the F1 generation were analyzed to determine the achieved concentration. The mean % nominal concentration should be between 90 to 110% and with a relative standard deviation (RSD) ≤5.0%. Results were within these criteria. Test article was not detected in the Group 1 control samples.
The analytical procedure was validated within Covance study 8280017.

Duration of treatment / exposure:

Males: 119 consecutive days (10 weeks prior to pairing, 2 weeks during pairing, and 35 days post-pairing) , Females: up to 121 days (10 weeks prior to pairing, during pairing, throughout gestation, and up to LD 21).

Frequency of treatment:

Daily, at approximately the same time each day +/- 3.5 hours.

Details on study schedule:

- F0 males were dosed for 119 consecutive days (10 weeks prior to pairing, 2 weeks during pairing, and 35 days post-pairing); females were dosed for up to 121 days (10 weeks prior to pairing, during pairing, throughout gestation or the post-pairing phase, and up to LD 21).
- All F1 offspring were dosed from PND 14 to 21; prior to this, pups received test article through the milk. Following selection into appropriate cohorts, the selected F1 animals were administered once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 males and 24 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Route of administration rationale: The oral route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type. The oral route is a potential route of accidental human exposure to be investigated in hazard and risk assessment.
- Dose selection rationale:
The high-dose level of 150 mg/kg/day AI was anticipated to induce minimal parental toxicity (i.e., changes in red blood cell parameters and local irritancy of the stomach) without causing death or affecting parturition, pup growth, and mating of the first generation.
The intermediate-dose level of 100 mg/kg/day AI was anticipated to be a no observed adverse effect level (NOAEL) for parental toxicity.
The low-dose level of 50 mg/kg/day AI was anticipated to be a no observed effect level (NOEL) for parental and offspring toxicity.
Background information:
* In a 28-day, repeat-dose toxicity study in the Sprague-Dawley rat (Safepharm Laboratories Limited Study number 625/051), animals were administered 15, 150, or 1000 mg/kg bw/day AI of the test article by oral gavage. Clinical observations of toxicity and functional incidents, such as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption, and reduced body weight gain, were observed in animals administered 1000 or 150 mg/kg bw/day AI. Test article-related changes in the liver and spleen were detected in animals administered 1000 or 150 mg/kg bw/day AI. Histopathological changes were observed in the kidneys, urinary bladder, and stomach of animals administered the high dose. Hematological changes (hemolytic anemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time) and elevations in plasma aspartate aminotransferase, alanine aminotransferase, and bilirubin were observed in animals administered 1000 or 150 mg/kg bw/day AI. Therefore, the no observed effect level (NOEL) for this study was considered to be 15 mg/kg bw/day AI.
* In an OECD 421 reproduction/developmental toxicity screening study in the Han Wistar rat (Harlan Laboratories Ltd Study 2833-003), administration of 15, 40, or 100 mg/kg bw/day AI did not result in any test article-related effects on reproduction or offspring development up to PND 5.
* In a 90-day repeat-oral dose toxicity study in the Sprague-Dawley rat (Covance Study 8280017), animals were administered 50, 150, or 500 mg/kg bw/day AI of the test article by oral gavage. One male administered 500 mg/kg bw/day AI was found dead during Week 12 and this was considered test article related. Clinical observations at this dose included salivation, staining around the mouth, and noisy breathing and, in combination with high water consumption values, this was indicative of an irritant or unpalatable test article formulation. Decreased hemoglobin, red blood cell counts, mean cell hemoglobin concentration, hemoglobin distribution width, and red cell distribution width and increased reticulocyte counts and percentage, mean cell, volume and mean cell hemoglobin were noted. Microscopic spleen changes of hemosiderin deposition were noted, together with an increase in spleen weight, in animals administered 500 mg/kg bw/day AI. Microscopic examination revealed adverse findings in animals administered 500 mg/kg bw/day AI, including squamous cell hyperplasia of the forestomach and transitional cell hyperplasia of the urinary bladder, which was considered a result of localized irritancy properties of the test article. Findings in animals administered 150 mg/kg bw/day AI included low body weight gains, and as no effect on food consumption was noted, this was considered nonspecific toxicity. Forestomach squamous cell hyperplasia was noted in a single male administered 150 mg/kg bw/day AI and was considered adverse. Test article-related red blood cell changes were noted but considered nonadverse. The only test article-related change observed in males administered 50 mg/kg bw/day AI were statistically relevant minor changes in the red blood cell parameters, which were considered test article-related but not adverse in nature; therefore, the no observed effect level (NOEL) for this study was considered to be 50 mg/kg bw/day AI.
* In a recent pre- and postnatal development dose range-finding study (Covance Study 8410554) performed in the pregnant female Han Wistar rat at dose levels of 75, 100, or 125 mg/kg bw/day AI, no unscheduled deaths occurred as a result of the test article. During gestation, incidences of mouth rubbing were noted after dosing in animals administered 100 or 125 mg/kg bw/day AI, although no test article effects on body weights or food consumption were evident. At sacrifice on GD 21, uterine contents were not adversely affected, although gravid uterus weights and total weight change were marginally higher in animals administered 125 mg/kg bw/day compared with controls. No effect on fetal or placenta weights were noted. Female fetal ano-genital distance was reduced for female fetuses from the group administered 125 mg/kg bw/day AI, compared with control (-13.5%), although the etiology of this is unclear. One fetus from a 125 mg/kg bw/day AI litter exhibited malformations of omphalocele, short trunk, malrotated hindlimb, and micromelia of the hind and forelimb. The fetus was also recorded with variations of edema of the head and neck and small genital tubercle. This was isolated to one fetus, remaining fetuses did not exhibit any variations or malformations, and the etiology of this finding is unclear; however, as no effects were noted in the pups following parturition in this group, this was considered an incidental finding. In the postnatal subgroup, isolated instances of mouth rubbing and excessive salivation were noted for females administered 125 mg/kg bw/day AI during lactation. One female administered 75 mg/kg bw/day AI had a total post-natal litter loss on LD 0. The litter, consisting of three pups, was confirmed to have been born alive, but had no milk in the stomach at necropsy. In isolation, this finding was considered to have arisen incidentally. No test article-related effects on litter data or pup weights were observed, and no test article-related clinical observations were noted. Ano-genital distance recorded on PND 4 was marginally lower for female pups maternally administered 125 mg/kg bw/day AI, compared with controls (-6%). At necropsy, one pup maternally administered 125 mg/kg bw/day AI was recorded with pelvic dilatation of the kidneys, and one was recorded with a tail sore. No other macroscopic observations were noted for the pups. For the adults, thyroid weights were lower in all test article-treated groups, compared with controls, although a dose response was not evident (-3, -20, or -10% for animals administered 75, 100, or 125 mg/kg bw/day AI). Pup thyroid weights, taken on PND 22 were increased by +16, -30, or +22% for absolute weights and +25, +24, or +27% for body weight relative, following maternal exposure of 75, 100, or 125 mg/kg bw/day AI. Test article-related macroscopic findings noted in the adult females were confined to a single incidence of pelvic dilatation of the right kidney noted for one female administered 125 mg/kg bw/day AI, and a uterine mass on the right uterine horn for one female administered 100 mg/kg bw/day AI.

