Magnesium, bis[2-(hydroxy-.kappa.O)benzoato-.kappa.O]-, ar, ar'-di-C14-18-alkyl derivs.

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: 21 Feb 2020 To: 12 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 70 consecutive days
- Basis for dose level selection: The dosage levels were selected based on the results of a number of previous studies on the target and read-across substances but ultimately on the dose range finder enhanced OECD 421 study in rats which was conducted to transition from gavage route to the diet as per ECHA decision (see for details section "Details on study design").
- Inclusion of extension of Cohort 1B: yes, not specified.
- Termination time for F2: no, not specified.
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: no, not specified.
- Exclusion of developmental immunotoxicity Cohort 3: no, not specified.
- Route of administration: not specified; dose levels were based on a previous OECD 421 study in which rats were administered the test substance via diet.
- Species: The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
- Number of animals: The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, Jun 2018, which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality in each generation of the study, 25 P animals/sex/group was an appropriate number of animals to achieve the desired number of animals in each generation.

Test material

Test material information

Constituent 1

Reference substance name:

Magnesium, bis(2-hydroxybenzoato-O1,O2)-, ar,ar'-di-C14-18alkyl derivs.

EC Number:

931-371-5

Cas Number:

171171-80-5

IUPAC Name:

Magnesium, bis(2-hydroxybenzoato-O1,O2)-, ar,ar'-di-C14-18alkyl derivs.

Details on test material:

Supplier: Infineum International LTD
Storage conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C, protected to light

Specific details on test material used for the study:

Supplier: Sponsor
Storage conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C, protected from light

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: 180 to 249 g (males), 140 to 191 g (females)
- Fasting period before study: no
- Housing: in solid-bottom cages containing heat-treated aspen bedding (Northeastern Products Corp.). On arrival (P animals) or following weaning (F1), animals were group housed (2 to 3 animals of the same sex) until cohabitation. During cohabitation, the animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. For enrichment, animals were provided items such as treats, a gnawing device, and/or nesting material, except when interrupted by study procedures/activities.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal), ad libitum, except during periods of fasting for clinical pathology assessments. The feed was analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water: tap water, ad libitum. Periodic analysis of the water is performed. It was considered that there were no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: yes, not specified
- Allocation to group: P generation animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals at extremes of body weight range were not assigned to groups.

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C to 26°C (target)
- Humidity: 30% to 70% (target)
- Air changes (per hr): not reported
- Photoperiod: 12-hour light/12-hour dark
IN-LIFE DATES: From: 10 Mar 2020 To: 03 Nov 2020

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

FORMULATION PROCEDURE
- Preparation of test substance: Dietary formulations were prepared at appropriate concentrations to meet exposure level requirements. Correction factors of 3.19 (lot E01324-129) or 3.25 (lot E01209-311) were applied. An appropriate amount of the test substance for each group was added to an appropriate amount of rodent feed on a weight/weight basis, and mixed to form a premix. The resulting premix was then mixed thoroughly with the remaining amount of feed in a Hobart mixer to achieve a total batch of homogeneous diet at the appropriate concentration/group.
- Frequency of preparation: Approximately weekly, and divided into aliquots for daily dispensation.
- Storage conditions: Stored at room temperature (18°C to 24°C) until use.
- Stability: Stability of the test substance in diet at concentrations ranging from 137 to 10,000 ppm at controlled room temperature for at least 8 days of storage has been determined (D’Sa, 2020, 00537044). Additionally, calibration standards and processed formulation samples were analyzed and then stored at room temperature for 8 days before re-analysis to assess test substance stability at 500 and 4500 ppm on this study.

ADMINISTRATION (CONCENTRATIONS)
- At a constant concentration (ppm) in the diet: P generation males for the entire study, to P generation females during the premating period and from the end of lactation to euthanasia, and to F1 males and females from PND 50 to euthanasia or confirmation of mating (Cohort 1B females).
- Based on historical control data: During the gestation and lactation periods. Adjusted body weights (to the estimated mid-next-week weight, corrected body weight) were calculated based on historical body weight data and dietary concentrations were calculated.
- Females with no evidence of mating: same group until euthanasia.

Details on mating procedure:

P generation and Cohort 1B
- M/F ratio per cage: 1:1 (on the first PND 104 for Cohort 1B)
- Length of cohabitation: 14 days
- Proof of pregnancy: by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no
- Parturition: The day parturition was initiated was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). All females were allowed to deliver naturally. Beginning on the day parturition was initiated, the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed (P generation and Cohort 1B). The dam and litter remained together until weaning on PND 21 (P generation). The dam and litter remained together until euthanasia on PND 4 (Cohort 1B).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analytical technique: high performance liquid chromatographic method with ultraviolet (HPLC/UV) detection
- Acceptance criteria: Measured concentration = theoretical concentration ± 30% (concentration and homogeneity); relative standard deviation of the mean concentration (RSD) = 10% (homogeneity).
- Sampling: for concentration analysis on a regular basis of all dose groups; for homogeneity assessment at the start of the study for all dose groups (P generation) and for the low dose group of the F1 generation (PND 21-28).

Analytical text from reports (plasma and milk) attached.

Duration of treatment / exposure:

P GENERATION
- Males: continuously in the diet for a minimum of 70 consecutive days prior to mating and continuing until euthanasia.
- Females: continuously in the diet for a minimum of 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, until euthanasia.

F1 GENERATION
- Offspring: Following weaning until euthanasia or the initiation of fasting prior to scheduled necropsy (PND 52 [Cohort 1 Surplus], PND 91 [Cohort 1A], and Lactation Day 4 [Cohort 1B females] or PND 132–146 [Cohort 1B males and females without evidence of mating or that failed to deliver]).
- F1 males and females: from PND 50 to euthanasia or confirmation of mating (Cohort 1B females).

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 ppm (nominal)

Remarks:

low dose group - equivalent to 30 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 500 ppm (nominal)

Remarks:

mid dose group - equivalent to 100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

4 500 ppm (nominal)

Remarks:

high dose group - equivalent to 300 mg/kg bw/day

No. of animals per sex per dose:

P: 25 per sex per dose level
F1: 20 per sex per dose level (Cohort 1A and 1B)

Control animals:

yes, plain diet

Details on study design:

DOSE SELECTION RATIONALE
The ECHA Final decision for this substance stated that the study should be an Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method OECD TG 443) in rats, oral route with the registered substance, and that it should specifically contain: 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 (Reproductive toxicity); Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation. The OECD guideline for the 443 study states the following concerning the dose level setting;
"Normally, the study should include at least three dose levels and a concurrent control. When selecting appropriate dose levels, the investigator should consider all available information, including the dosing information from previous studies, TK data from pregnant or non-pregnant animals, the extent of lactational transfer, and estimates of human exposure. If TK data are available which indicate dose dependent saturation of TK processes, care should be taken to avoid high dose levels which clearly exhibit saturation, provided of course, that human exposures are expected to be well below the point of saturation. In such cases, the highest dose level should be at, or just slightly above the inflection point for transition to nonlinear TK behaviour. 21. In the absence of relevant TK data, the dose levels should be based on toxic effects, unless limited by the physical/chemical nature of the test chemical. If dose levels are based on toxicity, the highest dose should be chosen with the aim to induce some systemic toxicity, but not death or severe suffering of the animals. "
There are no toxicokinetic data for this substance, so toxicity data was used from previous studies on the target and a read-across substance (14d DRF, 28d repeat dose, 90d repeat dose and 414 developmental tox, and 421 reproductive screen) to develop suitable dose levels to propose for this study in the communication with ECHA prior to the Final decision. The results of these can be summarised as follows:
¿ For OECD 408 and OECD 414 (target substance), Dosage levels selected based on the results of a 14-day dose range-finding study in Sprague-Dawley rats in which dosage levels of 0, 10, 30, 100, 300 and 450 mg/kg/day were administered via oral gavage. Significant increases in ALP and ALT were observed in the 300 and 450 mg/kg/day groups when serum chemistry was evaluated at the time of necropsy. On completion, Acceptance of these studies was agreed with ECHA and thus taken as evidence of dose levels being adequate to show systemic tox or developmental tox. Text from the ECHA letter (underlined):
Communication number: CCH-C-2114351633-52-01/F, Registration number: 01-2119489802-28-0000, Submission number subject to follow up evaluation: CY622296-13, Date of submission subject to follow up evaluation: 9 June 2016
NOTIFICATION OF INFORMATION OBTAINED AND CONCLUSIONS MADE AFTER COMPLETION OF DOSSIER EVALUATION
You are hereby notified of the information obtained and any conclusions made after completion of dossier evaluation in accordance with Article 42(2) of Regulation (EC) No 1907/2006 (REACH Regulation).
Pursuant to Article 42(1) of the REACH Regulation, the European Chemicals Agency (ECHA) has examined the information received on 9 June 2016 for the registration number 01-2119489802-28-0000 concerning the substance magnesium, bis(2-hydroxybenzoato-o1,o2)-, ar,ar'-di-c14-18alkyl derivs. The Registrant submitted this information in consequence of ECHA’s decision of 12 December 2014, CCH-D-2114288615-38-01/F.
The information received in the updated dossier corresponds to the information requirement(s) addressed in the decision and ECHA considers the dossier evaluation with regard to this decision as completed.
¿ 14d DRF calcium salicylate was conducted at doses of 0, 50, 125, 250, 500 and 1000 mg/kg/day to both male and female Sprague Dawley rats. Test article was dosed, uncorrected, from material supplied at 43% w/v Active Ingredient (AI). Effects included body weight or body weight changes only at the highest dose. Serum chemistry (primarily the liver) was affected in both sexes at doses = 125 mg/kg/day
¿ 28-day, repeat oral dose study (OECD 407) on calcium salicylate CAS #114959-46-5 - doses were set at 0, 50, 150 and 500 mg/kg/day. Effects (top dose); lower weight changes, longer prothrombin times and several elevated liver enzymes all in male animals. There were also increased liver weights (in both males and females) and increased thyroid weights in males.
¿ A reproduction/developmental toxicity screening study of CAS #114959-46-5 in rats was conducted at WIL Laboratories (Knapp, WIL-186048). Doses were set at 0, 50, 150 and 500 mg/kg/day. Effects on bodyweights but not on reproductive performance


