Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)
Remarks:
Cohort 1C and surplus cohorts were included. Surplus and chort 2B were not dosed.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 April 2018 - 31 January 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance to international guidelines and in accordance with GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2018
Deviations:
yes
Remarks:
Minor deviations listed in attachment under overall remarks. The deviations do not impact the validity of the study.
Qualifier:
according to
Guideline:
other: OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151
Version / remarks:
July 2013
Deviations:
yes
Remarks:
Minor deviations listed in attachment under overall remarks. The deviations do not impact the validity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The design of this study was based on the final decision on a compliance check of Hexahydro-1,3,5-trimethyl-1,3,5-triazine by ECHA (Decision no. CCH-D-2114371729-35-01/F, Date: 22 Sep 2017).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han rat
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. the testing facility has general and reproduction/developmental/neurological historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive, neurological toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: Not stated.
- Age at study initiation: 6 weeks old
- Weight at study initiation: Males: 138 - 167g & Females: 113 -142 g
- Fasting period before study: No
- Housing: On arrival, prior to mating and during the post-weaning period, animals were group housed (up to 5 animals of the same sex and same dosing group and cohort together) in polycarbonate cages (Macrolon type IV; height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (type III; height 18 cm).
During the post-mating phase, males were housed in type 2000P cages (61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21).
During locomotor activity monitoring, F1- Cohort 2A animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water for a maximum of 2 hours.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
-Food: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, F1- Cohort 2A animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, F1- Cohort 2A animals had no access to water for a maximum of 2 hours.
- Acclimation period: 5 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24 ºC (actual mean daily temperature was 20 to 22 ºC)
- Humidity (%): 40 to 70 % (actual mean daily Humidity was 43 -74%)
- Air changes (per hr): =>10 air changes/hour with 100% fresh air (no air recirculation).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
IN-LIFE DATES: From: 18th April 2018 To 05 Nov 2018. 


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 6 hours adding the vehicle to the test item, from 23 Apr 2018 until 25 Jun 2018 and from 17 Aug 2018 onwards.

From 26 Jun 2018 until 16 Aug 2018, the dosing formulations were also prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light; the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.

Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test item. Correction was made for the purity/composition of the test item. A factor of 2.435 was used to correct for the purity/composition (i.e. water content) of the test item.

DIET PREPARATION: N/A 
 - Rate of preparation of diet (frequency):
 - Mixing appropriate amounts with (Type of food): 
 - Storage temperature of food:
 
 VEHICLE
 - Justification for use and choice of vehicle (if other than water): Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure according to the pertaining Standard Operating Procedure. The test item was soluble in propylene glycol and stable for at least 6 hours at room temperature and 8 days in the refrigerator over concentration range of 2 to 20 mg/mL
- Concentration in vehicle: 2 to 20 mg/mL
- Amount of vehicle (if gavage): 2 to 20 mg/mL
- Lot/batch no. (if required): Not stated 
 - Purity: Not stated
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days

- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.

- Further matings after two unsuccessful attempts: yes as detailed deviation

- After successful mating each pregnant female was caged (how): Once mating had occurred, the males and females were separated, males were housed in type 2000P cages (61x43.5x21.5 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (type III, height 18 cm).
- Any other deviations from standard protocol:
Detection of mating was not confirmed in first instance for two females at 10 mg/kg/day (No. 141 and 142). For female No. 142, evidence of mating was obtained by palpation and indirectly by delivery of a litter. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. For female No. 141, evidence of mating was obtained by implantation sites recorded at necropsy; a mating date of this animal could not be estimated. Apparently, mating was overlooked in the assessment of the vaginal lavage of these animals, which explains the continuation of di-estrus during the mating in these females.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure (Test Facility Study No. 20146537). Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%.
During the course of this study at one occasion during the treatment phase, stability of the prepared formulation was determined at 6 hours at room temperature under normal light conditions and 8 days in a refrigerator.
Duration of treatment / exposure:
F0-female = 16 -18 weeks
F0-female which failed to deliver : 16 weeks
F0-male = 17 -18 weeks
F0-female no. 190& 194= Not dosed one occasion
F1- Cohort 1A = 10
F1- Cohort 1B= 11 weeks
F1- Cohort 1C = 4 weeks
F1- Cohort 2A = 8 Weeks
F1- Cohort 2B = Not Dosed
F1- Cohort surplus = Not Dosed
Frequency of treatment:
Once daily seven days a week.
Details on study schedule:
F0-males were treated for 17-18 weeks, including 10 weeks prior to mating (with the objective of covering at least one spermatogenic cycle) and during the mating, up to and including the day before scheduled necropsy. F0-females were treated for 16-18 weeks, including 10 weeks prior to mating, the variable time to conception, the duration of pregnancy and at least 21 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 16 weeks.
The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 was assumed the day of the animal being replaced).
F0-female nos. 190 and 194 (Group 4), were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
From weaning onwards (PND 21), F1-animals of Cohorts 1A, 1B, 1C and 2A were dosed up to and including the day before scheduled necropsy (i.e. for a minimum of 4 weeks). The F1-animals of Cohort 2B and Cohort Surplus were not dosed.
Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 was assumed the day of the animal being replaced).
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
For more information refer to table 6 in the additional information on methology section.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1
Dose / conc.:
10 mg/kg bw/day
Remarks:
Group 2
Dose / conc.:
30 mg/kg bw/day
Remarks:
Group 3
Dose / conc.:
100 mg/kg bw/day
Remarks:
Group 4
No. of animals per sex per dose:
F0 = 25
F1 Cohort 1A & B = 20
F1 Cohort 1C, 2A, 2B + surplus = 10 with exception of Group 2 Cohort 1C 5 M ale only
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:

The dose levels in this study were selected based on the results of a Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) in the rat (Test Facility Study No. 491807). In this OECD 422 study, rats received 0, 10, 30 or 100 mg/kg/day test item by daily oral gavage. Parental toxicity was noted at 100 mg/kg/day body weight/day and consisted of clinical signs (salivation, rales and/or piloerection), slightly decreased total protein and albumin, a thickened limiting ridge of the stomach at macroscopic examination and microscopic findings for the stomach (lymphogranulocytic inflammation of the glandular stomach and hyperplasia of the epithelium of the limiting ridge. No parental toxicity was observed at 10 and 30 mg/kg, and no reproductive/developmental toxicity was observed at any dose level.
The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is intended to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Rationale for animal assignment (if not random):

F0-animal was identified using earmark and tattoo.
F1-pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurs the identification, the pups were identified by tattoo on the feet.
From weaning (PND 21) onwards, the animals of Cohorts 1A, 1B, 1C and 2A were identified by ear and tail mark, and from approximately PND 42 onwards, these animals were identified by earmark and tattoo. The animals of Cohort 2B and Cohort Surplus were identified by tail mark.

Fasting period before blood sampling for clinical biochemistry:
The selected F0-animals and Cohort 1A animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of F0-animals and Cohort 1A animals1 housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available.

- Other:
On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, between 7.00 and 10.30 a.m. in the necropsy room, and samples were pooled per litter. If available, blood was collected from one male and one female pup per litter. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup. If the target volume of 0.5 mL could not be reached by poling two pups, a third culled pup was sampled, if available.
On PND 22, blood was collected from all Cohort Surplus animals (10/sex/group), if possible. Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes 
 - Time schedule: During clinical observations conducted prior to dosing and 0-30 minutes after dosing then twice daily up to the day prior to necropsy.
- Cage side observations checked in table [No.?] were included. Yes

 DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
 - Time schedule: Observations conducted prior to dosing and 0-30 minutes after dosing then twice daily up to the day prior to necropsy.

 BODY WEIGHT: Yes 
 - Time schedule for examinations:Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.
A terminal weight was recorded on the day of scheduled necropsy

 FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 
 - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
 - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, 14 and 21.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes 
 - Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

 OTHER: Not specified

Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0-females beginning 14 days prior to mating and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of scheduled necropsy, a vaginal lavage was also taken.
Sperm parameters (parental animals):
For all surviving males, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy. Sperm motility and progressive motility were assessed from all samples. Sperm smears for morphological evaluation were fixed from all samples and stained with haematoxylin and eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if possible) per animal was recorded. Evaluation was performed for all samples.
One epididymis (left) was removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the left epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for all samples.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.:
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Selective elimination of pups, e.g. based upon body weight or AGD, was not done.
Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: clinical observations, food and water consumption, clinical chemistry, coagulation, litter size, sex ratio, survival indices, ano-genital distance, body weight change, presence of nipple/areolae count in male offspring, sperm analysis, splenic lymphocyte subpopulation analysis, macroscopic pathology / abnormalities. Histopathology. Thyroid Hormone Analysis, Urinalysis, Functional Observation Battery, acoustic Startle response, Vaginal patency (vaginal opening), balanopreputial separation, Estrous stages.

