Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In life: 13 June 2018 - 24 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3. test method: EU B.56./OECD TG 443) in rats, oral route; with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation:
Ten weeks premating exposure duration is required because there is no substance specific information in the dossier supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment R.7a, chapter R.7.6 (version 4.1, October 2015).
- Dose level setting shall aim to induce some toxicity at the highest dose level:
The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1h)-one, sodium salt) via drinking water administration in rats, Test Facility Study No. 519710), and in an attempt to produce graded responses to the test item. In this preliminary study, treatment at 2000 ppm (corresponding to an average test item intake of 143 mg/kg/day in males and 195 mg/kg/day in females) resulted in a slight body weight loss in females during pre-mating and a reduced body weight gain (both sexes). Mean body weights were about 10% lower than controls. These effects were accompanied by a reduced food and water consumption (about 5% and 25%, respectively, after correction for body weight and when compared to controls). Moreover, the number of implantation sites at 2000 ppm was lower, which resulted in a lower mean litter size and a lower post-implantation survival index. The effects at 500 and 1000 ppm were limited to slightly lower body weights and body weight gains (both sexes), and a slightly lower food consumption in females at 1000 ppm during gestation only.
- Cohort 1A (Reproductive toxicity)
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation:
The conditions to include the extension of Cohort 1B are currently not met. Furthermore, no triggers for the inclusion of Cohorts 2A and 2B (developmental neurotoxicity) and Cohort 3 (developmental immunotoxicity) were identified. No information on other studies justified the inclusion of Cohorts 2A and 2B and Cohort 3.
- Route of administration:
The oral route is the most appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 4.1, October 2015) R.7a, chapter R.7.6.2.3.2. The administration of the first hydrolysis product NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1h)-one, sodium salt) via inclusion in the drinking water was considered to be the most suitable route based on analytical results and the corrosive effects of cyanuric chloride.

Test material

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

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
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. Charles River Den Bosch has general and reproduction/developmental internal data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 7 wks
- Weight at study initiation: (P) Males: 143-185 g; Females: 111-149 g
- 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. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam until termination or weaning (on PND 21).
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum except before blood sampling with a maximum of approximately 24 hours.
- Water: tap water (controls) or Drinking water preparations, containing the test substance (test groups)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 47-67
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 June 2018 To:24 December 2018

Administration / exposure

Route of administration:
oral: drinking water
Details on exposure:
JUSTIFICATION OF ROUTE:
The oral route of exposure was selected as specified in the final decision on a compliance check by ECHA. Due to its corrosive properties, oral gavage was not considered an appropriate route of expsoure for cyanuric chloride (CYC). Furthermore, It was technically not feasible to prepare diets with CYC, due to its hydrolytical instability. Therefore it was decided to perform a study with exposure via drinking water with the first hydrolysis product NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1h)-one, sodium salt).

PREPARATION OF DOSING SOLUTIONS:
The test item was diluted with the required amount of municipal tap water. Drinking water solutions were prepared at least weekly and were stored in the refrigerator for a maximum of 8 days if not used on the same day. The preparations were removed from the refrigerator and acclimatized for at least 30 minutes at room temperature before administration. From Week 3 of treatment onwards, after stability over 8 days at room temperature was confirmed, drinking water solutions were prepared at least weekly and stored at room temperature for a maximum of 6 days before use. No adjustment was made for specific gravity of the test item. A correction factor of 12.5 was used to correct for the purity/composition of the test item.
Details on mating procedure:
- M/F ratio per cage: 1:1 within the same treatment group, avoiding sibling mating after at least 10 weeks of treatment
- Length of cohabitation: until mating, maximally 2 weeks
- Proof of pregnancy: evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy

Detection of mating was not confirmed in first instance for two control females, one female at 16 mg/kg/day, one female at 55 mg/kg/day and two females at 160 mg/kg/day. Evidence of mating for these animals was obtained indirectly by delivery of a litter. The mating date of these animals was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. For one female at 16 mg/kg/day, evidence of mating was obtained by one implantation site recorded at necropsy; a mating date for 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:
Drinking water preparation samples were collected for analysis in week 1, 11 and 22. Test substance concentration was analysed for all groups, homogeneity was analysed in low and high dose group samples. Additional drinking water solutions were prepared in Week 1 of treatment, in order to conduct stability analysis over a concentration range that covers the complete study period; those drinking water preparations were not used for administration to the animals.
Analyses were performed using a validated analytical procedure (Test Facility Study No. 519709). 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 equal or less than 10%. During the course of this study at one occasion during the treatment phase, stability of the prepared drinking water containing test item was determined for 8 days at room temperature.
Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 519709) demonstrated that the test item is stable in tap water for 2 hours at room temperature and 8 days in the refrigerator when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
P-males: 11-12 weeks, including 10 weeks prior to mating.
P-females: 16-17 weeks, including 10 weeks prior to mating
P-females which failed to deliver: 14-16 weeks
F1 Cohort 1A: 9-11 weeks
F1 Cohort 1B: 10-11 weeks
F1 Cohort 1C: 2-5 weeks
F1 Cohort Surplus: not applicable
Cohort 1C is included to obtain a sufficient amount of animals for the assessment of sexual maturation (i.e. the same number of animals that would have been available for assessment of sexual maturation in case cohorts 2 and 3 were included).