In the recent Final Decision, (2019) ECHA agreed with the dose selection approach however as the route of administration was changing to dietary and the previous studies had been gavage it was proposed to include a new enhanced OECD 421 range finder to confirm the proposed dose levels based on the systemic toxicity agreed. However it was noted that there might be a potential build up or accumulation in the animals (as referenced by ECHA in the Final Decision) hence there was a need to accommodate a long premating dosing in anticipation of accumulation. This was due to the fact that there are indications that the internal dose for the registered substance will reach a steady state in the test animals only after an extended exposure because its logKow value is = 4.5 (logKow= 5.64 at 40°C) and absorption is expected to be slow/ inefficient via the gastrointestinal tract. In addition to the long pre-mating dosing, this meant doses needed to take into account the build up.
Further, ECHA noted a potential concern for Endocrine Disruption effects to be investigated in the 443: in the OECD TG 408 study conducted with the registered substance, statistically significant higher relative adrenal gland weights (56% and 35% in males and females, respectively) were noted in the high dose groups (300 mg/kg bw/day). Test substance-related microscopic findings (adrenal cortical cell hypertrophy) were also noted in the adrenal cortex of high dose females at necropsy. The OECD TG 408 study also provided an increase in relative (but not absolute) thyroid/parathyroid weight of 16% (in high dose females only) in the presence of general toxicity (increased liver weight, significant increase of serum liver enzymes of up to 579%, and test substance-related microscopic findings in the liver. All this supported that a top dose of around 300mg/kg bw/day would produce definite systemic toxicity (without deaths or stress) in the key organ systems and significantly including any potential Endocrine Disruption effects, and that these levels might indeed increase over time within the animals. Therefore this dose level was considered of interest to replicate in the diet as a high dose group.

A palatability study in the diet was conducted first with concentrations up to around 5000ppm in the diet (above 410mg/kg bw/day), where there were clear palatability effects and a drop in food consumptions and bodyweights in less than 10 days. (Report attached) This then led into the OECD 421 study conducted n the diet as a dose range finder. The dosage levels for the 443 were ultimately selected based on the results of this OECD 421 study in rats (Herberth, 2020, study ref 00537050). In that study, rats were administered the test diets at constant concentrations of 500, 1500, or 4500 ppm. There were no significant treatment effects in the parental animals however, mean body weights for the F1 pups in the 4500 ppm group during the postnatal phase were significantly lower than controls. This was likely due to an increased mg/kg bw/day dose during the lactation period for the P generation females and post-weaning period for the F1 pups as a result of increased food consumption during these periods. Based on the previous studies, the concentrations of diet were adjusted during these critical periods to maintain a more constant mg/kg bw/day dose. More details of F0 and F1 dosing and effects are as follows:
• F0 generation doses were:
¿ see attachment

¿ Effects noted:
¿ Test substance-related small thymus gland was noted in the 4500 ppm group females at the scheduled necropsy. The finding correlated with lower mean thymus gland weights and microscopically with decreased lymphoid cellularity.
¿ At 4500ppm, liver weights and related changes in parental animals.
¿ At 4500 ppm, mean alkaline phosphate concentration for F0 males and females was 177.4% and 200.8% higher, respectively, than the control group. In addition, mean alanine aminotransferase concentration for F0 males and females was 44.4% and 41.7% higher
¿ In the females, A spectrum of findings in the 4500 ppm group females provided weight of evidence for test substance-related stress response, including lower mean final body, ovaries/oviducts, thymus, and spleen weights; higher mean adrenal gland weights; and vaginal epithelial cell atrophy noted microscopically.
¿ In the males at 4500ppm, statistically significant mean organ weight differences were attributed to test substance-related lower mean final body weights, including higher mean brain and kidney weights relative to final body weight and lower mean seminal vesicle/coagulating gland weights (absolute and relative to brain weights)

• F1 generation were exposed to higher concentrations of the test substance (up to 560mg/kg per day) resulting in a big drop in bodyweights and other effects
¿ see attachment
¿ In the F1 generation, the most prominent test substance-related effects were noted on pup body weights and body weight gains during the preweaning and postweaning periods in the 1500 and 4500 ppm groups. In the 4500 ppm group, lower mean F1 pup body weight gains were noted throughout the preweaning period, resulting in mean F1 male and female pup absolute body weights that were up to 35.5% and 36.0% lower, respectively, than the control group during PND 7–21. The effects on body weight and body weight gain at 4500 ppm were considered test substance-related and adverse.
¿ Mean body weight gains at 4500 ppm were lower than the control group throughout the postweaning period (PND 21–42) for F1 males and during PND 21–31 and when the entire postweaning period was evaluated for F1 females. Corresponding lower mean food consumption was noted for F1 males and females at 4500 ppm throughout the postweaning period. As a result, mean absolute body weights at 4500 ppm were up to 46.2% and 43.2% lower than the control


In addition to this, consideration was given to the clear evidence of lactational transfer (in the additional work done in the enhanced OECD 421 screen) and the human exposure route of concern. This confirmed the decision to maximise the dose levels at the 300mg/kg bw/day equivalent in the diet.

ECHA review of EOGRTS OECD 443 studies (July 2021)
Recent publication of a review of some conducted OECD 443 studies has indicated that the guideline is not specific enough in the design of the protocols and that some criteria are not being met. While it is clearly too late to take the recommendations on board for this completed study, which followed the guideline, it is noted with regard to the dose selection process that ECHA themselves were very involved – see earlier comment - as they agreed with the dosing of previous studies and the use of these in dose selection for the DRF in this study (the OECD 421) as well as the general plan for dosing (albeit in the diet) for the 443 on this substance in the Final Decision. The dosing selection rationale above outlines why the top dose was chosen in continuity with previous studies, seeing definite systemic toxicity and potential endocrine changes concurrently, and these considering the potential for the substance loading in the body to build up over time. The lack of evidence fertility and reproductive toxicity in the 421 and in the 443 cannot be taken as evidence that dosing was not conducted at a high enough dose level or that a limit dose should have been employed – this would have meant severe parental toxicity (as seen in the bodyweight drops) and confounding of interpretation of the endocrine parameters of interest as it would be impossible to establish if this was secondary to the effects on the liver and other general effects on parental systems. The whole premise of dosing selection has to rely on information from preceding studies in order to reduce animal suffering, save use of animals, and to ensure we are targeting the right information - and this was done here. Given the lack of toxicity seen in general with this substance (and the read-across substance), it cannot be justified to require higher dose level toxicity testing and use of yet more animals when there is clearly no hazard profile to be investigated from this or any preceding study, and thus any point of departure to be clarified, with relation to human hazard labelling, or exposure and risk assessment.


CONSTITUTION OF THE F1 GENERATION
For the F1 generation, 3 F1 pups/sex/litter from all available litters (=20 litters/group) were randomly selected prior to weaning and were assigned to the following cohorts. Cohorts 1A and 1B were assigned to reproductive/developmental toxicity testing. Animals assigned to Cohort 1B were maintained on study for possible breeding when the animals were between 90 and 120 days of age to generate an F2 generation. Cohort 1 surplus was assigned to postweaning developmental landmarks. Assignment of same-sex littermates to a particular cohort was avoided whenever possible. In addition, if there were an insufficient number of pups to fill a designated cohort, the following prioritization plan was used (highest to lowest priority): Cohort 1A, Cohort 1B, Cohort 1 surplus.

F2 GENERATION
The F2 litters were terminated on PND 4. While it is noted that investigations on this generation were not identical to those of F1, at the time of this study the guideline being followed (443) did not specify this need except where there wad evidence in the preceding studies or generations of an effect which might require additional investigations to classify appropriately. This was not required in case of this substance where no such effects had been identified and the lab determined that these would not add any value to the study.

Examinations

Parental animals: Observations and examinations:

MORBIDITY AND MORTALITY (P and F1 generation): Yes
- Time schedule: twice daily, all animals.

DETAILED CLINICAL OBSERVATIONS (P and F1 generation): Yes
- Time schedule: once daily, all animals

BODY WEIGHT (P and F1 generation): Yes
- Time schedule: weekly, individually, beginning 1 week prior to test diet administration (P generation), or following weaning (F1 generation), and continuing throughout the study and prior to the scheduled necropsy (P generation and F1 generation). Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 3, 6, 9, 12, 15, 18, and 20 and on Lactation Days 1, 4, (P generation and Cohort 1B), and 7, 14, and 21 (P generation). Any animals that were fasted had body weights collected prior to and after fasting (P generation and Cohort 1A).