GROSS EXAMINATION OF DEAD PUPS:
Yes, where required or if applicable.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Yes

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Yes
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: F0 Males: Around weaning (a minimum of 10 weeks of treatment).
- Maternal animals:Females which delivered;LD 23-25
-F0 females failing to produce a viable litter:
With evidence of mating: Post-coitum Days 25-27 (Nos. 105, 120, 132, 134, 136, 144, 146, 156, 172, 175, 176, 179, 182 and 199).
Without evidence of mating: Approximately 24-26 days after the last day of the mating period (No. 141; evidence for mating was only obtained at necropsy when this females was found to have implantation sites).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, harderian, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, muscle skeletal, nerves (optic & sciatic), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferent)

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively. Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, muscle skeletal, nerves (optic & sciatic), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferent).
Postmortem examinations (offspring):
SACRIFICE
- Culled Pups (PND 4) – F1 -Generation: Post natal day (PND) 4
- Cohort 1A: PND 96 (nos. 456-459, 776-778) PND 100 (nos. 442-445) & PND 89-95 (other animals).
-Cohort 1B: PND ≥ 97
-Cohort 1C:After VP/BPS positive (VP = viaginal paptency, BPS = balanopreputial separation)
-Cohort 2A: PND 76-90
-Cohort 2B: PND 21-22
-Cohort Surplus: PND 22
-Spare F1-animals: PND 22-24

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, harderian, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, lymph node, muscle skeletal, nerves (optic, sciatic & tibial), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferens.
Specific tissues: Basal ganglia, Cerebellum, cerebral cortex, hippocampus, hypothalamus, medulla oblongata, midbrain, olfactory bulbs, pons, retina, root fibres, thalamus and tibial nerve calf muscle branches.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively. Bone (marrow & Sternum) Brain, Cervix, Epididymis, eye, Gland (adrenal, coagulation, harderian, mammary, parathyroid, pituitary, preputial, prostate, seminal vesicle, thyroid), Gross lesions/masses, heart, kidney, Large intestine (Cecum, colon & rectum), liver, lung, lymph node, muscle skeletal, nerves (optic, sciatic & tibial), Ovaries, oviducts, Skin, Small intestine (duodenum, ileum & jejunum), spinal cord, spleen, stomach, testes, Thymus, trachea, urinary bladder, uterus, vagina and vas deferens.
Specific tissues: Basal ganglia, Cerebellum, cerebral cortex, hippocampus, hypothalamus, medulla oblongata, midbrain, olfactory bulbs, pons, retina, root fibres, thalamus and tibial nerve calf muscle branches.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed according to sex and occasion. Descriptive statistics number, mean and standard deviation were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made:
Group 2 Vs. Group1
Group 3 Vs. Group1
Group 4 Vs. Group1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
The motor activity data set (at least 3 groups) was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group
An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Reproduction and Developmental Variables (indices): Mating index males & females (%), Precoital time, Fertility index males and females (%), Gestation index (%), Duration of gestation, Post-implantation survival index (%), Live birth index (%),
Offspring viability indices:
Offspring Variables (indices): Viability index (%), Weaning index (%) and Percentage live males and females at weaning (%).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was seen after dosing among animals of all groups, including controls, in a dose- related manner and with an earlier onset in the higher dose groups. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. mainly after dosing). This sign was considered to be a physiological response related to taste and/or irritating properties of the test item rather than a sign of systemic toxicity.

The type and incidence of any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post-coitum and lactation phase.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male at 100 mg/kg/day (No. 94) was euthanized in extremis on Day 99 of treatment, due to a swelling and wound (with scabs) at the left inguinal region and pale appearance. Veterinary examination on Day 91 and 95 of treatment confirmed an open, partly movable wound/swelling. Macroscopy confirmed a wound in the left inguinal region, which correlated histopathologically with a basal cell carcinoma of the skin of the left inguinal region. This neoplastic lesion was considered to be a spontaneous alteration unrelated to the treatment with the test item.

One female at 30 mg/kg/day (No. 168) was euthanized in extremis on Day 109 (Day 13 of Lactation) due to exophthalmos and enlargement of the left eye. Veterinary examination on Day 12 of lactation additionally showed corneal vascularisation, yellowish contents of the posterior chamber of the eye and vitreous humor, and consistent pupillary constriction. Necropsy confirmed findings of exophthalmos and enlargement and correlated histopathologically with increased fluid in the anterior and posterior chamber. This early sacrifice was regarded to be unrelated to treatment with the test item.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One control female (No. 571) was sacrificed in extremis due to an eye lesion (exophthalmos and opacity of the right eye), which was confirmed at necropsy and histopathologically by granulocytic inflammation of the cornea, extending to the anterior chamber and iris.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in treated males and/or females at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased absolute neutrophil count in males (1.55x; within historical control range). White blood cell counts (WBC) were also marginally higher than controls (1.22x; not statistically significant and within historical control range).
- Increased absolute eosinophil count in females (2.00x; exceeding the historical control range).
- Increased red blood cell count in females (1.08x; within historical control range).
- Increased absolute reticulocyte count in males (1.27x; concurrent control mean was at the lower range of the historical control range and the mean at 100 mg/kg/day was well within this range). In contrast, absolute reticulocyte counts in females were reduced (0.68x), and were also reduced at 10 (0.80x; not statistically significant) and 30 mg/kg/day (0.78x); the concurrent control mean was at the upper range of the historical control data range, and means at 10, 30 and 100 mg/kg/day remained within this range.
Other statistically significant differences in haematology parameters were considered not to represent a change of biological relevance as values remained within the normal range and/or occurred in the absence of a dose-related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in treated males or females at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses
- Decreased total protein level in males (0.93x; within historical control range).
- Decreased urea level in males (0.84x; within historical control range).
- Decreased creatinine level in males (0.92x; within historical control range).
- Increased potassium level in females (1.08x; within historical control range).
- Increased chloride level in males (1.02x; exceeding the historical control range).
- Decreased inorganic phosphate level in males (0.90x; within historical control range).
In addition, higher bile acid levels (4.64-5.77x) were noted in female Nos. 152 and 159 (at 30 mg/kg/day), and No. 180 (at 100 mg/kg/day), that were above the historical control range. In the absence of any corroborating findings, this was considered to be of no toxicological significance.
Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in treated males at 30 and 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased urinary volume (1.63x; within historical control range).
- Decreased specific gravity (0.99x; within historical control range, not statistically significant at 30 mg/kg). This was considered secondary to the higher urinary volume.
The statistically significantly lower mean ketone level in males at 100 mg/kg/day was of no toxicological relevance as an increase is expected in case of target organ toxicity, and since all individual values remained within the historical control range.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 30 mg/kg/day in males an increased incidence of lymphogranulocytic infiltrate was recorded at mini mal degree and in a few females hemorrhage (minimal-slight), erosion/ulceration (minimal-slight) and degeneration/regeneration mucosa (minimal in a single female).