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

VIABILITY CHECKS
- Time schedule: All animals were observed in the home cage twice daily at the beginning and end (nominal) of the working day for signs of ill health or overt toxicity.

CAGE SIDE AND DETAILED OBSERVATIONS
- General Appearance: Each animal was given a detailed physical examination once daily from the start of dosing. The examinations were performed after dosing (after returning the animal to the home cage) from the issue of Protocol Amendment 2 (Pre-Pairing Day 2 or 16). An individual record was maintained of the clinical condition of each animal.
- Postdose Observations: The first batch of animals (arrived on 17 December 2019) were observed daily for the first 7 days approximately 1 hours postdose. The second batch of animals (arrived on 02 January 2020) was observed daily for the first 7 days immediately after dosing, upon return to the home cage.
- Clinical observations: All animals were assessed by detailed clinical observations for approximately 30 seconds in an open field arena. Males were assessed once weekly from the start of dosing. Females were assessed once weekly during the pre-pairing and post pairing phases; on GD 0, 6, 14, and 20; and on LD 1, 7, 14, and 21.
- Maternal observations: Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered.

BODY WEIGHT
Frequency: Body weights were recorded once during acclimation (Day 1 of the predose phase [Day-3]) for all animals.
Male body weights were recorded weekly from the first day of dosing (Pre-Pairing Day 1).
Female body weights were recorded weekly from the first day of dosing (Pre-Pairing Day 1); on GD 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20; and on LD 1, 4, 7, 14, and 21. Body weights recorded during the pairing phase were not reported. Body weights for females with no confirmation of mating were recorded weekly during the post-pairing phase.

FOOD CONSUMPTION
Frequency
- Males: food consumption was recorded twice weekly during pre- and post-pairing.
- Females: food consumption was recorded twice weekly during pre-pairing; every 2 days during gestation; and from LD 1 to 4, 4 to 7, and 7 to 14.
- Food consumptions were not recorded during pairing, between GD 21 and LD 0, due to parturition, and from LD 14, due to the start of weaning of pups.

CLINICAL PATHOLOGY
- Sample collection: Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), clinical chemistry (1 x 0.6 mL [serum separator tubes], nominal) and thyroid hormone analysis (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta at necropsy for at least 10 randomly selected males and females within each group from Cohort 1A.
- Animals fasted: Samples were collected after animals were fasted overnight.

HEMATOLOGY and COAGULATION:
- Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time followed by at least 5 minutes on an automatic mixer. Blood samples for clinical chemistry were gently inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator.
- Hematology Tests: hemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, total and differential white cell count, platelet count
- Coagulation Tests: prothrombin time, activated partial thromboplastin time, fibrinogen
- Clinical Chemistry Tests: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transferase, total cholesterol, total bilirubin, total protein, albumin
globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, triglycerides, creatinine, urea, glucose

THYROID ANALYSES:
- During sample processing, each blood sample for thyroid hormone analysis was split into two equal aliquots. One aliquot was analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis were retained in storage until report finalization, after which, they will be discarded.
Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until placed in frozen storage. See Protocol Deviations.
- Each blood sample for thyroid hormone analysis was gently inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator, and was stored at room temperature (15 to 25°C) for at least 30 minutes prior to processing to allow samples to clot. Each sample was then centrifuged at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at < 10°C (nominal -20°C).
- Parameters: thyroxine (Total T4), thyroid stimulating hormone (TSH)

URINALYSIS
- Sample collection: Urine samples from 10 randomly selected males and females in each group from Cohort 1A were collected overnight the day prior to necropsy. Food was removed during collection.
- Parameters: volume, color, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, microscopy of sediment.

Oestrous cyclicity (parental animals):

Daily vaginal lavage (washings) were undertaken from females for 14 days prior to pairing, and until the confirmation of mating, and on the day of necropsy.

Sperm parameters (parental animals):

For each scheduled male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from the left epididymis (cauda epididymis and vas deferens). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from males in Groups 1 and 4 were read for morphological changes. The left epididymis was discarded following seminology investigations.

Litter observations:

VIABILITY CHECKS
- Preweaning: Litter size and pup sex were recorded on PND 0, 1, 4, 7, 14, and 21. Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition were given a macroscopic necropsy for cause of death and possible birth defects. All pups were observed daily from PND 14 to 21 immediately after dosing and upon return to the home cage.
- Postweaning: All animals were observed in the home cage twice daily at the beginning and end (nominal) of the working day for signs of ill health or overt toxicity.

CLINICAL OBSERVATIONS
- Preweaning: A detailed clinical examination was recorded for each pup on PND 1, 4, 7, 14, and 21.
- Postweaning: Each animal was given a detailed physical examination once daily from the start of dosing, performed immediately after dosing, upon return to the home cage. An individual record was maintained of the clinical condition of each animal. All animals were assessed by detailed clinical observations for approximately 30 seconds in an open-field arena. Animals were assessed once weekly from Maturation Day 22.