FOOD CONSUMPTION AND COMPOUND INTAKE (P and F1 generation): Yes
- Time schedule: beginning 1 week prior to test diet administration (P generation), or following weaning (F1 generation), except during the mating period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 3, 6, 9, 12, 15, 18, and 20 and Lactation Days 1, 4, (P generation and Cohort 1B), and 7, 14, and 21 (P generation).
- Food evaluation: The mean amounts of test substance consumed (mg/kg bw/day) by each sex per dose group were calculated from the mean food consumed (g/kg bw/day) and the appropriate target concentration of test substance in the food (mg/kg). Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported (P and F1 generation).

WATER CONSUMPTION: No

HEMATOLOGY (P generation and Cohort 1A): Yes
Blood samples were taken from the jugular vein of 10 animals/sex/group into tubes containing K2EDTA as anticoagulant.
- Time point: Day of necropsy (is PND 91 for Cohort 1A)
- Fasting: yes, overnight
- Parameters analysed: Total leukocyte count (WBC) Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (Platelet), Reticulocyte count, Percent (RETIC), Absolute (RETIC Absolute), Differential leukocyte count [Percent and absolute] (Neutrophil (NEU), Lymphocyte (LYMPH) , Monocyte (MONO) , Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC)), Red cell distribution width (RDW), Hemoglobin distribution width (HDW), Platelet estimate, Red cell morphology (RBC Morphology).

COAGULATION (P generation and Cohort 1A): Yes
Blood samples were taken from the inferior vena cava of 10 animals/sex/group at the time of euthanasia from animals euthanized via carbon dioxide inhalation. Sodium citrate was used as anticoagulant.
- Time point: Day of necropsy (is PND 91 for Cohort 1A)
- Fasting: yes, overnight
- Parameters analysed: Activated partial thromboplastin time (APTT), Prothrombin time (PT).

CLINICAL CHEMISTRY (P generation and Cohort 1A): Yes
Blood samples were taken from the jugular vein of 10 animals/sex/group into tubes without anticoagulant.
- Time point: Day of necropsy (is PND 91 for Cohort 1A)
- Fasting: yes, overnight
- Parameters analysed: Albumin, Total protein, Bile Acids, Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total BILI), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium,
Chloride, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase (SDH), Triglycerides (Triglyceride), Appearance.

BLOOD SAMPLES FOR FURTHER POSSIBLE ANALYSES (P generation and Cohort 1A): Yes
Blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped, and retained for possible future evaluation.

URINALYSIS (P generation and Cohort 1A): Yes
Urine was collected overnight using metabolism cages.
- Time point: Day of necropsy (is PND 91 for Cohort 1A)
- Fasting: yes, overnight
- Parameters analysed: Specific gravity (SG), pH, Urobilinogen (URO), Total volume (TVOL), Color (COL), Clarity (CLA), Protein (PRO), Glucose (GLU), Ketones (KET), Microscopy of sediment Bilirubin (BIL), Occult blood (BLD), Leukocytes (LEU).

THYROID HORMONE ANALYSIS (P generation and Cohort 1A): Yes
Blood samples were taken (prior to 12:00 hours) from10 animals/sex/group from the jugular vein into tubes without anticoagulants.
- Time point: Week 19 (P generation), PND 90 (Cohort 1A)
- Fasting: No
- Parameters analysed: thyroid hormone (T4) and thyroid stimulating hormone (TSH) levels.

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily, and the slides were evaluated microscopically, according to the following time schedule:
• beginning on the day vaginal opening was observed (Cohort 1A)
• daily for 14 days prior to cohabitation and continuing until evidence of mating was observed, or until the end of the mating period (P generation and Cohort 1A)
• on the day of necropsy (P generation and Cohort 1A)

Sperm parameters (parental animals):

FULL SEMINOLOGY (P generation and Cohort 1A)
- Groups examined: control and all dose groups
- Examinations: testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, and sperm morphology

Litter observations:

LITTER VIABILITY (F1 and F2 pups)
- Time schedule: twice daily, once in the morning and once in the afternoon
- Observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS (F1 and F2 pups): Yes
- Time schedule: on PND 1, 4 (F1 and F2 pups), 7, 14, and 21 (F1 pups)
- Observations: detailed clinical observations, and abnormalities in nesting and nursing behavior were recorded

SEX DETERMINATION (F1 and F2 pups): Yes
- Time schedule: individually on PND 0, 4 (F1 and F2 pups), 14, and 21 (F1 pups)

BODY WEIGHT (F1 and F2 pups): Yes
- Time schedule: individually on PND 1, 4 (before culling) (F1 and F2 pups), 7, 14, and 21 (F1 pups)

PREWEANING DEVELOPMENTAL LANDMARKS (F1 pups):
- Anogenital Distance: all pups on PND 1. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle
- Assessment of areolas/Nipple Anlagen Retention: All males on PND 13

SEXUAL DEVELOPMENT (F1 pups): Yes
- Balanopreputial Separation: at least 3 male F1 pups/litter (where possible), beginning on PND 35
- Vaginal Patency: at least 3 female F1 pups/litter (where possible), beginning on PND 25

THYROID HORMONE ANALYSIS (F1 pups): Yes
Time points: PND 4 and PND 21 (prior to 12:00 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels)
Blood sampling: via cardiac puncture of animals anesthetized by inhalation of isoflurane into tubes without anticoagulants. At PND 4, samples were collected from culled pups and pooled by litter until a total of 10 samples/dosage level were obtained. To the extent possible, samples from the first 10 litters at each dosage level with sufficient numbers of culled pups were used. At PND 21, samples collected from nonselected pups (10/sex/group)
Parameters analysed: Thyroxine (Total T4), Thyroid Stimulating Hormone (TSH)

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE (P and F1 generation)
All surviving animals, including females that failed to deliver, were euthanized by carbon dioxide inhalation, on study day 128-133 (P generation), PND 52 (Cohort 1 surplus), PND 91 (Cohort 1A), PND 132–146 (Cohort 1B males), Lactation day 4 (Cohort 1B females).

GROSS NECROPSY (P and F1 generation)
- Unscheduled: All animals that died on study or were euthanized, underwent necropsy and specified tissues were saved (P and F1 generation).
- Scheduled: All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy (P and F1 generation). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964) (F1 generation).

HISTOPATHOLOGY / ORGAN WEIGHTS (P and F1 generation)
The body weight of each animal euthanized as scheduled was recorded before euthanasia. For these animals, the organs (according to Guideline) were weighed at necropsy. Paired organs were weighed together (except for epididymides and testes). Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated. Tissue collection and preservation according to guideline.

LYMPHOCYTE SUBTYPING (Cohort 1A)
Half of the spleen of 10 males and 10 females per group from Cohort 1A animals were subject to a splenic lymphocyte subpopulation analysis (T lymphocytes, CD4+ and CD8+ T lymphocytes, B lymphocytes, Natural Killer (NK) cells and NKT cells).
The following formula was used in generating the absolute number of different subsets of lymphocytes in the spleen:
Absolute number of cells (x10EXP6 /tissue) = [(whole organ wt.)*(live cell concentration)*(volume of cells)] / partial organ wt. x (cell subset frequency)/100 x 0.000001

Postmortem examinations (offspring):

SACRIFICE
Pups not selected on PND 4 were sacrificed by exsanguination (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital (F1 and F2 generation), and pups not selected on PND 21 by exsanguination (those pups used for blood/thyroid collection) or carbon dioxide inhalation (F1 generation).

GROSS NECROPSY
- Unscheduled: Pups died PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (F1 and F2 generation). Pups died after PND 4 or were euthenized, underwent necropsy and specified tissues were saved (F1 generation).
- Scheduled: On PND 4, one culled pup/sex/litter was subjected to a complete necropsy examination. Pups were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). All remaining culled pups were discarded without examination (F1 and F2 generation). On PND 21, non-selected pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system (F1 generation).

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs (according to Guideline) were weighed at necropsy from 1 non-selected F1 pup/sex/litter on PND 21. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated. Representative samples of the tissues (according to Guideline) were collected from 1 pup/sex/litter on PND 4 of the F2 generation and preserved in 10% neutral buffered formalin. Tissue collection and preservation according to guideline.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals or cages (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit.
Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group by sex.
Male and female mating, fertility, copulation, and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Mean P generation and F1 adult (weekly, gestation, and lactation) and offspring data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-exposed groups to the control group. Mean litter proportions (percent per litter) of postnatal pup survival and pup sexes at birth (percentage of males per litter), percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-exposed groups to the control group. The mean number of ovarian primordial follicles was subjected to a parametric ANOVA test and Dunnett’s test as described above. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels (for details, see section 5 of the study report).