At 100 mg/kg/day most animals of both sexes showed lymphogranulocytic infiltrate (minimal-modera te). This was accompanied by hemorrhage (minimal-moderate), erosion/ulceration (males: minimal- moderate, females: minimal-slight), degeneration/regeneration mucosa (up to slight) and/or edema (up to slight). In addition, in males decreased number of parietal cells (minimal-slight) was seen in
some animals.
The degeneration/regeneration of the glandular mucosa was an alteration which was mainly seen in the area close to the limiting ridge and was characterized by increased basophilia of the mucosal c ells.
The findings recorded for the glandular stomach in the remaining dose groups including controls (lymp hogranulocytic infiltrate, hemorrhage and/or erosion/ulceration) were considered to be within backg round pathology for rats of this age and strain subjected to a repeated oral gavage study.
Adrenals glands: An increased incidence of vacuolation of the zona glomerulosa was recorded at 100 mg/kg/day. A background level of vacuolation of the zona glomerulosa was seen in the remaining do se groups including controls.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalen ce, severity, or histologic character of those incidental tissue alterations.
Male No. 94 (100 mg/kg) sacrificed after 109 days on test due to a basal cell carcinoma of the skin showed increased severity (moderate) of extramedullary hematopoiesis in the spleen. Microscopic
alterations which were in line with the microscopic findings in the remaining animals of this dose gro up consisted of moderate erosion/ulceration of the glandular mucosa of the stomach with edema and lymphogranulocytic inflammatory cell infiltrate. The basal cell carcinoma was regarded to be the cause of morbidity for this animal and this neoplastic lesion in a single animal was considered to be a spontaneous alteration and unrelated to the treatment with the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females had regular cycles of 4 to 5 days, except for one female at 10 mg/kg/day (No. 141), for which the estrous cycle could not be determined. At necropsy, this female was found to have 3 implantation sites only. Given the incidental nature and the absence of a dose-related incidence, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
At 100 mg/kg/day, a statistically significantly lower number of cells with a normal morphology was recorded (0.93x). As mean and individual values of abnormal sperm cells at this dose level generally remained within the control range and/or within the available historical control range , this slight difference was considered not to be related to treatment. At 10, 30 and 100 mg/kg/day, the mean number of cells with a coiled tail appeared higher than the concurrent control mean (16, 17 and 21, vs 11 in the control). Means remained well within the historical control range, did not achieve a level of statistical significance, occurred in the absence of a dose-related trend, and the concurrent control mean was slightly low compared to the historical control range.
Reproductive performance:
no effects observed
Description (incidence and severity):
Histopathological examination of reproductive tissues revealed no evidence of a treatment- related effect on reproduction.
Of the 25 couples of each dose group, two control couples, six couples at 10 mg/kg/day, three couples at 30 mg/kg/day and four couples at 100 mg/kg/day did not succeed in producing healthy offspring. The reproductive organs from one additional couple at 30 mg/kg/day (168/68) were examined because the female was euthanized in extremis on Day 13 of the lactation period. For male No. 5 (control) and male No. 75 (30 mg/kg), massive bilateral tubular atrophy of the testes with massive reduced sperm in the epididymides was regarded the cause of infertility of couples 105/5 and 175/75. No abnormalities were seen in the reproductive organs of the remaining couples, which could account for their lack of healthy offspring.
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. Except for male No. 5 (control) and No. 75 (30 mg/kg/day), the testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Details on results (P0)

F0-generation Data

Mortality: One male at 100 mg/kg/day (No. 94) was euthanized in extremis on Day 99 of treatment, due to a swelling and wound (with scabs) at the left inguinal region and pale appearance. Veterinary examination on Day 91 and 95 of treatment confirmed an open, partly movable wound/swelling. Macroscopy confirmed a wound in the left inguinal region, which correlated histopathologically with a basal cell carcinoma of the skin of the left inguinal region. This neoplastic lesion was considered to be a spontaneous alteration unrelated to the treatment with the test item.
One female at 30 mg/kg/day (No. 168) was euthanized in extremis on Day 109 (Day 13 of Lactation) due to exophthalmos and enlargement of the left eye. Veterinary examination on Day 12 of lactation additionally showed corneal vascularisation, yellowish contents of the posterior chamber of the eye and vitreous humor, and consistent pupillary constriction. Necropsy confirmed findings of exophthalmos and enlargement and correlated histopathologically with increased fluid in the anterior and posterior chamber. This early sacrifice was regarded to be unrelated to treatment with the test item.

Clinical Observations: No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation was seen after dosing among animals of all groups, including controls, in a dose- related manner and with an earlier onset in the higher dose groups. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. mainly after dosing). This sign was considered to be a physiological response related to taste and/or irritating properties of the test item rather than a sign of systemic toxicity. The type and incidence of any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose- related trend. At the incidence observed, these were considered to be unrelated to treatment.

Body Weights and Body Weight Gains: Body weights and body weight gain of treated animals were considered not affected by treatment. Any statistically significant changes in body weights and body weight gain were considered to be unrelated to treatment as no trend was apparent regarding dose and duration of treatment.

Food Consumption: Food consumption before or after correction for body weight was similar to the control level over the treatment period. Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment as no trend was apparent regarding dose and duration of treatment.

Haematology: The following statistically significant changes were noted in treated males and/or females at 100 mg/kg/day. Relative differences in mean values as compared to the control group are 4 indicated between parentheses. Relevant internal control data are provided by footnote .
- Increased absolute neutrophil count in males (1.55x; within internal control range). White blood cell counts (WBC) were also marginally higher than controls in males (1.22x; not statistically significant and within internal control range).
-  Increased red blood cell count in females (1.08x; within internal control range).
-  Increased absolute reticulocyte count in males (1.27x; concurrent control mean was slightly below the internal control range and the mean at 100 mg/kg/day was well within this range). In contrast, absolute reticulocyte counts in females were reduced (0.68x), and were also reduced at 10 (0.80x; not statistically significant) and 30 mg/kg/day (0.78x); the concurrent control mean was at the upper range of the internal control data range, and means at 10, 30 and 100 mg/kg/day remained within this range.
Increased absolute eosinophil count in females (2.00x; exceeding the internal control range) was primarily attributed to a high value for one female (No. 189), and therefore considered not to be related to treatment with the test item. Other statistically significant differences in haematology parameters were considered not to represent a change of biological relevance as values remained within the normal range and/or occurred in the absence of a dose-related response. 

Coagulation: Coagulation parameters of treated rats were considered not to have been affected by treatment.
The statistically significantly higher platelet counts in males at 100 mg/kg/day occurred in the absence of a clear dose-related trend, and the mean remained within the internal control range . This variation was therefore considered not to be related to treatment.
The low platelet count in one male at 30 mg/kg/day (No. 53) was considered not related to treatment given its incidental occurrence and absence of a dose-relationship.

Clinical Chemistry: The following statistically significant changes were noted in treated males or females at 100
mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses. Relevant internal control data are provided by footnote .
- Decreased total protein level in males (0.93x; within internal control range).
- Decreased urea level in males (0.84x; within internal control range).
- Decreased creatinine level in males (0.92x; within internal control range).
- Increased potassium level in females (1.08x; within internal control range).
- Increased chloride level in males (1.02x; exceeding the internal control range).
- Decreased inorganic phosphate level in males (0.90x; within internal control range). 
In addition, higher bile acid levels (4.64-5.77x) were noted in female Nos. 152 and 159 (at 30 mg/kg/day), and No. 180 (at 100 mg/kg/day), that were above the internal control range. In the absence of any corroborating findings, this was considered to be of no toxicological significance. Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose- related response. As such, these slight differences were considered not related to treatment. 
Serum levels of TSH (thyroid stimulating hormone) and T4 were considered not affected by 
treatment. 


Urinalysis: The following statistically significant changes were noted in treated males at 30 and 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses. Relevant internal control data are provided by footnote .
- Increased urinary volume (1.63x; within internal control range).
- Decreased specific gravity (0.99x; within internal control range, not statistically significant at 30 mg/kg). This was considered secondary to the higher urinary volume. 
The statistically significantly lower mean ketone level in males at 100 mg/kg/day was of no toxicological relevance as an increase is expected in case of target organ toxicity, and since all individual values remained within the internal control range. 
Urinalysis parameters of females were considered not affected by treatment. 


Organ Weights: There were no test item-related alterations in organ weights in the F0 animals.

Macroscopic Findings: Test item-related macroscopic alterations at the end of the treatment period were recorded for the stomach and were observed in the 30 and 100 mg/kg/day group males and females. Among surviving animals, these alterations consisted of:
- Thickening of the limiting ridge in 8/25 males and 4/24 females at 30 mg/kg/day and 23/24 males and 21/25 females at 100 mg/kg/day. 

- Dark red/reddish/black-brown/black focus/foci in the glandular mucosa in 4/24 females at 30 mg/kg/day and in 17/24 males and 17/25 females at 100 mg/kg/day. 
The remainder of the recorded macroscopic findings (including red foci in the glandular stomach in 1/25 males at 10 mg/kg/day and 3/25 females of the control group and thickening of the limiting ridge in 2/25 males at 10 mg/kg/day) were within the range of background gross observations encountered in rats of this age and strain. 