BODY WEIGHTS
- Preweaning: Pup body weights were recorded on PND 1, 4, 7, 13, 14, 16, 18, and 20.
- Postweaning: Body weights were recorded on Maturation Day 22, 24, 26, 28, 30, 32, 34, and 36, then weekly thereafter. In addition, body weights were recorded on the day balano preputial separation or vaginal opening occurred.

ANOGENITAL DISTANCE
- Preweaning: Prior to the culling procedure on PND 4, the ano-genital distance of all pups was measured.
Procedure:

NIPPLE PRESENCE - ALL MALE ANIMALS
- Preweaning: The number of nipples/areolae for all male pups was recorded on PND 13.

FOOD CONSUMPTION
- Frequency: Food consumption was recorded twice weekly from Maturation Day 22 until the day prior to necropsy for Cohort 1A and until the day of necropsy for Cohort 1B.
- Procedure: Consumption was calculated as g/animal/day.

SEXUAL MATURATION
- Animals were observed for vaginal opening (females) from Maturation Day 28 and cleavage of the balano-preputial gland (males) from Maturation Day 37.
- Observations lasted until these milestones were attained. Body weights were recorded on the day balano-preputial separation or vaginal opening occurred.

ESTROUS CYCLE EVALUATIONS in F1 generation
Daily vaginal lavage (washings) were undertaken for Cohort 1A and 1B females after vaginal opening until the first cornified smear was observed, then from Maturation Day 75 to 89, inclusive, and on the day of necropsy.

CLINICAL PATHOLOGY
- Sample collection: Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), clinical chemistry (1 x 0.6 mL [serum separator tubes], nominal) and thyroid hormone analysis (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta at necropsy for at least 10 randomly selected males and females within each group from Cohort 1A.
- Samples were collected after animals were fasted overnight.

HEMATOLOGY and COAGULATION:
- Blood samples for hematology and coagulation were fully inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time followed by at least 5 minutes on an automatic mixer. Blood samples for clinical chemistry were gently inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator.
- During sample processing, each blood sample for thyroid hormone analysis was split into two equal aliquots. One aliquot was analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis were retained in storage until report finalization, after which, they will be discarded.
- Hematology Tests: hemoglobin, red blood cell count, packed cell volume, mean cell volume
mean cell hemoglobin, mean cell hemoglobin concentration, reticulocyte count, red cell distribution width, total and differential white cell count, platelet count.
- Coagulation Tests: prothrombin time, activated partial thromboplastin time, fibrinogen
- Clinical Chemistry Tests: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transferase, total cholesterol, total bilirubin, total protein, albumin
globulin, albumin:globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, triglycerides, creatinine, urea, glucose.

THYROID ANALYSES:
- Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until placed in frozen storage.
- Each blood sample for thyroid hormone analysis was gently inverted several times (approximately 10), ensuring that the blood traveled all the way to the top and bottom of the tube each time to mix with the clot activator, and was stored at room temperature (15 to 25C) for at least 30 minutes prior to processing to allow samples to clot. Each sample was then centrifuged at 2300g for 10 minutes at approximately 4C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at < 10°C (nominal -20°C).

URINALYSIS
- Urine samples from 10 randomly selected males and females in each group from Cohort 1A were collected overnight the day prior to necropsy. Food was removed during collection.
- Parameters: volume, color, turbidity, specific gravity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, microscopy of sediment.

Postmortem examinations (parental animals):

SACRIFICE:
- Sacrifice / method of euthanasia: Males were sacrificed by isoflurane anesthesia on Post-Pairing Day 36 (Week 18) after an overnight period without food. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
Females were sacrificed by isoflurane anesthesia on LD 22 (those that littered) or 26 days post coitum (those with confirmation of mating that did not litter) or Post Pairing Day 36 (those with no confirmation of mating that did not litter). Females sacrificed on LD 22 had food removed overnight prior to necropsy.

- Unscheduled death: Rats that died or were euthanized before scheduled termination were examined for the cause of death or condition as soon as possible after the observation was made. The rats were examined for gross lesions.

- Scheduled euthanasia:
Females with the same day of mating/littering were sacrificed in a controlled randomization order, when possible. Females that did not litter were sacrificed in animal identification order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.

OVARIAN AND UTERINE EXAMINATIONS
- The number of implantations sites for all paired females were recorded. The uterus of any apparently non pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

NECROPSY
- Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded. Organ weights were recorded at each scheduled sacrifice, excluding decedents.

ORGAN WEIGHTS
- Organ weights were recorded at each scheduled sacrifice, excluding decedents. Organs were dissected free from fat and other contiguous tissue and were weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
- weighed organs: adrenal, brain, epididymis (right), heart, ovary, oviduct, pituitary, prostate, seminal vesicle with coagulating, spleen, testis, kidney, liver, thymus, thyroid with parathyroid and uterus with cervix

TISSUE COLLECTION AND PRESERVATION
- Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated: adrenal nerve, nerve, sciatic, bone marrow smear (femur), ovary, brain, oviduct, cecum, pituitary, colon, prostate, duodenum, rectum, epididymis (right), seminal vesicle with coagulating glands and fluid, eye, spinal cord, cervical, femur with bone marrow and femerotibial joint, spinal cord, lumbar, gross lesions, spinal cord, thoracic, head, spleen, heart, stomach, ileum, testis, jejunum, thymus, kidney, thyroid with parathyroid, liver, trachea, lungs with main stem, bronchi and bronchioles, urinary bladder, mammary gland, uterus with cervix, muscle, biceps femoris and vagina
- The uterus of any apparently non pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

HISTOLOGY
- All tissues (adrenal nerve, nerve, sciatic, bone marrow smear (femur), ovary, brain, oviduct, cecum, pituitary, colon, prostate, duodenum, rectum, epididymis (right), seminal vesicle with coagulating glands and fluid, eye, spinal cord, cervical, femur with bone marrow and femerotibial joint, spinal cord, lumbar, gross lesions, spinal cord, thoracic, head, spleen, heart, stomach, ileum, testis, jejunum, thymus, kidney, thyroid with parathyroid, liver, trachea, lungs with main stem, bronchi and bronchioles, urinary bladder, mammary gland, uterus with cervix, muscle, biceps femoris and vagina) from Groups 1 and 4 and all decedents, and the spleen from Groups 2 and 3, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
- All tissues ( animal identification, pituitary, epididymis (right), prostate, gross lesions, seminal vesicle, mammary gland, testis, ovary, uterus with cervix, oviduct and vagina) from males in Groups 2 and 3 that did not sire and females in Groups 2 and 3 that were not mated and/or not pregnant were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.
- Additional sections of testes and epididymides were also stained with periodic acid Schiff (PAS) for spermatogenic staging for all males from Groups 1 and 4 and all decedents.