Reproductive indices:

The following parameters were calculated:
Post-implantation loss: (Number of implantation sites - Number of live pups / Number of implantation sites) x 100
Mating index: (Number of mated animals / Number of paired animals) x 100
Fertility index: (Number of pregnant female partners / Number of mated pairs) x 100
Gestation index: (Number of females with live born pups / Number of pregnant females) x 100

Offspring viability indices:

LITTER VIABILITY (F1 and F2 pups)
Mean Live Litter Size = (Total Viable Pups on PND 0) / (No. Litters with Viable Pups on PND 0)
Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = (Sum of (Viable Pups/Litter on PND 0 or PND 4 /No. of Pups Born/Litter)) / (No. of Litters/Group) x 100
Postnatal Survival for All Other Intervals (% Per Litter) = (Sum of (Viable Pups/Litter at End of Interval N/Viable Pups/Litter at Start of Interval N)) / (No. of Litters/Group) x 100
Where N = PND 0–1 and 1–4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No exposure-related clinical findings were noted during the daily examinations. Findings noted in the test substance-exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All P generation animals in the control, low, mid and high groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

WEEKLY
In the high dose group males, lower mean body weight gains were observed generally throughout the entire generation compared to the control group; differences were generally statistically significant. As a result, mean absolute body weights in the high dose group males were statistically significantly lower (up to 11.5% lower) than the control group beginning on Study Day 14 and continuing until euthanasia. The effects on mean body weight and body weight gain in the high dose group males were considered test substance-related and adverse. Mean body weights and body weight gains in the low and mid dose group males were unaffected by test substance exposure and were comparable to the control group throughout the generation. Any statistically significant differences between males and the low and mid dose groups and the control group were transient and did not impact mean absolute body weights.
In the high dose group females, sporadically lower mean body weight gains resulted in a statistically significant lower mean body weight gain compared to the control group when the overall premating period (Study Days 0–70) was evaluated. As a result, mean absolute body weights in the high dose group females were lower (up to 9.3%) than the control group beginning on Study Day 14 and continuing throughout the remainder of the premating period, and were considered test substance-related and adverse. Mean body weights and body weight gains in the mid and high dose group females were comparable to the control group throughout the premating period; statistically significant differences were transient, did not impact mean absolute body weights, and therefore were not considered test substance-related.

GESTATION
In the high dose group, lower mean body weight gains were observed sporadically throughout the gestation period compared to the control group; differences were statistically significant during Gestation Days 0–3 and when the entire gestation period (Gestation Days 0–20) was evaluated. Due to the effects on mean body weights during the premating period, mean absolute body weights in the high dose group females were lower (8.3%) than the control group on Gestation Day 0 and remained lower (up to 10.5%) throughout gestation; differences were statistically significant. The effects on mean body weights in the high dose group during gestation were considered test substance-related and adverse.
Mean absolute body weights and body weight gains in the low and mid dose group females were unaffected by test substance exposure during gestation. Differences from the control group were slight and not statistically significant.

LACTATION
In the high dose group females, higher mean body weight gains or lower mean body weight losses were noted compared to the control group resulting in a statistically significantly higher mean body weight gain when the entire lactation period (Lactation Days 1-21) was evaluated. Mean absolute body weights in the high dose group were 11.3% lower (statistically significant) than the control group on Lactation Day 0 (due to effects noted during the premating and gestation periods) and only 1.2% lower on Lactation Day 21 suggesting an amelioration of the previous effects in this group during lactation.
In the low and mid dose group females, mean body weights and body weight gains were generally comparable to the control group throughout lactation, with the following exception. A statistically significantly higher mean body weight gain was noted in the mid dose group when the entire lactation period (Lactation Days 1–21) was evaluated; but had no impact on mean absolute body weights in this group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

WEEKLY
In the high dose group males, mean food consumption and food efficiency were generally lower than the control group throughout the premating and postmating periods compared to the control group. The differences were often statistically significant and correlated with the effects on mean body weights and body weight gains in this group, and therefore were considered test substance-related and adverse. Mean food consumption in the low and mid dose group males was unaffected by test substance exposure and was comparable to the control group throughout the generation.
In the high dose group females, mean food consumption and food efficiency were generally lower than the control group throughout the premating period. The differences were often statistically significant and correlated with the effects on mean body weights and body weight gains in this group, and therefore were considered test substance-related and adverse. Mean food consumption in the low and mid groups were unaffected by test substance exposure and were generally comparable to the control group throughout the premating period. Any statistically significant differences were transient and not associated with effects on mean body weights or body weight gains.

GESTATION
Test substance-related lower mean food consumption was noted in the high dose group compared to the control group throughout gestation (Gestation Days 0–20); differences were statistically significant and correlated with effects on mean body weights and body weight gains in this group.
In the mid dose group, mean food consumption was sporadically lower compared to the control group but was not associated with effects on mean body weights or body weight gains in this group and therefore, not considered test substance-related. Mean food consumption in the low dose group and mean food efficiency in the low, mid and high groups were comparable to the control group throughout gestation.

LACTATION
P generation mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance exposure during lactation. Differences between the control, low, mid and high groups were slight not statistically significant, with the following exception. Statistically significantly higher mean food efficiency was noted in the mid and high dose groups when the entire lactation period (Lactation Days 1–21) was evaluated.

TEST SUBSTANCE CONSUMPTION
Mean compound consumptions (mg/kg bw/day) were based on theoretical dietary concentrations of the test substance and are presented in Table 1 of the section "Any other information on results incl. tables".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See the section "Food consumption and compound intake (if feeding study)"

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology and coagulation parameters. Statistically significant differences noted in P generation males were minimal in magnitude, not observed in a dose-responsive manner, and/or attributed to individual animal variability (hematology and coagulation parameters), or within the Charles River Ashland historical control range (coagulation parameters).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Dose-dependent higher mean liver alkaline phosphatase (ALP) and sorbitol dehydrogenase (SDH) in both P generation males and females, see Table 3 of the section "Any other information on results incl. tables". The differences in the high dose group were statistically significant, and correlated with increased relative liver weights and microscopic liver findings and were hence considered test substance-related and adverse. There were no other test substance-related effects on serum chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on urinalysis parameters.

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):

The incidence of selected histopathologic findings in the P generation is shown in Table 5 of the section "Any other information on results incl. tables".
Liver findings of minimal to mild necrosis were considered adverse in the high dose group and were characterized by focal to multifocal areas of hepatocyte loss with disrupted cytoplasm integrity focally associated with presence of cell debris and/or mixed inflammatory cell infiltrates. Additional findings included evidence of well-defined areas of hepatocellular cytoplasm with clear round vacuoles; and/or increased incidence of single cell necrosis observed in individual hepatocytes often hyper eosinophilic and with presence of nuclear debris. These findings were present above control levels in males and females in all dose groups apart from single cell necrosis in females for which the increased incidence/severity when compare to control values was limited to the high dose group. They correlated with increased relative liver weights; dose dependent increases in liver alkaline phosphatase (ALP) in males and females and increases in sorbitol dehydrogenase (SDH), also in males and females, mostly observed in the high dose group; and white macroscopic liver discoloration in one individual male in the high dose group.
Additionally, males in the high dose group also had increased incidence of renal intratubular pigment. There were no findings in the reproductive organs, including ovaries and testes evaluation that were attributable to the administration of the test substance.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Two, 3, and 5 males and females in the control, mid and high dose groups, respectively, were suspected of reduced fertility. Reproductive organs were evaluated due to reduced fertility; however, there were no microscopic findings to explain failure to mate, sire, conceive or deliver. Additionally, ovarian follicular counts were performed for these P generation females animals and no significant findings were observed, with the variability present being interpreted to be within the expected range for this age in this strain of rats.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no neoplastic histopathological findings.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean lengths of estrous cycles in the test substance exposed groups were similar to the control group value. There were no statistically significant differences.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test substance related effects were observed on P generation sperm parameters (mean epididymal sperm numbers and sperm motility, progressive motility, and morphology) in males at any dosage concentration. Differences from the control group were slight and were not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

MATING AND FERTILITY DATA
P generation male and female reproductive parameters are presented in Table 2 of the section "Any other information on results incl. tables".
No test substance related effects on P generation reproductive performance were observed at any exposure concentration. No statistically significant differences were noted between the control and test substance exposed groups. Two, 3, and 5 mating pair(s) in the control, mid and high dose groups, respectively, did not produce a litter. There were no macroscopic or microscopic findings to explain the failure to mate, conceive, sire, or deliver.
The mean numbers of days between pairing and coitus in the test substance exposed groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
No test substance related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean P generation gestation lengths in the test substance exposed groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the low, mid and high dose groups were 21.6, 21.8, and 21.6 days, respectively, compared to mean gestation lengths of 21.9 days in the concurrent control group and 21.8 days in the Charles River Ashland historical control data. No signs of dystocia were noted at any exposure level.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity (bodyweight loss and systemic toxicity)

Effect level:

>= 1 500 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive/ developmental toxicity

Effect level:

>= 4 500 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

no effects observed

Description (incidence and severity):

F1 GENERATION FOLLOWING WEANING
No test substance related clinical findings were noted during the generation at the daily examinations. Findings noted in the test substance exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