Histopathology: Test item-related microscopic findings were noted in the stomach of both sexes starting at 30 mg/kg/day and the adrenal glands of males at 100 mg/kg/day; Stomach: Microscopic alterations in the stomach were seen in the glandular part and consisted of a combination of findings. At 30 mg/kg/day in males an increased incidence of lymphogranulocytic infiltrate was recorded at minimal degree and in a few females hemorrhage (minimal-slight), erosion/ulceration (minimal-slight) and degeneration/regeneration mucosa (minimal in a single female).
At 100 mg/kg/day most animals of both sexes showed lymphogranulocytic infiltrate (minimal-moderate). This was accompanied by hemorrhage (minimal-moderate), erosion/ulceration (males: minimal-moderate, females: minimal-slight), degeneration/regeneration mucosa (up to slight) and/or edema (up to slight). In addition in males decreased number of parietal cells (minimal-slight) was seen in some animals.
The degeneration/regeneration of the glandular mucosa was an alteration which was mainly seen in the area close to the limiting ridge and was characterized by increased basophilia of the mucosal cells.
The findings recorded for the glandular stomach in the remaining dose groups including controls (lymphogranulocytic infiltrate, hemorrhage and/or erosion/ulceration) were considered to be within background pathology for rats of this age and strain subjected to a repeated oral gavage study. Adrenal glands: An increased incidence of vacuolation of the zona glomerulosa was recorded at 100 mg/kg/day. A background level of vacuolation of the zona glomerulosa was seen in the remaining dose groups including controls. There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Male No. 94 (100 mg/kg) sacrificed after 109 days on test due to a basal cell carcinoma of the skin showed increased severity (moderate) of extramedullary hematopoiesis in the spleen. Microscopic alterations which were in line with the microscopic findings in the remaining animals of this dose group consisted of moderate erosion/ulceration of the glandular mucosa of the stomach with edema and lymphogranulocytic inflammatory cell infiltrate. The basal cell carcinoma was regarded to be the cause of morbidity for this animal and this neoplastic lesion in a single animal was considered to be a spontaneous alteration and unrelated to the treatment with the test item.

Reproductive performance: Histopathological examination of reproductive tissues revealed no evidence of a treatment- related effect on reproduction
Of the 25 couples of each dose group, two control couples, six couples at 10 mg/kg/day, three couples at 30 mg/kg/day and four couples at 100 mg/kg/day did not succeed in producing healthy offspring. The reproductive organs from one additional couple at 30 mg/kg/day (168/68) were examined because the female was euthanized in extremis on Day 13 of the lactation period. For male No. 5 (control) and male No. 75 (30 mg/kg/day), massive bilateral tubular atrophy of the testes with massive reduced sperm in the epididymides was regarded the cause of infertility of couples 105/5 and 175/75. These males also had no motile/progressive sperm cells. No abnormalities were seen in the reproductive organs of the remaining couples, which could account for their lack of healthy offspring. Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. Except for male No. 5 (control) and male No. 75 (30 mg/kg/day) the testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Sperm Analysis: Sperm motility, concentration and morphology were considered not affected by treatment. At 100 mg/kg/day, a statistically significantly lower number of cells with a normal morphology was recorded (0.93x). As mean and individual values of abnormal sperm cells at this dose level generally remained within the control range and/or within the available internal control range , this slight difference was considered not to be related to treatment. At 10, 30 and 100 mg/kg/day, the mean number of cells with a coiled tail appeared higher than the concurrent control mean (16, 17 and 21, vs 11 in the control). Means remained well within the internal control range, did not achieve a level of statistical significance, occurred in the absence of a dose-related trend, and the concurrent control mean was slightly low compared to the internal control range. Male No. 5 (control) and male No. 75 (30 mg/kg/day), both of which had massive bilateral tubular atrophy of the testes with massive reduced sperm in the epididymides, had no motile or progressive sperm. This was regarded the cause of infertility of couples 105/5 and 175/75.

Estrous Cycle: Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of 4 to 5 days, except for one female at 10 mg/kg/day (No. 141), for which the estrous cycle could not be determined. At necropsy, this female was found to have 3 implantation sites only. Given the incidental nature and the absence of a dose- related incidence, this finding did not indicate a relation with treatment.

Mating Index: Mating index was not affected by treatment. All females showed evidence of mating.

Precoital Time: Precoital time was considered not affected by treatment. All females showed evidence of mating within 4 days, except for one female at 10 mg/kg/day (No. 131) and two females at 100 mg/kg/day (Nos. 190 and 194), for which mating took 5, 8 and 12 days, respectively. As this variation in precoital time remained within the normal range of biological variation9 and occurred at low incidence, this was considered to be unrelated to treatment.

Number of Implantation Sites: Number of implantation sites was not affected by treatment. One female at 10 mg/kg/day (No. 141), for which mating was not detected, had 3 implantation sites only. As this occurred in absence of a dose response relationship, this was considered to be unrelated to treatment.

Fertility Index: Fertility index was considered not to be affected by treatment. The fertility indices were 92, 80, 88 and 84% for the control, 10, 30 and 100 mg/kg/day groups, respectively. The number of non-pregnant females versus mated females was 2/25, 5/25, 3/25 and 4/25 in the control, 10, 30 and 100 mg/kg/day groups, respectively. As these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive toxicity, this was considered not to be related to treatment.

Developmental
Data
 Gestation Index and Duration: Gestation index and duration of gestation were unaffected by treatment. All pregnant females had live offspring, except for one female at 10 mg/kg/day (No. 141), that had 3 implantation sites only. The gestation index was 100% for the control and 30 and 100 mg/kg/day groups, and 95% for the 10 mg/kg/day group. The failed pregnancy of female No. 141, without related histopathology changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.

Parturition/Maternal Care: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 95, 89, 89 and 93% for the control, 10, 30 and 100 mg/kg/day groups, respectively. For one female at 30 mg/kg/day (No. 154), the number of pups was slightly higher than the number of implantations. . This phenomenon is observed from time to time, and no toxicological relevance was attached to this finding in the current study.

Litter Size: Litter size was not affected by treatment. Live litter sizes were 11.7, 11.8, 11.2 and 12.1 living fetuses/litter for the control, 10, 30 and 100 mg/kg/day groups, respectively.

Sex Ratio: Sex ratio was considered not affected by treatment.