HISTOPATHOLOGY
All processed tissues were examined microscopically by the Study Pathologist. Qualitative assessments of stages of spermatogenesis and interstitial testicular cell structure were also performed for males from Groups 1 and 4.

Postmortem examinations (offspring):

SACRIFICE:
- Method of Euthanasia:
-Pups culled on PND 4, unselected pups on PND 22, and pups in a moribund condition were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, major blood vessels were severed to exsanguinate the animal. Sacrifices were carried out in group order, where possible.
- Cohort A: Animals were sacrificed by isoflurane anesthesia on Maturation Day 90 to 93 after an overnight period without food. Sacrifices were carried out in a controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
- Cohort B: Animals were sacrificed by isoflurane anesthesia on Maturation Day 108 to 113 after an overnight period without food. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.

- Unscheduled death: With the exception of fasting, these procedures were also followed for unscheduled sacrifices and deaths.

MALE REPRODUCTIVE ASSESSMENT
For each scheduled male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from the left epididymis (cauda epididymis and vas deferens). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from males in Groups 1 and 4 were read for morphological changes. The left epididymis was discarded following seminology investigations.

NECROPSY
Pups: Full macroscopic examinations, with particular attention to the reproductive genitals, were conducted for all decedents and pups sacrificed on PND 4 or PND 22.
Cohort A and B: Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

ORGAN WEIGHTS
Organ weights were recorded at each scheduled sacrifice, excluding decedents. Organs were dissected free from fat and other contiguous tissue and were weighed before fixation. Left and right organs were weighed together.
- Cohort A: adrenal, brain, epididymis (right), heart, kidney, liver, lymph node (mesenteric and mandibular), ovary, oviduct, pituitary, prostate, seminal vesicle with coagulating, spleen, testis
thymus, thyroid with parathyroid and uterus with cervix.
- Cohort B: epididymis, ovary, oviduct, pituitary, prostate, seminal vesicle, testis and uterus with cervix

TISSUE COLLECTION AND PRESERVATION
Tissues were retained in 10% neutral buffered formalin, unless otherwise indicated.
- Cohort A: adrenal, muscle, biceps femoris, nerve (optic and sciatic), bone marrow smear (femur),
brain, ovary, cecum, oviduct, colon, pituitary, duodenum, prostate
epididymis (right), rectum, eye, seminal vesicle with coagulating glands and fluid, femur with bone marrow and femerotibial joint, spinal cord, cervical, gross lesions, spinal cord, lumbar, head spinal cord, thoracic, heart, spleen, ileum, stomach, jejunum, testis, kidney, thymus, liver, thyroid with parathyroid, lungs with main stem bronchi and bronchioles, trachea, lymph node, mesenterice, urinary bladder, lymph node, mandibulare, uterus with cervix, mammary gland and vagina
- Cohort B: pituitary, epididymis, prostate, gross lesions, seminal vesicle, mammary gland, testis, ovary, uterus with cervix, oviduct and vagina

HISTOLOGY 
- Cohort A:
- All tissues (adrenal, muscle, biceps femoris, nerve (optic and sciatic), bone marrow smear (femur), brain, ovary, cecum, oviduct, colon, pituitary, duodenum, prostate
epididymis (right), rectum, eye, seminal vesicle with coagulating glands and fluid, femur with bone marrow and femerotibial joint, spinal cord, cervical, gross lesions, spinal cord, lumbar, head spinal cord, thoracic, heart, spleen, ileum, stomach, jejunum, testis, kidney, thymus, liver, thyroid with parathyroid, lungs with main stem bronchi and bronchioles, trachea, lymph node, mesenterice, urinary bladder, lymph node, mandibulare, uterus with cervix, mammary gland and vagina) from Groups 1 and 4 and all decedents, and the spleen from Groups 2 and 3, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin. See Protocol Deviations.
- For at least 10 animals/sex from Groups 2 and 3, the mandibular and mesenteric lymph nodes, adrenals, thymus, spleen, and bone marrow (femur) were processed for immunotoxicity assessments. All macroscopic lesions and the spleen were processed for remaining animals.
- Additional sections of testes and epididymides were also stained with periodic acid Schiff (PAS) for spermatogenic staging for all males from Groups 1 and 4 and all decedents.
- Pre- and post-natally induced immunotoxic effects were evaluated by assessment of the mandibular and mesenteric lymph nodes, adrenals, thymus, spleen, and bone marrow (femur) from at least 10 animals/sex/group from all Cohort 1A dose groups. Approximately half the spleen was used for flow cytometry analysis. The excised sample was weighed and the weight recorded. Absolute values were calculated from the recorded weights. The samples were placed in an appropriate cell culture media, then prepared into a single cell suspension as soon as possible by means of mechanical disruption of the spleen section.
- Cohort B: All tissues (pituitary, epididymis, prostate, gross lesions, seminal vesicle, mammary gland, testis, ovary, uterus with cervix, oviduct and vagina) from all Cohort 1B animals, including decedents, were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
All processed tissues were examined microscopically by the Study Pathologist. Qualitative assessments of stages of spermatogenesis and interstitial testicular cell structure were also performed for males from Groups 1 and 4.
Qualitative assessment of the stages of follicular development to regression from small growing follicles to corpora lutea was performed in the ovaries.