F1 GENERATION FOLLOWING WEANING
There were no test substance-related effects on survival at any exposure level. One and 2 females in the control and low dose groups, respectively, were found dead or euthanized in extremis during the F1 generation following weaning. In the low dose group, one female was found dead on PND 68 with no significant clinical observations noted prior to death and no recorded gross observations at necropsy. Notable microscopic findings for this animal included minimal lung edema and hemorrhage, presence of minimal pigment in the Harderian gland and minimal decreased spleen marginal zone cellularity, however the cause of death was not determined based on the histopathological evaluation. One female (Cohort 1B) was euthanized in extremis on Gestation Day 9 due to a large, scabbed mass on the ventral trunk that correlated microscopically with an ulcerated mammary adenocarcinoma characterized by capsule invasion and presence of intratumoral necrosis and hemorrhage; 15 early resorptions were noted in the uterus. These deaths in the low dose group were not considered test substance-related because there was no mortality or moribundity for F1 animals in the mid and high dose groups. In the control group, one female was found dead on PND 104 in the absence of remarkable clinical observations or effects on body weight or food consumption. At necropsy, this female was noted with red fluid in the thoracic cavity; the cause of death was not determined based on gross or histopathological evaluation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION FOLLOWING WEANING
At the time of weaning, on PND 21, mean absolute body weights for males and females in the high dose group were 15.0% and 13.8% lower, respectively than the control group; differences were generally statistically significant.
For F1 males in the high dose group, lower mean body weight gains were noted sporadically throughout the generation when compared to the control group; differences were occasionally statistically significant. As a result, mean absolute body weights for males in this group remained 7.4% to 14.4% lower (statistically significant) than the control group throughout the generation; these differences were considered test substance related and adverse. In contrast, mean body gains for F1 females in the high dose group were generally comparable to the control group during the generation (PND 21-90) although statistically significantly lower mean absolute body weights compared to the control group were noted during PND 21–35; these differences were considered to be a continuation of the body weight effects noted during the pre-weaning period. Mean body weight gains for females in this group were generally comparable to the control group throughout the postweaning period.
In the mid and high dose group F1 males, sporadically lower mean body weight gains were noted throughout the generation when compared to the control group, resulting in lower (up to 5.9% and 6.7%, respectively) mean absolute body weights; differences were occasionally statistically significant, however, these effects were considered non-adverse due to the minimal magnitude of the change. Mean body weights and body weight gains for F1 females in the low and high dose groups were similar to the control group throughout the generation; statistically significant changes were transient and not considered test substance-related.

MATERNAL BODY WEIGHT
COHORT 1B: F1 mean maternal body weights and body weight gains were unaffected by test substance exposure during gestation and during lactation. Any statistically significant differences between the control, low, mid and high dose groups were transient and/or not dose-responsive.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION FOLLOWING WEANING
WEEKLY
In the high dose group F1 males, statistically significantly lower mean food consumption was noted compared to the control group throughout the generation. In the low and mid dose group F1 males, sporadically lower mean food consumption was noted compared to the control group. The lower mean food consumption in the low, mid and high dose group males correlated with the effects on body weight and body weight gain in these groups and was considered test substance-related at all exposure levels and adverse only in the high dose group, given the minimal magnitude of the change in the low and mid dose groups. Mean food efficiency in these groups was generally comparable to the control group. Statistically significant differences in the high dose group were attributed to the effects on mean body weights.
In the F1 females, mean food consumption and food efficiency in the low, mid and high dose groups were generally comparable to the control group throughout the post-weaning period. Statistically significant differences were transient and not considered test substance-related.

TEST SUBSTANCE CONSUMPTION
Mean compound consumptions (mg/kg bw/day) were based on theoretical dietary concentrations of the test substance and are presented in Table 6 of the section "Any other information on results incl. tables".

COHORT 1B
WEEKLY
F1 male mean food consumption during the postmating period (PND 119–140) was lower in the low, mid and high dose groups compared to the control group; differences were generally statistically significant and corresponded with the effects on mean body weight and body weight gains noted in these groups. Mean food efficiency for F1 males in the test substance-exposed group was comparable to or slightly higher than the control group throughout the postmating period.

GESTATION AND LACTATION
F1 mean maternal food consumption, evaluated as g/animal/ day, and food efficiency were unaffected by test substance exposure during gestation and lactation. Any statistically significant differences between the control, low, mid and high dose groups were transient and/or not dose-responsive.

TEST SUBSTANCE CONSUMPTION
Mean compound consumptions (mg/kg bw/day) were based on theoretical dietary concentrations of the test substance and are presented in Table 8 of the section "Any other information on results incl. tables".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See the section "Food consumption and compound intake (if feeding study)"

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

COHORT 1A
There were no test substance-related effects on hematology and coagulation parameters. Differences from controls were slight, not statistically significant, and/or did not occur in a clear dose responsive manner.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A
Test substance-related serum chemistry values of Cohort 1A are presented in Table 7 of the section "Any other information on results incl. tables". Statistically significantly higher levels of alanine aminotransferase (ALT) in F1 males and alkaline phosphatase (ALP) in F1 males and females in the high dose group correlated with increased liver weights and microscopic liver findings noted in this group. There were no other test substance-related effects on serum chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

COHORT 1A
There were no test substance-related effects on urinalysis parameters.

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Description (incidence and severity):

COHORT 1A
There were no test substance-related splenic immunophenotyping changes noted at any dose level.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A
Test substance-related organ weight changes of Cohort 1A are presented in Table 10 of the section "Any other information on results incl. tables". Higher mean liver weights (relative to final body weight in males and absolute and relative for females) were noted in the high dose group. Some organ weight differences, including higher mean relative to final body weight male adrenal, brain, epididymides, cauda epididymides, and testes weights; lower absolute and/or relative to brain heart, thyroid/parathyroid, and kidney weights, that were statistically significant when compared to the control group were considered to be a result of a test substance-related effect on final body weight.

COHORT1B
For Cohort 1B, some organ weight differences, including higher mean relative to final body weight seminal vesicles and testes; lower absolute and/or relative-to-body epididymides and pituitary weights observed in males, that were statistically significant when compared to the control group, were considered to be a result of a test substance-related effect on final body weight.
There were no other test substance-related effects on organ weights. However, some statistically significant differences were observed when the control and test substance-exposed groups were compared.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

COHORT 1A
Review of the gross necropsy observations for Cohort 1A revealed no observations that were considered to be associated with administration of the test substance.

COHORT 1B
One male in Cohort 1B in the mid dose group had a white area in the liver, however microscopic evaluation was not performed for this cohort and its relationship to treatment cannot be ascertained. There were no test substance-related macroscopic findings in the Cohort 1 surplus animals. For Cohort 1B, no test substance-related effects were observed on the number of pups born, the number of corpora lutea, the number of former implantation sites, and the number of unaccounted-for sites. The differences between the control and test substance-exposed groups were slight and not statistically significant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A
Incidence of selected histopathologic findings of Cohort 1A are presented in Table 11 of the section "Any other information on results incl. tables". Liver findings above control levels were observed in Cohort 1A males in the high dose group and consisted of minimal focal necrosis and increased incidence of single cell necrosis. Potentially associated to test substance was evidence of hepatocellular vacuolation in males, because a similar incidence was observed in P generation males for this finding. Additionally, one F1 female in the high dose group also had evidence of hepatocellular necrosis, however a similar incidence for this change was also observed in 1 control female from the P generation, and therefore a relationship to treatment cannot be assumed. These changes correlate with increased liver weights, and increased liver ALT (males) and ALP (males and females) in the high dose group.

One male in the high dose group had multi-organ evidence of myelomonocytic leukemia that affected the liver, spleen, meninges, and mandibular lymph node and correlated with macroscopic findings of swollen and enlarged liver, swollen and enlarged spleen with white areas, and thickened and red discolored meninges. This isolated neoplasm was considered incidental and unrelated to treatment with the test substance.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no neoplastic histopathological findings.

Other effects:

no effects observed

Description (incidence and severity):

BALANOPREPUTIAL SEPARATION
Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of balanopreputial separation were 43.3, 43.2, and 43.7 days in the low, mid and high dose groups, respectively, when compared to 43.0 days in the control group. Mean body weights at the age of attainment were 236.7 g, 236.4 g, and 226.2 g in the same respective groups compared to 239.3 g in the control group. None of the differences from the control group were statistically significant.

VAGINAL PATENCY
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of vaginal patency were 35.1, 34.5, and 35.6 days in the low, mid and high dose groups, respectively, when compared to 33.9 days in the control group. Mean body weights at the age of attainment were 141.4 g, 136.8 g, and 136.8 g in the same respective groups compared to 134.4 g in the control group. The mean age of attainment for the high dose group was slightly higher (not statistically significant) than the concurrent control group. However, the value was within the range of Charles River Ashland historical control data and the mean body weight on day of attainment was similar to the control group indicating that the delay was likely secondary to the body weight effects in this group around this time.

OVARIAN FOLLICLE COUNTS
Cohort 1A: There were no test substance-related effects on ovarian follicle counts in the control and high dose group females. Any variation in counts between those groups was due to normal biological variability.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Cohort 1A
The mean ages at the first occurrence of estrus in the low, mid and high dose groups (38.1, 37.3, and 37.4 days, respectively) were generally comparable to the control group (37.7 days). The duration from vaginal opening to first estrus in these same respective groups (4.5, 3.9, and 2.5 days) was slightly lower compared to the control group (4.6 days) likely due to the slightly higher age of attainment of vaginal opening in these groups. None of the differences were statistically significant.
The mean lengths of estrous cycles in the test substance-treated groups from PND 75-91 and the percentage of females cycling were comparable to the control group. None of the differences were statistically significant.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

COHORT 1A
No exposure related effects were observed on F1 sperm parameters (mean epididymal sperm numbers and sperm motility, progressive motility, and morphology) in males at any exposure concentration. Differences from the control group were slight and were not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
F1 male and female reproductive parameters are presented in Table 9 of the section "Any other information on results incl. tables".
No test substance related effects on F1 reproductive performance were observed at any exposure concentration. No statistically significant differences were noted between the control and test substance exposed groups. One, 2 , 2, and 1 mating pair(s) in the control, low, mid, and high dose groups, respectively, did not produce a litter.
The mean numbers of days between pairing and coitus in the test substance exposed groups were similar to the control group value. The mean lengths of estrous cycles in these groups were slightly higher than the concurrent control group; however, the differences did not occur in an dose responsive manner, and therefore were not considered test substance related. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
No test substance related effects were noted on mean gestation lengths or the process of parturition at any exposure concentration. Mean F1 gestation lengths in the test substance exposed groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the low, mid and high dose groups were 21.6, 21.6, and 21.5 days, respectively, compared to mean gestation lengths of 21.6 days in the concurrent control group and 21.8 days in the Charles River Ashland historical control data. No signs of dystocia were noted at any exposure concentration.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

1 500 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive/developmental toxicity

Effect level:

4 500 ppm (nominal)

Based on:

test mat.