Live Birth Index:The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 100% for the control, 30 and 100 mg/kg/day groups, and 99% for the 10 mg/kg/day group. One pup at 10 mg/kg/day (litter No. 142) was sacrificed in extremis on PND 1, based on a pale appearance and a wound at the right hindleg. One pup of the control group (litter No. 112) and two pups at 10 mg/kg/day (one in each of litter Nos. 126 and 131) were found dead at first litter check. These dead/missing pups were considered unrelated to treatment since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Viability Index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 (viability index) was considered not affected by treatment. Viability indices were 99, 99, 100 and 98% for the control, 10, 30 and 100 mg/kg/day groups, respectively.
Two pups of the control group (one in each of litter Nos. 101 and 111), three pups at 10 mg/kg/day (one in each of litter Nos. 126 and 133), one pup at 30 mg/kg/day (litter No. 161) and four pups at 100 mg/kg/day (one in each of litter Nos. 180, 185, 186 and 195) were found dead or missing between PND2 and 4. In addition, one pup at 100 mg/kg/day (litter No. 188) was sacrificed in extremis on PND 1, based on absence of milk in the stomach and fissures at the flews (cleft lip). Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Weaning Index: The number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. One pup at 10 mg/kg/day (litter No. 126) was found dead on PND 21 (cannibalised) and all 8 pups of litter No. 168 (30 mg/kg/day) were necropsied together with the dam that was sacrificed in extremis on PND 13 , resulting in weaning indices of 100, 99, 95 and 100% for the control, 10, 30 and 100 mg/kg/day groups, respectively. No toxicological relevance was attributed to the dead/sacrificed pups as the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on repoductive parameters
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was seen after dosing among animals of all test item-treated groups, in a dose-related manner and with an earlier onset in the higher dose groups. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. mainly after dosing). This sign was considered to be a physiological response related to taste of the test item and/or irritating properties of the test item rather than a sign of systemic toxicity.
One male at 100 mg/kg/day (No. 449) was recorded to have overgrown teeth on a single day. Hunched posture, piloerection and lean appearance recorded for this animal on the same day were considered secondary to overgrown teeth. As such, these findings were considered not to be related to treatment.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
A total 13 F1 animals that were dosed in the post-weaning phase were found dead or were sacrificed in extremis during the first 4 days of treatment, mostly after dosing. This concerned 2/120 control animals, 3/105 animals at 10 mg/kg, 3/120 animals at 30 mg/kg, and 5/120 animals at 100 mg/kg.
Based on the nature of the necropsy findings recorded for these animals in the respiratory tract (pale discolouration of the lungs, lungs not collapsed, foamy contents of the lungs or trachea), the cause of moribundity/death of animals showing these lesions was considered to be related to the gavage administration procedure, possibly in combination with reflux. None of these animals showed clinical signs prior to death/sacrifice. For two of these females (Nos. 601 and 689), tissues were examined histopathologically. For female no. 689, the cause of death was not evident from the sections examined microscopically. For female No. 601, a histopathological cause of death was indicated as marked ulceration of the cecum (see also 9.5.16 Histopathology).
For one male at 10 mg/kg/day (No. 301), a possible cause of death could not be established due to cannibalism, but it could not be excluded that this animal had died due to similar respiratory lesions. No clinical signs were shown by this animal prior to death.
One female at 100 mg/kg/day (No. 789) was sacrificed due to clinical signs (pallor, piloerection, labored respiration) and since its cage mates were biting this animal. Its deteriorated condition may also have been influenced by an incident occurring on the previous day where this animal had dropped on the floor. Necropsy findings for this female (many dark-red foci on the glandular mucosa of the stomach) was distinct to what was previously seen for other sacrificed animals.
One control female (No. 571) was sacrificed in extremis due to an eye lesion (exophthalmos and opacity of the right eye), which was confirmed at necropsy and histopathologically by granulocytic inflammation of the cornea, extending to the anterior chamber and iris.
Overall, the mortality incidence recorded over the dose groups did not show a dose-related trend. None of the F1 animals that survived until their scheduled necropsy showed similar lesions in the respiratory tract.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
One control female (No. 571) was sacrificed in extremis due to an eye lesion (exophthalmos and opacity of the right eye), which was confirmed at necropsy and histopathologically by granulocytic inflammation of the cornea, extending to the anterior chamber and iris.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in animals at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased white blood cell (WBC), neutrophil and monocyte count in males (1.31x, 1.50x and 2.00x, respectively; within historical control range).
- Increased reticulocyte counts in males (1.23x; within historical control range) and in females (1.37x; within historical control range).
Haematological values at 10 and 30 mg/kg/day were considered not affected by treatment.
Coagulation parameters of treated rats were considered not affected by treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes were noted in animals at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses
- Decreased total protein levels in males (0.96x; within historical control range).
- Increased cholesterol levels in females (1.58x; within historical control range).
Any other statistically significant changes in clinical chemistry parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend (lower total bilirubin level in males at 30 mg/kg) or since an increase would be expected in case of target organ toxicity (lower alkaline phosphatase activity (ALP) in females at 100 mg/kg).
Serum levels of TSH (thyroid stimulating hormone) and T4 were considered not affected by treatment.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The following statistically significant changes were noted at 30 and/or 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased urinary volume in males at 100 mg/kg/day (1.71x; within historical control range).
- Decreased specific gravity in males at 100 mg/kg/day (0.99x; within historical control range). This was considered secondary to the higher urinary volume.
- Increased urinary pH in females at 30 and 100 mg/kg/day (1.08x and 1.10x, respectively; within historical control range, and control mean at the lower range of the historical control range).
The statistically significantly lower protein level in males at 10 mg/kg/day occurred in the absence of a dose-related trend and an increase is expected in case of target organ toxicity. Therefore, this change was considered not related to treatment.
Any statistically significant changes in urinary parameters were considered to have arisen as a result of slightly low control values, were only minor in magnitude and/or lacked a dose relationship. These were therefore considered not to be toxicologically relevant.
Sexual maturation:
no effects observed
Description (incidence and severity):
Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrus and time between vaginal opening and first estrus in females was considered not to be affected by treatment.
Mean vaginal opening time at 100 mg/kg/day was statistically significantly higher than controls (PND 33 vs. PND 32 in controls), but the mean was similar as recorded at 10 and 30 mg/kg. Mean body weight on the day of vaginal opening was also statistically significantly higher at 100 mg/kg, as well as at 10 mg/kg/day (without a dose-related trend). Individual values remained within the concurrent control range and mean values remained within the historical control range . The day of acquiring first estrous was statistically significantly higher at 10 and 30 mg/kg. However, means did not show a dose-related trend and remained within the available control range . Therefore, these variations in vaginal opening time and day of acquiring first estrous were considered not to be related to treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 100 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A (PND 89-95): At 100 mg/kg/day, a statistically significantly higher liver weight was recorded for females (absolute and relative to body weight, 1.13x and 1.07x, respectively). Means remained within the internal control data range. For males at 100 mg/kg/day, a statistically significant marginally higher liver to body weight ratio was recorded (1.06x), but a clear dose-related trend (also in absolute body weights) was absent. Therefore, liver weights in males were considered not affected by treatment. There were no other test item-related organ weight changes. The statistically significant higher absolute ovary weight at 30 mg/kg/day occurred in the absence of a dose-related trend and as such was considered to be unrelated to treatment..

Cohort 1B (≥ PND 97): Organ weights and organ to body weight ratios were considered not affected by treatment.
The statistically significantly higher absolute and relative ovary weights at 100 mg/kg/day remained within the historical control data range, and no clear dose-related response was apparent. Moreover, ovary weights did not show a similar change in Cohort 1A animals that were necropsied on a similar time point. Therefore, this change was considered not to be related to treatment

Cohort 2A (PND 76-100): Fixed brain weights were considered not affected by treatment.

Cohort 2B (PND 21-22): Fixed brain weights were considered not affected by treatment.

Cohort Surplus (PND 22): Brain and spleen weight were considered not affected by treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A (PND 89-95): Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1A were recorded for the stomach and were observed in the 100 mg/kg/day group males and females. These alterations consisted of:
- Thickening of the limiting ridge in 18/20 males and 16/20 females.
- Dark red/reddish focus/foci in the glandular mucosa in 7/20 males and 2/20 females.
The remainder of the recorded macroscopic findings (including red foci in the glandular stomach in 1/20 males of the control group and thickening of the limiting ridge in 2/20 males at 30 mg/kg/day) were within the range of background gross observations encountered in rats of this age and strain.

Cohort 1B (≥ PND 97): Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1B were recorded for the stomach and were observed in the 100 mg/kg/day group males and females. These alterations consisted of:
- Thickening of the limiting ridge in 16/20 males and 19/20 females.
- Dark red/reddish focus/foci in the glandular mucosa in 7/20 males and 5/20 females.

Cohort 1C (males: ≥ PND 35; females: ≥ PND 25: There were no gross observations in animals of Cohort 1C.

Cohort 2A (PND 76-100): There were no test item-related gross observations in animals of Cohort 2A.

Cohort 2B (PND 21-22): There were no gross observations in animals of Cohort 2B.

Cohort Surplus (PND 22): There were no test item-related gross observations in animals of Cohort Surplus: Necropsy findings were confined to pale, gray-white discoloration of the lungs in one control male (No. 279) and thickening of the spleen in one male at 100 mg/kg/day (No. 512). These findings were considered not related to treatment as they did not show a clear dose-related trend or were observed in the control group only.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: Test item-related microscopic findings in Cohort 1A were noted in the stomach of both sexes at 100 mg/kg/day.
Microscopic alterations in the glandular stomach of the F1 Cohort 1A-animals were in general consistent with the findings in the F0-animals. However, these microscopic alterations in F1 -animals were recorded at a slightly lower incidence and severity and only regarded test item-related at 100 mg/kg/day. These findings included mixed or lymphocytic inflammatory cell infiltrate (minimal-slight), hemorrhage (minimal), erosion (minimal-slight) and degeneration/regeneration of the glandular mucosa (slight).
The inflammatory cell infiltrate recorded for the glandular stomach in the remaining dose groups including controls were considered to be within background pathology for rats of this age and strain subjected to a repeated oral gavage study.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Spermatogenesis-staging (Cohort 1A): Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Ovarian Follicle Counts (Cohort 1A): There were no test item-related effects on the ovarian follicle counts in the F1 females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between
group mean counts represented biological variability and were not statistically significant.
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Fixed brain weights of Cohort 2A animals (PND 76-90) and Cohort 2B animals (PND 21-22) were considered not affected by treatment.
Brain dimensions (length and width of brain) of Cohort 2A animals (PND 76-90) and Cohort 2B animals (PND 21-22) were considered not affected by treatment.
In the F1 animals (Cohort 2A) at PND 76-90, there were no test item-related effects on the
H&E stained sections of brain or peripheral nervous system in the control or high-dose group males or females.
In the F1 animals (Cohort 2B) at PND 21-22, there were no test item-related effects on the
H&E stained sections of brain in the control or high-dose group males or females.
Any variations observed during morphometric analysis of the brain of Cohort 2B animals (PND 21-22) and Cohort 2A animals (PND 76-90) were most likely unrelated to treatment with the test item.
Morphometric analysis of the brain at PND 21-22 revealed statistically significantly higher hippocampus thickness measurements in the 100 mg/kg/day group males when compared with the control group. Morphometric analysis of the brain at PND 76-90 revealed statistically significantly lower frontal cortex thickness, parietal cortex thickness, and hippocampus thickness measurements in the 100 mg/kg/day group males and higher frontal cortex thickness measurements in the 100 mg/kg/day group females.
Trends between sexes were lacking, individual animal values were often within the range of the control group, and there were no differences that were observed at both time points or appeared worse with time. Additionally, there were no correlating changes in organ weights, histopathological findings, or clinical signs. Given these circumstances and the inconsistency of differences across brain regions, the differences were most likely unrelated to the test item.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Slightly lower T-cytotoxic cell splenic subpopulations were observed for males and females at 100 mg/kg. A slightly higher Th/Tc ratio was observed for males at 30 and 100 mg/kg/day and slightly lower T-cell splenic subpopulations were observed for females at 100 mg/kg. As these shifts were slight in nature, did not reach statistical significance and occurred in the absence of corroborative findings in the spleen, these shifts were considered to represent biological variability and considered not to represent an effect of the test item.