Statistics:

See: Any other information on materials and methods incl. tables

Reproductive indices:

- Female Mating index = (Number of mated females / Number of females cohabited (excluding females sacrificed during Cohabitation)) X 100
- Male mating index = (Number of males mating with at least 1 female / Number of males cohabitated with at least 1 female) X 100
- Female fecundity index = (Number of pregnant females / Number of females mated (excluding females with an undetermined pregnancy status)) X 100
- Male fecundity index = (Number of males impregnating at least 1 female / Number of males mating with at least 1 female) X 100
- Female fertility index = (Number of pregnant females / Number of females cohabited (excluding females sacrificed during Cohabitation or with an undetermined pregnancy status)) X 100
- Male fertility index = (Number of males impregnating at least 1 female / Number of males cohabitated with at least 1 female) X 100
- % post-implantation loss = ((Number of implantations - number of live embryos) / Number of implantations) X 100
- Gestation index = (Number of females with live born pups / Number of pregnant females) X 100

Offspring viability indices:

- Livebirth index = (Number of live born pups / Number of pups born) X 100
- Day 4 viability index = (Number of pups alive on PND 4 precull / Number of pups born alive) X 100
- Weaning index = (Number of pups alive at weaning / Number of pups alive on PND 4 post cull) X 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Nonadverse, test article-related clinical postdose observations of mouth rubbing were noted for all males and females administered 150 mg/kg/day AI.
A high incidence of excessive salivation was also noted for 16 of 24 males and 14 of 24 females from this dose group. Isolated instances of paddling behavior were also recorded for three females prior to pairing (Animal R0701 on Day 44 and Animals R0716 and R0724 on Day 52) and were also evident for two females during lactation (Animal R0708 on LD 3 and 8, and Animal R0707 on LD 4).
Test article-related, clinical postdose observations of mouth rubbing were also observed for all males and females administered 100 mg/kg/day AI, with isolated instances of excessive salivation reported for seven males. An isolated incidence of paddling behavior was also noted in three males from this dose group (Animals R0214 and R0218 on Day 62 and Animal R0219 on Day 70). An isolated incidence of paddling behavior was recorded for three females during gestation (Animals R0601 and R0612 on GD 1 and Animal R0601 on GD 4).
Isolated instances of postdose mouth rubbing were also observed 14 of 24 males and 3 of 24 females administered 50 mg/kg/day AI.
Such observations are common in gavage studies and are considered due to the unpalatable taste of the test article formulation, rather than an indication of the toxicity of the test article; therefore, they were considered nonadverse.
High incidences of abnormalities of the pelage (discoloration, hair loss, and thin hair) were observed for 6 of 24 males administered 150 mg/kg/day AI during the pre-pairing phase, although this was not so obvious as the study progressed; as such, these findings were considered to have arisen incidentally and were not test article-related.
No test article-related observations were noted during the weekly detailed examinations.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test article-related mortality occurred.
One female administered 150 mg/kg/day AI (Animal R0719) was sacrificed on Lactation Day 0 following a complete litter loss, which consisted of one stillborn pup and one pup death (born alive). In isolation, this was considered to have arisen incidentally with no relationship to the test article.
One control male (Animal R0010) was found dead on Day 17. The animal was recorded with a sore/lesion below the right ear and dark focus on the thymus. One control female (Animal R0410) was found dead and cannibalized on Day 20 and reported with a cannibalized uterus following microscopic examinations. One control female (Animal R0415) was found dead on Day 29. At macroscopic examinations, the animal was reported with a missing tail tip. The cause of demise was not confirmed for these animals, however, as only the control article (vehicle) was administered to the controls, no association with the test article occurred.
One control male (Animal R0011) was sacrificed on Post-Pairing Day 27 after the animal was observed with loss of limb movement, swelling, and blue coloration of the left hindlimb. Macroscopic examinations revealed a hindlimb fracture below the stifle joint and mottled lobes of the liver. The cause of demise was a physical injury.
One female administered 150 mg/kg/day AI (Animal R0717) was found dead on Day 49. Macroscopic examinations revealed an esophageal rupture and abnormal contents in the thoracic cavity. These findings were consistent with a dosing trauma and not test article related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects on mean body weight or body weight gain were noted for males throughout the study or for females during the pre-pairing, gestation, or lactation phases.
Transient instances of statistically significant differences in mean body weight gains were observed throughout the dose groups, with no relationship to the test article; as such, they were considered to have arisen incidentally.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test article-related effects on mean food consumption were observed for males throughout the study duration or for females during the pre-pairing, gestation, or lactation phases.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.

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):