Remarks:

equivalent to 300 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 PUPS
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance exposure.

F1 GENERATION FOLLOWING WEANING
No test substance related clinical findings were noted during the generation at the daily examinations. Findings noted in the test substance exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure related.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

LITTER SIZE
The mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–14, 14–21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection)–21 were unaffected by the test substance at all dosage concentrations. Differences from the control group were slight, were not statistically significant, and/or did not occur in an exposure related manner.

UNSCHEDULED DEATHS F1 PUPS
Six pups (from 5 litters) in the control group, 7 pups (from 7 litters) in the low dose group, 10 pups (from 5 litters) in the mid dose group, and 5 pups (from 2 litters) in the high dose group, were found dead or euthanized in extremis; the euthanasia of 1 control group pup was due to a mechanical injury. Two pups (from 2 litters), 5 pups (from 2 litters), 7 pups (from 6 litters), and 2 pups (from 2 litters) in the same respective groups were missing and presumed to have been cannibalized.

F1 GENERATION FOLLOWING WEANING
There were no test substance-related effects on survival at any exposure level. One and 2 females in the control and low dose groups, respectively, were found dead or euthanized in extremis during the F1 generation following weaning. In the low dose group, one female was found dead on PND 68 with no significant clinical observations noted prior to death and no recorded gross observations at necropsy. Notable microscopic findings for this animal included minimal lung edema and hemorrhage, presence of minimal pigment in the Harderian gland and minimal decreased spleen marginal zone cellularity, however the cause of death was not determined based on the histopathological evaluation. One female (Cohort 1B) was euthanized in extremis on Gestation Day 9 due to a large, scabbed mass on the ventral trunk that correlated microscopically with an ulcerated mammary adenocarcinoma characterized by capsule invasion and presence of intratumoral necrosis and hemorrhage; 15 early resorptions were noted in the uterus. These deaths in the low dose group were not considered test substance-related because there was no mortality or moribundity for F1 animals in the mid and high dose groups. In the control group, one female was found dead on PND 104 in the absence of remarkable clinical observations or effects on body weight or food consumption. At necropsy, this female was noted with red fluid in the thoracic cavity; the cause of death was not determined based on gross or histopathological evaluation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 PUPS
Mean male and female birth weights (PND 1) in all test substance-exposed groups were similar to the control group. Lower mean body weight gains were noted in the high dose male and female pups throughout the pre-weaning period (PND 1–21); changes were statistically significant during PND 4–21. As a result, mean absolute body weights for males and females in this group were 8.4% to 14.8% and 8.1% to 14.2% lower, respectively than the control group during PND 7–21; differences were generally statistically significant. These changes were considered test substance-related and adverse.
Mean body weights and body weight gains in the low and mid dose group male and female pups were comparable to the control group throughout the pre-weaning period (PND 1–21).

F1 GENERATION FOLLOWING WEANING
At the time of weaning, on PND 21, mean absolute body weights for males and females in the high dose group were 15.0% and 13.8% lower, respectively than the control group; differences were generally statistically significant.
For F1 males in the high dose group, lower mean body weight gains were noted sporadically throughout the generation when compared to the control group; differences were occasionally statistically significant. As a result, mean absolute body weights for males in this group remained 7.4% to 14.4% lower (statistically significant) than the control group throughout the generation; these differences were considered test substance related and adverse. In contrast, mean body gains for F1 females in the high dose group were generally comparable to the control group during the generation (PND 21-90) although statistically significantly lower mean absolute body weights compared to the control group were noted during PND 21–35; these differences were considered to be a continuation of the body weight effects noted during the pre-weaning period. Mean body weight gains for females in this group were generally comparable to the control group throughout the postweaning period.
In the mid and high dose group F1 males, sporadically lower mean body weight gains were noted throughout the generation when compared to the control group, resulting in lower (up to 5.9% and 6.7%, respectively) mean absolute body weights; differences were occasionally statistically significant, however, these effects were considered non-adverse due to the minimal magnitude of the change. Mean body weights and body weight gains for F1 females in the low and high dose groups were similar to the control group throughout the generation; statistically significant changes were transient and not considered test substance-related.

MATERNAL BODY WEIGHT
COHORT 1B
F1 mean maternal body weights and body weight gains were unaffected by test substance exposure during gestation and during lactation. Any statistically significant differences between the control, low, mid and high dose groups were transient and/or not dose-responsive.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION FOLLOWING WEANING
WEEKLY
In the high dose group F1 males, statistically significantly lower mean food consumption was noted compared to the control group throughout the generation. In the low and mid dose group F1 males, sporadically lower mean food consumption was noted compared to the control group. The lower mean food consumption in the low, mid and high dose group males correlated with the effects on body weight and body weight gain in these groups and was considered test substance-related at all exposure levels and adverse only in the high dose group, given the minimal magnitude of the change in the low and mid dose groups. Mean food efficiency in these groups was generally comparable to the control group. Statistically significant differences in the high dose group were attributed to the effects on mean body weights.
In the F1 females, mean food consumption and food efficiency in the low, mid and high dose groups were generally comparable to the control group throughout the post-weaning period. Statistically significant differences were transient and not considered test substance-related.

TEST SUBSTANCE CONSUMPTION
Mean compound consumptions (mg/kg bw/day) were based on theoretical dietary concentrations of the test substance and are presented in Table 6 of the section "Any other information on results incl. tables".

COHORT 1B
WEEKLY
F1 male mean food consumption during the postmating period (PND 119–140) was lower in the low, mid and high dose groups compared to the control group; differences were generally statistically significant and corresponded with the effects on mean body weight and body weight gains noted in these groups. Mean food efficiency for F1 males in the test substance-exposed group was comparable to or slightly higher than the control group throughout the postmating period.

GESTATION AND LACTATION
F1 mean maternal food consumption, evaluated as g/animal/ day, and food efficiency were unaffected by test substance exposure during gestation and lactation. Any statistically significant differences between the control, low, mid and high dose groups were transient and/or not dose-responsive.

TEST SUBSTANCE CONSUMPTION
Mean compound consumptions (mg/kg bw/day) were based on theoretical dietary concentrations of the test substance and are presented in Table 8 of the section "Any other information on results incl. tables".

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

See section "Food consumption and compound intake (if feeding study)".

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

COHORT 1A
There were no test substance-related effects on hematology and coagulation parameters. Differences from controls were slight, not statistically significant, and/or did not occur in a clear dose responsive manner.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A
Test substance-related serum chemistry values of Cohort 1A are presented in Table 7 of the section "Any other information on results incl. tables". Statistically significantly higher levels of alanine aminotransferase (ALT) in F1 males and alkaline phosphatase (ALP) in F1 males and females in the high dose group correlated with increased liver weights and microscopic liver findings noted in this group. There were no other test substance-related effects on serum chemistry parameters.

Urinalysis findings:

no effects observed

Description (incidence and severity):

COHORT 1A
There were no test substance-related effects on urinalysis parameters.

Sexual maturation:

no effects observed

Description (incidence and severity):

BALANOPREPUTIAL SEPARATION
Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of balanopreputial separation were 43.3, 43.2, and 43.7 days in the low, mid and high dose groups, respectively, when compared to 43.0 days in the control group. Mean body weights at the age of attainment were 236.7 g, 236.4 g, and 226.2 g in the same respective groups compared to 239.3 g in the control group. None of the differences from the control group were statistically significant.

VAGINAL PATENCY
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of vaginal patency were 35.1, 34.5, and 35.6 days in the low, mid and high dose groups, respectively, when compared to 33.9 days in the control group. Mean body weights at the age of attainment were 141.4 g, 136.8 g, and 136.8 g in the same respective groups compared to 134.4 g in the control group. The mean age of attainment for the high dose group was slightly higher (not statistically significant) than the concurrent control group. However, the value was within the range of Charles River Ashland historical control data and the mean body weight on day of attainment was similar to the control group indicating that the delay was likely secondary to the body weight effects in this group around this time.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Mean anogenital distances (absolute and relative to the cube root of pup body weight) in the low, mid and high dose groups were unaffected by parental test substance administration when evaluated on PND 1. Differences between the test substance-exposed groups and the control group were slight and not statistically significant, with the following exception. A statistically significantly lower mean absolute anogenital distance was noted in the high dose group males compared to the control group. However, there was no difference in the mean anogenital distance relative to cube root of body weight in this group and the absolute anogenital distance was within the Charles River Ashland historical control data range and therefore, the difference was not considered test substance-related.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen retention in F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. No nipples were present for males in any group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 PUPS
No direct test substance related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for F1 pups on PND 21 at any exposure level. Statistically significant differences were attributed to the lower mean absolute body weights in the high dose group when compared to the control group or did not occur in a dose related manner.