Details on results (F1)

F1-pups Results:
No clinical signs occurred among pups that were considered to be related to treatment. For some pups of all groups (including control) that were found dead/missing, pallor and/or absence of milk in the stomach was noted. At the incidence observed and in the absence of a dose-related trend, no toxicological relevance was attributed to these signs. The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age and were therefore considered not to be toxicologically relevant.

Body weights of pups were considered not affected by treatment.

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not affected by treatment.

Serum T4 levels in male and female pups culled at PND 4 were not affected by treatment. In addition, serum thyroid stimulating hormone (TSH) and T4 levels of male and female pups of Cohort Surplus at PND 22 were considered not affected by treatment.

No macroscopic findings were noted among pups sacrificed at the end of the lactation period that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment. Brain and spleen weight of surplus pups were considered not affected by treatment.

F1-generation Data (Cohort 1A, 1B, 1C, 2A)
No mortality occurred that was considered to be directly attributable to treatment with the test item. A total 13 F1 animals that were dosed in the post-weaning phase were found dead or were sacrificed in extremis during the first 4 days of treatment, mostly after dosing (see also table below). This concerned 2/120 control animals, 3/105 animals at 10 mg/kg, 3/120 animals at 30 mg/kg, and 5/120 animals at 100 mg/kg. Based on the nature of the necropsy findings recorded for these animals in the respiratory tract (pale discolouration of the lungs, lungs not collapsed, foamy contents of the lungs or trachea), the cause of moribundity/death of animals showing these lesions was considered to be related to the gavage administration procedure, possibly in combination with reflux. None of these animals showed clinical signs prior to death/sacrifice. For two of these females (Nos. 601 (control group) and 689 (30 mg/kg)), tissues were examined histopathologically. For female No. 689, the cause of death was not evident from the sections examined microscopically. For female No. 601, a histopathological cause of death was indicated as marked ulceration of the cecum. For one male at 10 mg/kg/day (No. 301), a possible cause of death could not be established due to cannibalism, but it could not be excluded that this animal had died due to similar respiratory lesions. No clinical signs were shown by this animal prior to death. One female at 100 mg/kg/day (No. 789) was sacrificed due to clinical signs (pallor, piloerection, labored respiration) and since its cage mates were biting this animal. Its deteriorated condition may also have been influenced by an incident occurring on the previous day where this animal had dropped on the floor. Necropsy findings for this female (many dark-red foci on the glandular mucosa of the stomach) was different to what was previously seen for other sacrificed animals. One control female (No. 571) was sacrificed in extremis due to an eye lesion (exophthalmos and opacity of the right eye), which was confirmed at necropsy and histopathologically by granulocytic inflammation of the cornea, extending to the anterior chamber and iris. Overall, the mortality incidence recorded over the dose groups did not show a dose-related trend. None of the F1 animals that survived until their scheduled necropsy showed similar lesions in the respiratory tract.

Clinical Observations: No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation was seen after dosing among animals of all test item-treated groups, in a dose- related manner and with an earlier onset in the higher dose groups. This was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. mainly after dosing). This sign was considered to be a physiological response related to taste of the test item and/or irritating properties of the test item rather than a sign of systemic toxicity. One male at 100 mg/kg/day (No. 449) was recorded to have overgrown teeth on a single day. Hunched posture, piloerection and lean appearance recorded for this animal on the same day were considered secondary to overgrown teeth. As such, these findings were considered not to be related to treatment. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Body Weights and Body Weight Gains: No changes in body weight or body weight gain were noted that were considered to be related to treatment. Any statistically significant changes in body weight and/or body weight gain occurring during treatment across all dose groups were considered not related to treatment as a dose-related trend was absent and changes were not consistently recorded with continuing treatment.

Food Consumption: No toxicologically relevant changes in food consumption (before and/or after correction for body weight) were recorded across the dose groups. All statistically significant changes in food consumption (before and/or after correction for body weight) were very minor and did not show a dose-relationship. The initially (statistically significantly) lower food intake may have been caused by variation in delivery dates of dams and subsequent allocation of the pups to the respective cages.

Functional Tests: Acoustic startle response was considered not affected by treatment. Any statistically significant changes in acoustic startle response parameters were considered not related to treatment as these were minimal and/or occurred in the absence of a dose- related trend. Detailed clinical observations revealed no symptoms that were considered to be related to treatment. The clinical symptoms that were observed were considered to be within the normal range of behavioural findings for this type of study and were generally also observed in control animals. These findings were therefore considered not to be related to treatment. At 100 mg/kg, mean rectal temperature of males was statistically significantly reduced (0.98x; mean remained within the internal control data range). Mean rectal temperature at 10 and 30 mg/kg/day (both sexes) and at 100 mg/kg/day (females) were similar to the control mean. At 100 mg/kg, a lower motor activity was recorded for females compared to the control means (both for total movements (0.69x) and ambulations (0.66x), statistically significant only for ambulations). Means remained within the internal control data range. Motor activity of males treated up to 100 mg/kg/day and of females up to 30 mg/kg/day was considered not affected by treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Functional observation parameters (hearing ability, pupillary reflex, foot splay and grip strength) were considered not affected by treatment. A statistically significantly lower mean foot splay value was recorded for males at 30 and 100 mg/kg. However, a dose-related response was absent, i.e. the mean at 100 mg/kg/day was higher than the mean at 30 mg/kg/day and comparable to the mean at 10 mg/kg. As such, this variation was considered to be unrelated to treatment. Hearing ability and pupillary reflex were normal in all examined animals. Grip strength was considered not affected by treatment.

Sexual Maturation: Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrous and time between vaginal opening and first estrous in females was considered not to be affected by treatment. Mean vaginal opening time at 100 mg/kg/day was statistically significantly higher than controls (PND 33 vs. PND 32 in controls), but the mean was similar as recorded at 10 and 30 mg/kg. Mean body weight on the day of vaginal opening was also statistically significantly higher at 100 mg/kg, as well as at 10 mg/kg/day (without a dose-related trend). Individual values remained within the concurrent control range and mean values remained within the internal control range. The day of acquiring first estrous was statically significantly Balanopreputial separation (prepuce opening) in males and vaginal patency (vaginal opening), occurrence of first estrous and time between vaginal opening and first estrous in females was considered not to be affected by treatment. Mean vaginal opening time at 100 mg/kg/day was statistically significantly higher at 10 and 30 mg/kg. However, means did not show a dose-related trend and remained within the available control range. Therefore, these variations in vaginal opening time and day of acquiring first estrous were considered not to be related to treatment.

Estrous Cycle – F1-Generation (Cohort 1A): Length and regularity of the estrous cycle were not affected by treatment. For all females for which estrous cycle regularity could be determined, regular cycles of 4 to 5 days were recorded between PND 75 and 88. No cycle classification was possible for 1/19 females at 10 mg/kg/day (No. 602), 1/19 females at 30 mg/kg/day (No. 695) and 1/20 females at 100 mg/kg/day (No. 763). In addition, one female at 100 mg/kg/day (No. 764) was acyclic. Given the incidental nature and the absence of a dose-related incidence, this single occurrence of an acyclic estrous cycle was considered not to indicate a relation to treatment.