Nonadverse, dose-related test article-related changes in the red cell parameters were evident at all dose levels investigated at the end of the dosing phase for parental animals.
Dose-related reductions in mean cell hemoglobin concentration (MCHC), hemoglobin and red cell distribution and red blood cell count (RBC) were observed for males administered at all dose levels, compared with controls. Statistical significance was attained for all these parameters for males administered 100 or 150 mg/kg/day AI, and extended to 50 mg/kg/day AI males for RBC and MCHC (P < 0.001). These changes were accompanied by statistically significantly increased reticulocyte counts and percentage reticulocytes (P < 0.05 to P < 0.001) compared with controls. Statistically significant increases in mean cell volume (MCV) was also evident at all dose levels (P< 0.001) and mean cell hemoglobin levels (MCH) were also increased for males administered 100 or 150 mg/kg/day AI (P < 0.05 or P < 0.001, respectively), compared with controls.
Similar changes in red cell mass were evident for the females. RBC, RDW, and MCHC were reduced for females administered at all dose levels, with statistical significance attained for females administered 100 or 150 mg/kg/day AI, (P < 0.05 to P < 0.001) compared with controls. MCH, MCV reticulocytes (counts and percentages) were also increased for females at all dose levels with a clear dose relationship with increasing dose (from not statistically significant to P< 0.001) compared with controls.
Monocyte counts were reduced (P < 0.01), and percentage monocytes were decreased (P < 0.05) for males administered 150 mg/kg/day AI, compared with controls. Large unstained cell (LUC) was reduced for males administered 50 or 150 mg/kg/day AI, although a dose relationship was not evident. The absence of similar changes observed for the females and no associated supporting data to suggest that these intergroup differences were attributable to test article administration, these minor changes in the white cell parameters were considered to have arisen incidentally and were of no toxicological importance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Nonadverse test article-related reductions in potassium levels were evident following administration of 100 or 150 mg/kg/day AI, compared with controls.
Potassium levels were significantly reduced for males (P < 0.01) and females (P < 0.001) administered 150 mg/kg/day AI, compared with controls, with the reduction extending into females administered 100 mg/kg/day AI, compared with controls (P < 0.05). No such effect was evident for males administered 100 mg/kg/day or animals of either sex administered 50 mg/kg/day AI.
Alkaline phosphatase activity was reduced for females administered 50 or 100 mg/kg/day AI, compared with controls. The significance attained was minimal (P < 0.05), and in the absence of a similar change observed at the highest dose levels and the lack of a similar response observed for males, these reductions were considered to have arisen incidentally with no relationship to the test article.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects on thyroxine or thyroid stimulating hormone were observed.
A small but statistically significant increase in T4 was evident for males administered 50 mg/kg/day AI, compared with controls (P < 0.05). In the absence of a dose relationship, and in isolation, this increase was considered to have arisen incidentally.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test article-related effects were noted for the urine parameters assessed for males or females.
Statistical analysis of the urine volume or specific gravity data did not reveal any significant intergroup differences.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Upon microscopic examination, test article-related findings were recorded for the spleen.
A dose-related increase in the severity of extramedullary hemopoiesis and pigmented macrophages were noted for F0 animals administered 50, 100, or 150 mg/kg/day AI, compared with controls, more notably in males. Extramedullary hemopoiesis was characterized by increased numbers of hemopoietic cells from all three cell lines within the splenic red pulp. Pigmented macrophages were characterized by increased numbers of macrophages with golden-brown cytoplasmic pigment within the splenic red pulp.
No other test article-related microscopic findings were recorded.
Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings rats of this strain, age, and stage of reproduction.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects on estrous cycle assessments were noted for the mean number and duration of estrous cycles prior to pairing.
Statistically significant increases in mean length of the cycles was noted for groups administered 100 or 150 mg/kg/day AI, compared with controls. However, a dose relationship was not evident, and no impact on mating or fertility occurred; as such, these statistically significant intergroup differences were considered to have arisen incidentally and not as a consequence of test article administration.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects on the seminology data were observed.
Sperm concentration was significantly lower for males administered 100 or 150 mg/kg/day AI, compared with controls; however, a dose relationship was not observed (P < 0.01 or P < 0.05, respectively). All other parameters were unaffected; in the absence of an effect on male fertility in this study, these intergroup differences were considered to have arisen incidentally.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Mating, fecundity, or fertility indices were unaffected by administration of the test article.
Most paired animals mated within 5 days of pairing.
Fecundity and fertility indices were increased in the group administered 150 mg/kg/day AI, compared with controls, which was considered to have arisen incidentally.
Of the 21, 21, 22, or 22 paired females administered 0, 50, 100, or 150 mg/kg/day AI, respectively, 20, 20, 19, or 22 females were pregnant.
No test article-related effects on the litter parameters was evident.
Of the 20, 20, 19 or 22 pregnant females administered 0, 50, 100 or 150 mg/kg/day AI, one animal administered 150 mg/kg/day AI (Animal R0702) had no viable fetuses. Another animal from this group (Animal R0719) was sacrificed on Lactation Day 0 (Section 4.2.1) due to total litter loss (one pup born alive and one stillborn). Both events were considered to have arisen incidentally with no relationship to the test article. All remaining pregnant females had surviving litters on Lactation Day 21.
No test article-related effect on gestation length was noted.
Litter size and survival index was unaffected. Sex ratio was unaffected by test article administration at any dose level.
An increased incidence of stillborn pups was noted following administration of 150 mg/kg/day AI, compared with controls. Animal R0719 had one pup born alive and one stillborn pup. Animal R0719 also had a gestation length of 25 days, with a high post-implantation loss of 12 implants, compared with one stillborn and one pup born alive, which resulted in the total litter loss. The remaining litters with stillborn pups did not exhibit large post-implantation losses or larger than expected litters. A marginally higher post-implantation loss and reduced gestation index was noted for females administered 150 mg/kg/day AI, compared with controls. This was attributable to the in utero litter loss (Animal R0702), which, in isolation, was considered to have arisen incidentally. Seven stillborn pups were evident from five litters administered 150 mg/kg/day AI, compared with four stillborn pups from three litters for controls. This was slightly higher that the historical control day range (HCD; 0 to 6 stillborn pups). However, the percentage of stillbirths in this dose group (0.33%) was within the HCD (0 to 3%), the mean number of pups born in each litter was also within the HCD range (4.7 to 15 pups), and no effect on mean post implantation loss occurred; as such, the marginally higher number of stillborn pups was considered to have arisen incidentally, as it was not supported by any other changes in offspring survival.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Pup Clinical Observations: Test article-related clinical observations were confined to the presence of mouth rubbing, observed immediately after direct dosing from PND 14 for some animals administered 100 or 150 mg/kg/day Al, and a dose response was evident. This was not observed for pups administered 50 mg/kg/day AI or the control article (vehicle) alone.
The remaining clinical observations noted for the pups during the lactation phase were observed throughout the dose groups, including controls, with no dose relationship; as such, they were considered to have arisen incidentally.