COHORT 1A
Test substance-related organ weight changes of Cohort 1A are presented in Table 10 of the section "Any other information on results incl. tables". Higher mean liver weights (relative to final body weight in males and absolute and relative for females) were noted in the high dose group. Some organ weight differences, including higher mean relative to final body weight male adrenal, brain, epididymides, cauda epididymides, and testes weights; lower absolute and/or relative to brain heart, thyroid/parathyroid, and kidney weights, that were statistically significant when compared to the control group were considered to be a result of a test substance-related effect on final body weight.

COHORT1B
For Cohort 1B, some organ weight differences, including higher mean relative to final body weight seminal vesicles and testes; lower absolute and/or relative-to-body epididymides and pituitary weights observed in males, that were statistically significant when compared to the control group, were considered to be a result of a test substance-related effect on final body weight.
There were no other test substance-related effects on organ weights. However, some statistically significant differences were observed when the control and test substance-exposed groups were compared.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

UNSCHEDULED DEATHS
- F1 pups: No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead or euthanized in extremis. Aside from the presence or absence of milk in the stomach, 1 pup in the control group was noted with a laceration and 1 pup was noted with fractured parietal, parietal suture, and parietal interparietal suture with red contents surrounding the fracture. No other internal findings were noted.

SCHEDULED SACRIFICES
- F1 pups: No internal findings at the necropsy of culled pups euthanized on PND 4 nor on PND 21. At the PND 21 necropsy of F1 weanlings selected for organ weights, no internal findings could be attributed to parental test substance exposure. Internal findings included a small thymus for one pup in the control group and one pup in the mid dose group, and a small testes in one pup in the low dose group. No other internal findings were noted.
- Cohort 1A: No observations that were considered to be associated with administration of the test substance.
- Cohort 1B: One male in Cohort 1B in the mid dose group had a white area in the liver, however microscopic evaluation was not performed for this cohort and its relationship to treatment cannot be ascertained. There were no test substance-related macroscopic findings in the Cohort 1 surplus animals. For Cohort 1B, no test substance-related effects were observed on the number of pups born, the number of corpora lutea, the number of former implantation sites, and the number of unaccounted-for sites. The differences between the control and test substance-exposed groups were slight and not statistically significant.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A
Incidence of selected histopathologic findings of Cohort 1A are presented in Table 11 of the section "Any other information on results incl. tables". Liver findings above control levels were observed in Cohort 1A males in the high dose group and consisted of minimal focal necrosis and increased incidence of single cell necrosis. Potentially associated to test substance was evidence of hepatocellular vacuolation in males, because a similar incidence was observed in P generation males for this finding. Additionally, one F1 female in the high dose group also had evidence of hepatocellular necrosis, however a similar incidence for this change was also observed in 1 control female from the P generation, and therefore a relationship to treatment cannot be assumed. These changes correlate with increased liver weights, and increased liver ALT (males) and ALP (males and females) in the high dose group.

One male in the high dose group had multi-organ evidence of myelomonocytic leukemia that affected the liver, spleen, meninges, and mandibular lymph node and correlated with macroscopic findings of swollen and enlarged liver, swollen and enlarged spleen with white areas, and thickened and red discolored meninges. This isolated neoplasm was considered incidental and unrelated to treatment with the test substance.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There were no test substance-related effects on ovarian follicle counts in F1 females from Cohort 1A in the control and high dose group females. Any variation in counts between those groups was due to normal biological variability.

Cohort 1B: organs and tissues processed and retained were not subjected to histppathology as there were no equivocal results from the other Cohort, and the test substance is not suspected of being a reproductive toxicant or endocrine disruptor, as per Guideline 443.

Similarly, in response to the ECHA review document recently published, there are no significant treatment-related changes in the low and mid dose level groups and therefore full histopathology was not required.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

THYROID HORMONE ANAYLYSIS
- Pups: There were no test substance-related effects on thyroid hormones in PND 4 culled pups nor in PND 21 pups selected for hormone analysis. Differences from controls were slight, did not achieve statistical significance and were not noted in a dose dependent manner.
- Cohort 1A: There were no test substance-related effects on thyroid hormones. A statistically significantly lower mean T4 value was noted in the high dose group F1 males compared to the control group however, there were no corresponding effects in F1 females, TSH levels, or P generation males in the high dose group, and therefore, this difference was attributed to normal animal variability.

IMMUNOLOGICAL FINDINGS
- Cohort 1A: There were no test substance-related splenic immunophenotyping changes noted at any dose level.

OVARIAN FOLLICLE COUNTS
- Cohort 1A: There were no test substance-related effects on ovarian follicle counts in the control and high dose group females. Any variation in counts between those groups was due to normal biological variability.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity and Neonatal toxicity

Generation:

F1

Effect level:

1 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive/ developmental toxicity

Generation:

F1 (cohort 1B)

Effect level:

4 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4 500 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical findings) of all F2 pups in this study was unaffected by test substance exposure.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

LITTER SIZE
The mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4, and from birth to PND 4 were unaffected by the test substance at all exposure concentrations. Differences from the control group were slight, were not statistically significant, and/or did not occur in an exposure related manner.

UNSCHEDULED DEATHS
Five pups (from 4 litters) in the control group, 12 pups (from 5 litters) in the low dose group, 7 pups (from 5 litters) in the mid dose group, and 6 pups (from 5 litters) in the high dose group, were found dead. Two pups (from 2 litters), 3 pups (from 1 litter), 2 pups (from 2 litters), and 0 pups in the same respective groups were missing and presumed to have been cannibalized.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean F2 male and female birth weights (PND 1) were comparable across all groups. Lower mean body gains were noted in the high dose group F2 male and female pups compared to the control group during PND 1–4; the difference was statistically significant for males. As a result, mean absolute pup body weights for males and females in this group were 9.2% and 3.9% lower, respectively, than the control group on PND 4; the differences were not statistically significant. The effects in the high dose group were considered test substance related but not adverse due to different patterns being seen in the F1 and F2 generations and between males and females in this generation.

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:

effects observed, non-treatment-related

Description (incidence and severity):

UNSCHEDULED DEATHS
No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

SCHEDULED SACRIFICES
No internal findings that could be attributed to parental exposure with the test substance were noted at the necropsy of pups euthanized on PND 4. A single pup in the mid dose group was observed with an accessory liver lobule at necropsy. No other internal findings were noted.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Generation:

F2

Effect level:

4 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS

Homogeneity assessment of the diet done at the start of the study for all dose groups showed mean concentrations ranging from 74.2 to 89.0%, which was within the acceptability range of 70% to 110% of theoretical concentration. The relative standard deviation RSD) of the mean concentration ranged from 1.9 to 4.7% (back-up sample of the high dose level), which met the acceptability criterion of being ≤ 10%. The diet sample analysed for homogeneity of the F1 generation showed a mean concentration of 100% and an RSD of 3.8% for the females rodent meal, and 101% and 2.1% for the males rodent meal, thereby meeting the acceptability criteria.

The analyzed diet admix formulations used for test substance administration met the applicable protocol-specified acceptance criteria for test substance concentration acceptability, with six exceptions. Of these exceptions, back-up samples were analysed, and overall mean concentrations were measured of 67.8%, 121%, 126%, 114%, 120%, and 114% of target. The analyses of the back-up samples confirmed the initial analysis. No cause or error could be found. The out of range formulation results did not negatively impact the objectives of the study as the deviations were minimally outside of acceptance criteria. In addition, the above-range results noted in the low- and mid-dose formulations did not result in unexpected toxicity in these groups or impact establishment of appropriate NOAELs based on the objectives. 

The 8-day room temperature stability assessment performed for 4 concentrations showed that after 8 days the overall percent of time zero concentrations were ranging between 92.2 and 121%, thereby showing the substance is stable in the test diet for 8 days at room temperature. 

 

Table 1: Mean Calculated Test Substance Consumption (mg/kg bw/day^a) – P Generation

Dietary Concentration Group (ppm)	Mean Test Substance Consumption (mg/kg bw/day)
	Males		Females
	Prior to Mating	After Mating	Prior to Mating	Gestation^b	Lactation^b
500	32	22.8	34.8	26.9	27.5
1500	94.9	67.8	101	88.1	95.5
4500	279.7	203.7	295.5	260.9	281.8

^a Summation: (Mean test substance consumption for the specified interval)/(Number of days or intervals assessed).

^b Food consumption generally increases during gestation and lactation due to the increased caloric demands of milk production and direct consumption of diet by offspring in the latter portion of the lactation period. To compensate for the increased food consumption during gestation and lactation, the dietary concentrations of test substance were adjusted, based on historical control food consumption and body weight data, to maintain the target doses in the treated groups.

 

Table 2: Results of P generation Reproductive Performance

Parameter	Dietary Concentration (ppm)				CRL HC^a Mean (Range)
	0	500	1500	4500
Male Mating Index (%)	100.0	100.0	100.0	96.0	98.5 (93.3–100.0)
Female Mating Index (%)	100.0	100.0	100.0	96.0	98.5 (93.3–100.0)
Male Fertility Index (%)	92.0	100.0	88.0	80.0	93.9 (80.0–100.0)
Female Fertility Index (%)	92.0	100.0	88.0	80.0	94.1 (80.0–100.0)
Male Copulation Index (%)	92.0	100.0	88.0	83.3	95.3 (80.0–100.0)
Female Conception Index (%)	92.0	100.0	88.0	83.3	95.6 (80.0–100.0)
Estrous Cycle Length (days)	4.0	4.3	4.5	4.2	4.1 (3.9–4.4)
Pre-Coital Interval (days)	2.6	3.0	3.1	3.0	2.8 (2.3–3.4)

^a Charles River Ashland historical control data (version 2021.01).