Sperm Analysis – F1-Generation (Cohort 1A): Sperm motility, concentration and morphology were considered not affected by treatment. At 100 mg/kg/day, a statistically significantly lower number of cells with normal morphology was recorded (0.93x). No clear dose-related trend occurred as the mean was similar to that at 10 mg/kg, and the means remained within the internal control range. At both 10 and 100 mg/kg, the number of cells with coiled tail appeared higher than the control mean (not statistically significant), but again no dose-related trend was apparent and means remained within the internal control range. Also, the control mean number of cells with coiled tail was considered to be slightly low compared to the internal control mean. The statistically significantly higher epididymal sperm count at 30 mg/kg/day occurred in the absence of a dose-related trend. Therefore, these variations were considered not to represent an effect of the test item.
Haematology – F1-Generation (Cohort 1A): The following statistically significant changes were noted in animals at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased white blood cell (WBC), neutrophil and monocyte count in males (1.31x, 1.50x and 2.00x, respectively; within internal control range).
- Increased reticulocyte counts in males (1.23x; within internal control range) and in females (1.37x; within internal control range).
Haematological values at 10 and 30 mg/kg/day were considered not affected by treatment.

Coagulation – F1-Generation (Cohort 1A): Coagulation parameters of treated rats were considered not affected by treatment.

Clinical Chemistry – F1-Generation (Cohort 1A): The following statistically significant changes were noted in animals at 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
-Decreased total protein levels in males (0.96x; within internal control range).
-Increased cholesterol levels in females (1.58x; within internal control range).
Any other statistically significant changes in clinical chemistry parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend (lower total bilirubin level in males at 30 mg/kg) or since an increase would be expected in case of target organ toxicity (lower alkaline phosphatase activity (ALP) in females at 100 mg/kg).

Thyroid hormone analyses – F1-Generation (Cohort 1A): Serum levels of TSH (thyroid stimulating hormone) and T4 of Cohort 1A were considered not affected by treatment.

Urinalysis – F1-Generation (Cohort 1A): The following statistically significant changes were noted at 30 and/or 100 mg/kg/day. Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased urinary volume in males at 100 mg/kg/day (1.71x; within internal control range).
-Decreased specific gravity in males at 100 mg/kg/day (0.99x; within internal control range). This was considered secondary to the higher urinary volume.
-Increased urinary pH in females at 30 and 100 mg/kg/day (1.08x and 1.10x, respectively; within internal control range, and control mean at the lower range of the internal control range).
The statistically significantly lower protein level in males at 10 mg/kg/day occurred in the absence of a dose-related trend and an increase is expected in case of target organ toxicity. Therefore, this change was considered not related to treatment. Any statistically significant changes in urinary parameters were considered to have arisen as a result of slightly low control values, were only minor in magnitude and/or lacked a dose relationship. These were therefore considered not to be toxicologically relevant.

Splenic Lymphocyte Subpopulation – F1-Generation (Cohort 1A): Splenic lymphocyte subpopulations were considered not affected by treatment. Slightly lower T-cytotoxic cell splenic subpopulations were observed for males and females at 100 mg/kg. A slightly higher Th/Tc ratio was observed for males at 30 and 100 mg/kg/day and slightly lower T-cell splenic subpopulations were observed for females at 100 mg/kg. As these shifts were slight in nature, did not reach statistical significance and occurred in the absence of corroborative findings in the spleen, these shifts were considered to represent biological variability and considered not to represent an effect of the test item.

Gross Pathology – F1-Generation:
Cohort 1A - test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1A were recorded for the stomach and were observed in the 100 mg/kg/day group in both males and females. These alterations consisted of:
-Thickening of the limiting ridge in 18/20 males and 16/20 females.
-Dark red/reddish focus/foci in the glandular mucosa in 7/20 males and 2/20 females.
The remainder of the recorded macroscopic findings (including red foci in the glandular stomach in 1/20 males of the control group and thickening of the limiting ridge in 2/20 males at 30 mg/kg/day) were within the range of background gross observations encountered in rats of this age and strain.
Cohort 1B: Test item-related macroscopic alterations at the end of the treatment period in animals of Cohort 1B were recorded for the stomach and were observed in the 100 mg/kg/day group males and females. These alterations consisted of:
-Thickening of the limiting ridge in 16/20 males and 19/20 females.
-Dark red/reddish focus/foci in the glandular mucosa in 8/20 males and 5/20 females.
Cohort 1C: There were no gross observations in animals.
Cohort 2A: There were no gross observations in animals.
Cohort 2B: There were no gross observations in animals.
Cohort surplus: There were no gross observations in animals. Necropsy findings were confined to pale, grey-white discoloration of the lungs in one control male (No. 279) and thickening of the spleen in one male at 100 mg/kg/day (No. 512). These findings were considered not related to treatment as they did not show a clear dose-related trend or were observed in the control group only.

Organ weights – F1-Generation
Cohort 1A: At 100 mg/kg/day, a statistically significantly higher liver weight was recorded for females (absolute and relative to body weight, 1.13x and 1.07x, respectively). Means remained within the internal control data range. For males at 100 mg/kg/day, a statistically significant marginally higher liver to body weight ratio was recorded (1.06x), but a clear dose-related trend (also in absolute body weights) was absent. Therefore, liver weights in males were considered not affected by treatment. There were no other test item-related organ weight changes. The statistically significant higher absolute ovary weight at 30 mg/kg/day occurred in the absence of a dose-related trend and as such was considered to be unrelated to treatment.
Cohort 1B: Organ weights and organ to body weight ratios were considered not affected by treatment. The statistically significantly higher absolute and relative ovary weights at 100 mg/kg/day remained within the internal control data range and no clear dose-related response was apparent. Moreover, ovary weights did not show a similar change in Cohort 1A animals that were necropsied on a similar time point. Therefore, this change was considered not to be related to treatment.
Cohort Surplus: Brain and spleen weight were considered not affected by treatment.

Histopathology – F1-Generation:
Cohort 1A: Test item-related microscopic findings in Cohort 1A were noted in the stomach of both sexes at 100 mg/kg/day Microscopic alterations in the glandular stomach of the F1 Cohort 1A-animals were in general consistent with the findings in the F0-animals. However, these microscopic alterations in F1 - animals were recorded at a slightly lower incidence and severity and only regarded test item- related at 100 mg/kg/day. These findings included mixed or lymphocytic inflammatory cell infiltrate (minimal-slight), hemorrhage (minimal), erosion (minimal-slight) and degeneration/regeneration of the glandular mucosa (slight). The inflammatory cell infiltrate recorded for the glandular stomach in the remaining dose groups including controls were considered to be within background pathology for rats of this age and strain subjected to a repeated oral gavage study. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Spermatogenesis-staging (Cohort 1A): Stage-dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Ovarian Follicle Counts (Cohort 1A): There were no test item-related effects on the ovarian follicle counts in the F1 females (Cohort 1A) at 100 mg/kg/day when compared to control group females. Any variation between group mean counts represented biological variability and were not statistically significant.

Neuropathology and Morphometry– F1-Generation Cohorts 2A and 2B
Fixed brain weights of Cohort 2A animals and Cohort 2B animals were considered not affected by treatment. Brain dimensions (length and width of brain) of Cohort 2A animals and Cohort 2B animals were considered not affected by treatment. In the F1 animals (Cohort 2A), there were no test item-related effects on the H&E stained sections of brain or peripheral nervous system in the control or high-dose group males or females. In the F1 animals (Cohort 2B), there were no test item-related effects on the H&E stained sections of brain in the control or high-dose group males or females. Any variations observed during morphometric analysis of the brain of Cohort 2B animals and Cohort 2A animals were considered unrelated to treatment with the test item. Morphometric analysis of the brain of Cohort 2B animals revealed statistically significantly higher hippocampus thickness measurements in the 100 mg/kg/day group males when compared with the control group. Morphometric analysis of the brain of Cohort 2A animals revealed statistically significantly lower frontal cortex thickness, parietal cortex thickness, and hippocampus thickness measurements in the 100 mg/kg/day group males and higher frontal cortex thickness measurements in the 100 mg/kg/day group females. Trends between sexes were lacking, individual animal values were often within the range of the control group, and there were no differences that were observed at both time points or appeared worse with time. Additionally, there were no correlating changes in organ weights, histopathological findings, or clinical signs. Given these circumstances and the inconsistency of differences across brain regions, the differences were most likely unrelated to Hexahydro- 1,3,5-Trimethyl-1,3,5-Triazine.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproductive and developemntal parameters
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 12. Summary Test Item-Related Microscopic Finding F0 -Stomach

Dose level (mg/kg/day):

Males

Females

0

10

30

100

0

10

30

100

Stomacha

25

25

25

25

25

25

25

25

Lymphogranulocytic infiltrate glandular mucosa,

 

 

 

 

 

 

 

 

     Minimal

2

4

9

5

-

1

2

8

     Slight

-

-

-

16

-

1

-

11

     Moderate

-

-

-

4

-

-

-

1

Hemorrhage glandular mucosa 

 

 

 

 

 

 

 

 

     Minimal

-

1

-

7

1

-

3

7

     Slight

-

-

-

5

-

-

1

4

     Moderate

-

-

-

2

-

-

-

1

Erosion/ulceration glandular                                    mucosa

 

 

 

 

 

 

 

 

     Minimal

-

-

-

4

2

-

3

2

     Slight

-

-

-

7

-

1

1

1

     Moderate

-

-

-

1

-

-

-

-

Degeneration/regeneration

glandular mucosa

 

 

 

 

 

 

 

 

     Minimal

-

-

-

-

-

-

1

6

     Slight

-

-

-

3

-

-

-

2

Decreased number of parietal cells

 

 

 

 

 

 

 

 

     Minimal

-

-

-

2

-

-

-

-

     Slight

-

-

-

5

-

-

-

-

a = Number of tissues examined from each group.