F1 Clinical Observations: Nonadverse test article-related clinical observations were noted after dosing at all dose levels.
Transient and test article-related clinical observations of mouth rubbing were observed for all animals administered 100 or 150 mg/kg/day AI (Cohorts 1A and 1B). Isolated instances of mouth rubbing were also recorded for 14 of 40 males (two from Cohort 1A and 12 from Cohort 1B) and 12 of 40 females (five from Cohort 1A and seven from Cohort 1B) administered 50 mg/kg/day AI. A dose response was evident.
Isolated instances of paddling behavior were recorded for three females administered 150 mg/kg/day AI (Cohort 1A: Animal R1513 on PND 81; Cohort 1B: Animal R1533 on PND 102 and Animal R1535 on PND 104). Isolated instances of paddling behavior were also recorded on PND 101 and 102 for one Cohort 1B male administered 150 mg/kg/day AI (Animal R1135), and this was also evident for two Cohort 1B males administered 100 mg/kg/day AI (Animal R1028 on PND 29 and Animal R1133 on PND 101 and 102).
Excessive salivation was recorded on PND 67 and 80 for one Cohort 1A male (Animal R1106) administered 150 mg/kg/day AI. This was also reported on four occasions between PND 97 and 102 for one Cohort 1B male (Animal R1131) administered 150 mg/kg/day AI. Animal R1132, administered 150 mg/kg/day AI, was recorded with this on PND 102, and Animal R1137, administered 150 mg/kg/day AI, was reported with this finding on PND 96 and 97. One female (Animal R1435) administered 100 mg/kg/day AI also exhibited excessive salivation on PND 102. Animal R1534, administered 150 mg/kg/day AI, exhibited this finding on PND 104, and Animal R1539, administered 150 mg/kg/day AI, exhibited excessive salivation between PND 97 and 102.
An instance of jaw chomping was recorded on PND 31 for one Cohort 1B male (Animal R1124) administered 150 mg/kg/day AI. In isolation, this was considered to have arisen incidentally and not test article related.
The remaining clinical observations were observed throughout the dose groups, including the control; as such, they were considered to represent low incidence findings in animals of this age and arisen incidentally.
No observations were noted during the weekly detailed clinical examinations at any dose level.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test article-related unscheduled deaths have occurred. The following deaths were considered to have arisen incidentally.
One Cohort 1B male administered 100 mg/kg/day AI (Animal R1038) was sacrificed on PND 51. The animal was recorded with hunched posture, decreased activity, piloerection, thin and pale, cold to the touch, and with excessive red colored lacrimation from both eyes. Pale adrenals and thymus were recorded. The spleen was recorded as thin. The liver was recorded as mottled and pale. The jejunum included abnormal contents and was distended. The ileum was impacted. The pancreas had an abnormal texture with adhesion to the stomach. The abdominal cavity was fluid filled. A cause of demise could not be determined for this animal based on the macroscopic observations recorded. In the absence of similar mortality in the intermediate or in the high dose group, this death was considered to have occurred incidentally.
One Cohort 1A female administered 150 mg/kg/day AI (Animal R1506) was sacrificed on PND 75 following the presence of protruding eyes. In the absence of similar observations in test article treated animals, this death was considered to have occurred incidentally.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Pup Body Weights: No test article-related effect on body weights was noted.
Mean body weights were essentially similar across all dose groups at the end of the pre weaning phase, and statistical analysis of the data did not reveal any statistically significant intergroup differences.

Pup Organ Weights: Nonadverse test article-related increases in spleen weights were evident following 50 or 150 mg/kg/day AI, compared with controls.
Spleen:body weight ratios were higher for F1 female pups from dams administered 50 mg/kg/day and F1 pups of both sexes from dams administered 150 mg/kg/day AI, compared to F1 control pups (P < 0.05).This was not observed for pups administered 100 mg/kg/day AI, compared with controls. Test article-related effects on the spleen were prevalent in this study; as such, the increase in spleen weight was considered test article related, although these increases were considered not to represent an adverse effect.


F1 Body Weights: No test article-related body weight or body weight changes have been noted.
Mean body weights for test article-treated groups were essentially similar to controls.
A small statistically significant reduction in mean body weight gain was evident for Cohort 1A males administered 150 mg/kg/day AI, compared with controls, between PND 22 to 24 (P<0.05). This intergroup difference was not observed again for the Cohort 1A males and was not evident for the Cohort 1B males administered at the same dose level; as such, this was considered to have arisen incidentally with no relationship to test article administration.

F1 Organ Weights: Test article-related changes in splenic weights and weight ratios were recorded for F1 Cohort 1A animals from all test article-treated groups.
A generally dose-related increase in group mean unadjusted splenic weights, spleen:body weight, and spleen:brain weight ratios was recorded for F1 Cohort 1A animals from all test article treated groups, compared with controls, which generally correlated with the macroscopic and microscopic findings. A minor, statistically significant increase in liver weights and weight ratios was recorded for F1 Cohort 1A females administered 150 mg/kg/day AI, but this occurred without a corresponding microscopic finding.
No test article-related changes in reproductive organ weights or weight ratios were recorded for Cohort 1B animals. Minor organ weight and organ weight ratio changes were attributed to biological variation.
All other organ weight and organ weight ratio changes were attributed to biological variation and were considered not test article related as they were small in magnitude, not dose-dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test article-related effects on mean food consumption was evident for test article treated groups, compared with controls.
Statistical analysis of the data did not reveal any significant intergroup differences.

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):

Nonadverse test article-related changes in red cell parameters were evident for males and females throughout the dose groups.
Hemoglobin, mean cell hemoglobin concentration, and red cell counts were reduced for males and females administered 150 mg/kg/day AI, compared with controls and mean cell hemoglobin, mean cell volume, and reticulocyte counts and % reticulocytes were increased (P < 0.001), compared with controls. Red cell distribution was reduced for males administered 150 mg/kg/day AI (P < 0.001), and packed cell volume was also reduced for females administered 150 mg/kg/day AI (P< 0.05), compared with controls.
Hemoglobin and red cell count were reduced for males and females administered 100 mg/kg/day AI (P < 0.001). Mean cell hemoglobin concentration was reduced for males (P < 0.001) and packed cell volume and mean cell hemoglobin concentration was also reduced for females (P < 0.01), compared with controls. Mean cell hemoglobin, mean cell volume, reticulocyte counts, and % reticulocytes were increased (P < 0.001), compared with controls.
Hemoglobin and mean cell hemoglobin concentration, red cell count, and red cell distribution was reduced for males administered 50 mg/kg/day AI (P < 0.05 to P < 0.001), compared with controls. Red blood cell counts and mean cell hemoglobin concentration were reduced for females administered 50 mg/kg/day AI (P < 0.01). Mean cell hemoglobin, mean cell volume, reticulocyte counts, and % reticulocytes were increased for males and females administered 50 mg/kg/day AI (P < 0.001), compared with controls.
Remaining statistically significant intergroup differences were observed in one sex or did not exhibit a dose relationship; as such, they were considered to have arisen incidentally.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Nonadverse test article-related clinical chemistry changes of decreased glucose, triglyceride, and globulin levels (P < 0.05 to P< 0.001) with associated increases in A:G ratio (P < 0.001) were observed for males administered 150 mg/kg/day AI, compared with controls. No associated organ changes were noted to suggest that these changes resulted in adversity; therefore, these changes were of little toxicological significance.
No such changes were evident for females administered 150 mg/kg/day AI or animals of both sexes administered 50 or 100 mg/kg/day AI.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test article-related effects were noted for the urine parameters assessed for males or females at any dose level investigated.
Statistical analysis of the urine volume or specific gravity data did not reveal any significant intergroup differences.