 

Table 3: Test Substance-Related Serum Chemistry Values – P Generation

	Males				Females
Group	1	2	3	4	1	2	3	4
Dietary Concentration (ppm)	0	500	1500	4500	0	500	1500	4500
ALP (U/L)	90	106	123	185**	66	62	89	100**
SDH (U/L)	2	7	6	9	7	4	8	11

U/L = International unit/liter

* Based upon statistical analysis of group means, values are significantly different from control group – p<0.05; refer to data tables for actual significance levels and tests used. (**= significantly different from the control group at 0.01).

 

Table 4: Test Substance-Related Organ Weight Changes – P Generation

	Males				Females
Group	1	2	3	4	1	2	3	4
Dietary Concentration (ppm)	0	500	1500	4500	0	500	1500	4500
No. Animals per Group	25	25	25	25	25	25	25	25
Liver (No. Weighed) (g)	(25)	(25)	(25)	(25)	(25)	(25)	(25)	(25)
Absolute	18.39	19.44	20.03	18.39	10.61	10.69	11.24	11.03
% difference from control	-	5.7	8.9	0	-	0.8	5.9	4.0
Relative to Final Body Weight	2.905	3.118*	3.179**	3.256**	3.463	3.589	3.781*	3.954**
Relative to Brain Weight	826.7	877.5	905.6	840.1	535.7	534.2	564.5	552.6

* Based upon statistical analysis of group means, values are significantly different from control group – p<0.05; refer to data tables for actual significance levels and tests used (*=significantly different from the control group at 0.05, **= significantly different from the control group at 0.01).

 

Table 5: Incidence of Selected Histopathologic Findings: P Generation

	Males				Females
Group	1	2	3	4	1	2	3	4
Dietary Concentration (ppm)	0	500	1500	4500	0	500	1500	4500
No. Animals per Group	25	25	25	25	25	25	25	25
Liver	25	25	25	25	25	25	25	25
Necrosis	(0)^a	(2)	(0)	(3)	(1)	(2)	(2)	(2)
Minimal	0	2	0	2	1	2	2	1
Mild	0	0	0	1	0	0	0	1
Vacuolation	(2)	(5)	(2)	(6)	(2)	(3)	(2)	(3)
Minimal	1	2	1	5	1	0	1	0
Mild	1	3	1	1	1	3	1	3
Single cell Necrosis	(1)	(5)	(7)	(6)	(3)	(3)	(3)	(4)
Minimal	1	5	7	6	3	3	3	3
Mild	0	0	0	0	0	0	0	1

^a Number of tissues examined from each group.

 

Table 6: Mean Calculated Test Substance Consumption (mg/kg bw/day^a,b) – F₁ Generation

Dietary Concentration Group (ppm)	Mean Test Substance Consumption (mg/kg bw/day)
	Males PND 21–90	Females PND 21–84
500	31.5	31.8
1500	96.7	99.8
4500	292.7	302.8

^a Summation: (Mean test substance consumption for the specified interval)/(Number of days or intervals assessed).

^b During the postweaning period, to compensate for the higher food efficiency and growth rates, dietary concentrations of the test substance were adjusted across all treated groups, beginning on PND 21, and continuing through PND 49, based on historical control food consumption and body weight data.

 

Table 7: Test Substance-Related Serum Chemistry Values – F₁ Generation

	Males				Females
Group	1	2	3	4	1	2	3	4
Dietary Concentration (ppm)	0	500	1500	4500	0	500	1500	4500
ALP (U/L)	145	144	173	285**	106	91	104	218**
ALT (U/L)	42	38	44	50*	38	31	33	38

U/L = International unit/liter

*     Based upon statistical analysis of group means, values are significantly different from control group – p<0.05; refer to data tables for actual significance levels and tests used. (* = significantly different from the control group at 0.05; **= significantly different from the control group at 0.01)

 

Table 8: Mean Calculated Test Substance Consumption (mg/kg bw/day^a) – Cohort 1B

Dietary Concentration Group (ppm)	Mean Test Substance Consumption (mg/kg bw/day)
	Males	Females
	After Mating	Gestation^b	Lactation^b
500	22	26.6	23
1500	69.3	86.3	79
4500	215.3	255	238

^a   Summation: (Mean test substance consumption for the specified interval)/(Number of days or intervals assessed).

^b  Food consumption generally increases during gestation and also during lactation due to the increased caloric demands of milk production. To compensate for increased food consumption during gestation and lactation, the dietary concentrations of test substance were adjusted, based on historical control food consumption and body weight data, to maintain the target doses in the treated groups.

 

Table 9: Results of F₁ Reproductive Performance

Parameter	Dosage Level (ppm)				CRL HC^a Mean (Range)
	0	500	1500	4500
Male Mating Index (%)	94.7	100.0	94.7	100.0	98.5 (93.3–100.0)
Female Mating Index (%)	94.7	100.0	95.0	100.0	98.5 (93.3–100.0)
Male Fertility Index (%)	94.7	90.0	94.7	94.7	93.9 (80.0–100.0)
Female Fertility Index (%)	94.7	90.0	90.0	95.0	94.1 (80.0–100.0)
Male Copulation Index (%)	100.0	90.0	94.4	94.7	95.3 (80.0–100.0)
Female Conception Index (%)	100.0	90.0	94.7	95.0	95.6 (80.0–100.0)
Estrous Cycle Length (days)	4.2	4.5	5.0	4.7	4.1 (3.9–4.4)
Pre-Coital Interval (days)	3.4	3.1	2.8	3.3	2.8 (2.3–3.4)

^a Charles River Ashland historical control data.

 

Table 10: Test Substance0Related Organ Weight Changes – Cohort 1A

	Males				Females
Group	1	2	3	4	1	2	3	4
Dietary Concentration (ppm)	0	500	1500	4500	0	500	1500	4500
No. Animals per Group	20	20	20	19	20	20	20	20
Liver (No. Weighed) (g)	(20)	(20)	(20)	(19)	(20)	(20)	(20)	(20)
Absolute	19.54	19.32	18.79	19.03	9.44	10.09	10.00	10.72**
% difference from control	-	-1.1	-3.8	-2.6	-	6.9	5.9	13.6
Relative to Final Body Weight	3.824	3.773	3.736	4.172	3.546	3.640	3.807	4.031**
Relative to Brain Weight	891.5	886.1	848.8	880.7	469.2	531.5	495.7	536.1*

* Based upon statistical analysis of group means, values are significantly different from control group – p<0.05; refer to data tables for actual significance levels and tests used. (*=significantly different from the control group at 0.05, **= significantly different from the control group at 0.01)

 

Table 11: Incidence of Selected Histopathologic Findings: F₁ Generation

	Males				Females
Group	1	2	3	4	1	2	3	4
Dietary Concentration (ppm)	0	500	1500	4500	0	500	1500	4500
No. Animals per Group	20	20	20	19	20	20	20	20
Liver	20	20	20	19	20	20	20	20
Necrosis	(0)^a	(0)	(0)	(1)	(0)^a	(1)	(0)	(1)
Minimal	0	0	0	1	0	1	0	1
Vacuolation	(0)	(2)	(3)	(1)	(2)	(1)	(0)	(1)
Minimal	0	1	2	1	0	0	0	0
Mild	0	1	1	0	2	1	0	1
Single cell Necrosis	(3)	(2)	(3)	(8)	(3)	(1)	(2)	(2)
Minimal	3	2	3	8	3	1	2	2

^a Number of tissues examined from each group.

 

 

Applicant's summary and conclusion

Conclusions:

An dietary Extended One-Generation Reproductive Toxicity Study (EOGRTS) was performed with test item EXP1503090, in rats. The study was performed in compliance with GLP and according to OECD Guideline No. 443.
The test item was administered daily via the diet at dose levels of 500, 1500 and 4500 ppm to sexually-mature male and female rats (parental (P) generation), continuously from 10 weeks before mating and through mating, gestation and weaning of the pups (F1 generation). These dose levels corresponded with 30, 100 and 300 mg/kg bw/day. The test diet was administered to the offspring selected to become the F1 generation from weaning until premating (including during and following reproductive assessments - Cohort 1B) or entire generation (Cohort 1A). Pups were assigned to cohorts for reproductive/developmental toxicity testing (Cohorts 1A and 1B, including the production of a F2 generation).

Based on the adverse effects on body weight and/or food consumption noted in the P and F1 generations and microscopic liver findings noted in the P and F1 generations at 4500 ppm, an exposure level of 1500 ppm (100 mg/kg bw/day) was considered to be the no-observed-adverse- effect level (NOAEL) for P and F1 systemic and F1 neonatal toxicity of EXP1503090 when administered continuously in the diet to Crl:CD(SD) rats. Based on the lack of effects on reproductive parameters, intrauterine growth and survival, and splenic immunophenotyping changes and the lack of adverse effects on F2 body weights, an exposure level of 4500 ppm (300 mg/kg bw/day) was considered the NOAEL for P and F1 reproductive toxicity, F2 neonatal toxicity, and F1 immunotoxicity.