Table 13. Summary Test Item-Related Microscopic Findings F0– male adrenal gland 

 

Males

Dose level (mg/kg/day):

0

10

30

100

 

 

 

 

 

Adrenal glanda

25

25

25

25

  Vacuolation zona glomerulosa

 

 

 

 

     Minimal

3

5

7

13

     Slight

1

-

-

2

a = Number of tissues examined from each group.

 

Table 14. Correlation of Histopathology Findings with In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups.

Group

Dose level

mg/kg/day

Female/Male nos.

In-Life Reason

Histopathology

1

0

105/5

Not pregnant

Male 5[sb1] : massive bilateral tubular atrophy testes and reduced sperm eididymides

 

 

120/20

Not pregnant

No histopathological correlate

2

10

132/32

Not pregnant

No histopathological correlate

 

 

134/34

Not pregnant

No histopathological correlate

 

 

136/36

Not pregnant

No histopathological correlate

 

 

141/41

Implantation sites only

No histopathological correlate

 

 

144/44

Not pregnant

No histopathological correlate

 

 

146/46

Not pregnant

No histopathological correlate

3

30

156/56

Not pregnant

No histopathological correlate

 

 

168/68

Euthanized on Day 13 of lactation

Delivered pups, no findings suggesting lack of fertility

 

 

172/72

Not pregnant

No histopathological correlate

 

 

175/75

Not pregnant

Male 75: massive bilateral tubular atrophy testes and reduced sperm epididymides

4

100

176/76

Not pregnant

No histopathological correlate

 

 

179/79

Not pregnant

No histopathological correlate

 

 

182/82

Not pregnant

No histopathological correlate

 

 

199/99

Not pregnant

No histopathological correlate

Further results are included in background materials.

Applicant's summary and conclusion

Conclusions:
Based on the criteria set in Regulation (EC) No 1272/2008, hexahydro-1,3,5 -trimethyl-1,3,5 -triazine is not classified for reproductive or developmental toxicity.

Executive summary:

OECD 443 (2019): The pre- and postnatal effects of the test item was evaluated in Wistar Han rats at dose levels of 10, 30 and 100 mg/kg/day, based on the results of aCombined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422) in the rat (Test Facility Study No. 491807). The following parameters and end points were evaluated included mortality/ moribundity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm and splenic lymphocyte subpopulation analysis, organ weights and histopathological examinations. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood were conducted. The integrity and performance of the adult male and female reproductive systems  and developmental neurotoxicity  were also assessed.

F0-Generation:

Treatment resulted in adverse macroscopic and microscopic alterations in the stomach of both sexes at 30 and 100 mg/kg/day and non-adverse microscopic findings in the adrenal glands of males at 100 mg/kg/day. The stomach findings consisted of a combination of inflammatory and degenerative findings in the glandular stomach in the F0-animals at 30 and 100 mg/kg/day, consisting of an increased incidence and/or severity of lymphogranulocytic infiltrate, hemorrhage, erosion/ulceration, degeneration/regeneration mucosa, edema and/or decreased number of parietal cells. These findings were considered to be the result of irritating properties of the test item, and correlated to necropsy findings in these dose groups (foci in the glandular stomach; thickening of the limiting ridge observed at necropsy had no clear histopathological correlate).

Reproduction results – F0-generation:

No reproductive toxicity was observed up to the highest dose level tested (100 mg/kg/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. sperm motility, concentration and morphology, mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis).

Developmental results – F0 generation / F1 generation (pre-weaning):

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, live birth, viability and weaning indices, duration of gestation, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, thyroid hormone levels (T4 of PND 4 and 22 pups and TSH of PND 22 pups), macroscopic examination and brain and spleen weight

Developmental results – F1 generation (post-weaning):

No mortality occurred that was considered to be directly attributable to treatment with the test item. A total 13 F1 animals were found dead or were sacrificed in extremis during the first 4 days of treatment. The mortality incidence recorded over the dose groups did not show a dose-related trend. Based on the nature of the necropsy findings recorded for most of these animals in the respiratory tract (pale discolouration of the lungs, lungs not collapsed, foamy contents of the lungs or trachea), the cause of moribundity/death of animals showing these lesions was considered to be related to the gavage administration procedure, possibly in combination with reflux. None of the F1 animals that survived until their scheduled necropsy showed similar lesions, eg. in the respiratory tract.

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations, and body weight and food intake was considered not affected by treatment.

Treatment resulted in adverse macroscopic and microscopic alterations in the stomach of both sexes at 100 mg/kg/day (Cohort 1A-animals, PND 96 (nos. 456-459, 776-778), PND 100 (nos. 442-445) or PND 89-95 (other animals). These were in general consistent with the findings in the F0-animals, although these microscopic alterations in F1 -animals were recorded at a slightly lower incidence and severity and only occurred at 100 mg/kg/day.

These findings included mixed or lymphocytic inflammatory cell infiltrate (minimal-slight), hemorrhage (minimal), erosion (minimal-slight) and degeneration/regeneration of the glandular mucosa (slight).

Macroscopically, these findings were supported by thickening of the limiting ridge and foci in the glandular mucosa, which were also observed at similar incidences in Cohort 1B animals (≥ PND 97; no histopathological examination was conducted for Cohort 1B animals). These findings were considered to be the result of irritating properties of the test item, and correlated to necropsy findings in these dose groups (foci and in the glandular stomach and thickening of the limiting ridge).

Serum levels of TSH (thyroid stimulating hormone) and T4 were considered not affected by treatment.

No treatment-related effects were recorded for developmental parameters including balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, and sperm motility, concentration and morphology. Histopathologically, no test-item related effects were noted at stage-dependent qualitative evaluation of spermatogenesis, ovarian follicle and corpora lutea counts of females of Cohort 1A and morphology of reproductive organs.

No adverse changes in in-life or post-mortem developmental neurotoxicity endpoints were recorded for Cohort 2 animals. For Cohort 2A animals, these endpoints consisted of acoustic startle response, detailed clinical observations, rectal temperature, hearing ability, pupillary reflex, foot splay, grip strength and motor activity. For both Cohort 2A and 2B animals, developmental neurotoxicity endpoints consisted of fixed brain weights, brain dimensions (length and width of brain), routine sections of brain or peripheral nervous system and morphometric analysis of the brain. Non-adverse changes at 100 mg/kg/day consisted of a lower mean rectal temperature for males and lower motor activity for females. Mean rectal temperature of males was marginally reduced (0.98x). Given the minor degree of this change that occurred in the absence of any other corroborative changes, this was considered not adverse.

The lower motor activity for females (both for total movements and ambulations) was not supported by clinical observations or other functional observation tests, remained wthin the normal range for rats of this age and strain, and had no supportive morphological correlates in examined neuronal tissues.

Also, all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Overall these changes were considered not to represent an adverse effect on neurobehaviour.

No treatment-related changes in developmental immunotoxicity endpoints were recorded, i.e. splenic lymphocyte subpopulations, lymphoid histopathology and organ weights.

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohorts 1 and 2), the following no-observed-adverse-effect levels (NOAEL) of Hexahydro-1,3,5-trimethyl-1,3,5-triazine were established:

General toxicity (F0 and F1): 30 mg/kg/day (based on lesions in the glandular stomach at 100 mg/kg/day in F0 animals, and F1 animals).

Developmental neurotoxicity: at least 100 mg/kg/day.

Reproduction:       at least 100 mg/kg/day.

Development:       at least 100 mg/kg/day.