Sexual maturation:

no effects observed

Description (incidence and severity):

No test article-related effect on sexual maturation was noted.
Statistical analysis of the data did not reveal any significant intergroup differences.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test article-related effect on ano-genital distance was noted following administration of the test article at any dose level, compared with controls.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No presence of nipple retention was noted for any male pup following administration at any dose level.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test article-related changes in splenic weights and weight ratios were recorded for F1 Cohort 1A animals from all test article-treated groups.
A generally dose-related increase in group mean unadjusted splenic weights, spleen:body weight, and spleen:brain weight ratios was recorded for F1 Cohort 1A animals from all test article treated groups, compared with controls, which generally correlated with the macroscopic and microscopic findings. A minor, statistically significant increase in liver weights and weight ratios was recorded for F1 Cohort 1A females administered 150 mg/kg/day AI, but this occurred without a corresponding microscopic finding.
No test article-related changes in reproductive organ weights or weight ratios were recorded for Cohort 1B animals. Minor organ weight and organ weight ratio changes were attributed to biological variation.
All other organ weight and organ weight ratio changes were attributed to biological variation and were considered not test article related as they were small in magnitude, not dose-dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pup Macroscopic Observations: Nonadverse, test article-related enlarged spleens were recorded for one male pup each from a dam administered 50 or 150 mg/kg/day.
This was not observed for male pups administered 100 mg/kg/day AI or female pups at any dose level. Test article-related effects on the spleen were observed; as such, the incidences of enlarged spleens were considered to be test article related, although these increases were considered not to represent an adverse effect.
Other tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rat pups of this strain and age.

F1 Macroscopic Observations: Upon macroscopic examination, test article-related findings were noted in the spleen.
Large spleen was recorded for occasional F1 Cohort 1A males from all test article treated groups, which generally correlated with microscopic findings. Large spleen was also recorded for one F1 Cohort 1B male (Animal R1135) administered 150 mg/kg/day AI.
Other tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Upon microscopic examination, test article-related findings were recorded for the spleen.
An increased incidence and/or severity of extramedullary hemopoiesis and pigmented macrophages was noted for F1 Cohort 1A animals administered 50, 100, or 150 mg/kg/day AI. No other test article-related microscopic findings were recorded. No effects attributable to the test article were noted in the examined lymphoid tissues.
Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Pup Thyroid Hormone Analysis: No test article-related changes in thyroxine or thyroid stimulating hormones were evident for pups on PND 22.
Statistical analysis of the data did not reveal any statistically significant intergroup differences.

F1 Thyroid Hormone Analysis: No test article-related thyroxine or thyroid stimulating hormones changes were observed.
Males administered all dose levels attained statistically significant reductions in T4 levels, compared with controls (P < 0.05), although no convincing dose response was noted. Values were within the historical control data ranges (46-100 nmol/L), no correlating increase in TSH levels was observed, and a similar change was not evident for females; as such, this was considered to have arisen incidentally and unrelated to the test article.

F1 Estrous Cycle: No test article-related effects on the duration or mean number of estrous cycles was noted.The mean number and length of the estrous cycles was comparable across groups and statistical analysis of the data did not reveal any significant intergroup differences.

F1 Seminology Evaluations: No test article-related effects were observed for the sperm parameters investigated. The statistically significant intergroup differences observed did not exhibit a dose relationship or associated changes with other parameters; as such, they were considered to have arisen incidentally.

F1 Ovarian Follicle Counts: No test article-related changes were noted for ovarian follicles from test article-treated F1 females, compared with controls.
Statistical analysis did not reveal any statistically significant intergroup differences.
 

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

effects observed, treatment-related

Description (incidence and severity):

Nonadverse moderate increases in the absolute concentration of CD3+, CD3+CD4+, and CD3+CD8+ were noted in F1 generation Cohort 1A females administered 50 or 100 mg/kg/day AI; these increases were paralleled by a marginal increase in the relative frequencies of these immune cell populations.
No variation in the absolute concentration or relative frequency of CD45RA+ cells was observed for test article-treated F1 generation Cohort 1A animals.
Dose-dependent increases in the absolute concentration of CD3-CD161a+ cells, matched by negligible variations in frequency, were observed for test article-treated F1 generation Cohort 1A males.
Due to the variability in the absolute concentration and relative frequency observed in control and test article-treated F1 generation Cohort 1A animals, the contribution of the test article to the variations reported was deemed minor, and the test article was unlikely to contribute to immune toxicities; as such, it was considered nonadverse.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Applicant's summary and conclusion

Conclusions:

Once-daily oral gavage administration of 142.5, 285, or 427.5 mg/kg/day (equivalent to 50, 100, or 150 mg/kg/day AI) test substance resulted in test article-related systemic effects at all dose levels investigated and in both generations. Test article-related effects consisted of clinical observations noted after dosing, which were attributable to test article palatability and did not represent systemic toxicity. Changes in red cell parameters were also observed, together with an increased incidence and/or severity of extramedullary hemopoiesis and pigmented macrophages in the spleen, which corresponded with incidences of large spleens and increased spleen weights. These findings, however, did not have an impact on the overall health of the animals; as such, the no observed adverse effect level (NOAEL) for systemic toxicity is 150 mg/kg/day AI.
No adverse effects on mating performance, fertility, fecundity, gestation, parturition, or lactation were noted; as such, the no observed effect level (NOEL) for reproductive toxicity is 150 mg/kg/day AI in this study.
No effects on the developing offspring was noted at any dose level; as such, the NOEL for offspring growth and development is 150 mg/kg/day AI.