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, via spilled water from the water bottles or when they commenced drinking from the water bottles themselves.
From weaning onwards (PND 21), F1-animals received drinking water with test item up to and including the day before scheduled necropsy (Cohort 1A) or until and including the day of necropsy (Surplus, Cohorts 1B and 1C).
Frequency of treatment:
The amount of test item incorporated in drinking water was kept at a constant level in terms of ppm, throughout one specified phase of the study period. After termination, the actual test item intake was estimated based on the body weight and water consumption values. The drinking water solutions were refreshed at least every other day at approximately the same time.
Doses / concentrationsopen allclose all
Dose / conc.:
200 ppm
Remarks:
Equal to actual mean test item intakes of 16 and 22 mg/kg bw/day for males and females, respectively; Target dose level 16 mg/kg bw/day
Dose / conc.:
600 ppm
Remarks:
Equal to actual mean test item intakes of 51 and 74 mg/kg bw/day for males and females, respectively; Target dose level 55 mg/kg bw/day
Dose / conc.:
2 000 ppm
Remarks:
Equal to actual mean test item intakes of 166 and 239 mg/kg bw/day for males and females, respectively; Target dose level 160 mg/kg bw/day
No. of animals per sex per dose:
P-animals: 25
F1-animals: 20 for each cohort (1A, 1B, 1C), 10 as surplus
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose levels in this study were selected based on the results of a preliminary reproductive toxicity study (reproduction/developmental toxicity screening test) with NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1h)-one, sodium salt) via drinking water administration in rats (Test Facility Study No. 519710), and in an attempt to produce graded responses to the test item.
In this preliminary study, treatment at 2000 ppm (corresponding to an average test item intake of 143 mg/kg/day in males and 195 mg/kg/day in females) resulted in a slight body weight loss in females during pre-mating and a reduced body weight gain (both sexes). Mean body weights were about 10% lower than controls. These effects were accompanied by a reduced food and water consumption (about 5% and 25%, respectively, after correction for body weight and when compared to controls). Moreover, the number of implantation sites at 2000 ppm was lower, which resulted in a lower mean litter size and a lower post-implantation survival index. The effects at 500 and 1000 ppm were limited to slightly lower body weights and body weight gains (both sexes), and a slightly lower food consumption in females at 1000 ppm during gestation only.
Based on the findings of the preliminary study, the highest concentration in the main study was established at 2000 ppm, and was expected to induce some degree of toxicity, but not to compromise the study objectives. The lowest concentration, 200 ppm, was expected to represent a NOAEL.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to first administration and at least once daily from start of administration onwards, up to the day prior to necropsy. In addition, clinical observations were conducted in a standard arena once before the first administration of the test item and at weekly intervals during the treatment period

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to administration), 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 : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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, 4-7, 7-11, 11-14, 14-17 and 17-20 post-coitum and during lactation over Lactation Days (LD) 1-4, 4-7, 7-14 and 14-21.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was quantitatively measured at least over every two days, from start of administration onwards up to the day of necropsy, except for males and females which were housed together for mating and for females without evidence of mating. Water consumption was also measured on the days the concentration of test item in the drinking water was changed. For mated females, water consumption was measured daily during post-coitum and lactation. During lactation, water consumption was determined daily.

OTHER:
General Reproduction Data – P-Generation: From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
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 sample 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. As for two animals abnormalities were noted in the left epididymis, the left side organ(s) was fixed in modified Davidson's solution, and the right side organ was used for evaluation of sperm numbers.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); surplus pups were killed and discarded (after blood collection in some pups, see below).
- On PND 4 at culling, blood was collected from two surplus pups per litter (from all litters, if possible) by decapitation, 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. As for several litters the target volume of 0.5 mL was not reached by pooling two pups, a third surplus pup of the same litter was added.

PARAMETERS EXAMINED
The following parameters were performed for the F1 pups until weaning (PND 21):
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups,

On PND 4, the pups scheduled for culling (in case of > 8 pups per litter) were euthanized by decapitation. Sex was determined both externally and internally. Pups were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development. Descriptions of all external abnormalities were recorded.
Spare F1-animals which were not assigned to one of the Cohorts were sacrificed between PND 23-25 by oral administration of sodium pentobarbital (Euthasol® 20%). Animals were externally examined, with particular attention to the external reproductive genitals to examine signs of altered development, and sex was determined (both externally and internally). Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director.

GROSS EXAMINATION OF DEAD PUPS:
Stillborn pups and pups found dead between birth and PND 13 were sexed (both externally and internally) and externally examined with emphasis on developmental morphology.
Descriptions of all external abnormalities were recorded. External abnormalities were collected and fixed in 10% buffered formalin at discretion of the Study Director. The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

ASSESSMENT OF REPRODUCTIVE/DEVELOPMENTAL ENDPOINTS:
The in-life procedures, observations, and measurements listed below were performed for all F1-animals from weaning (PND 21) onwards, except for the animals of Cohort Surplus and spare F1-animals as these were terminated on PND 22-24 or 23-25:
Mortality, clinical oibservations, including arena observations, body weights, food consumption, water consumption, vaginal patency (females), balanopreputial separation (males), stage of estrus determination (females).
Cohort 1A: Estrous cycle determination (females),

Blood and urine samples
Blood of 10 selected animals/sex/group of P-animals and Cohort 1A animals was collected on the day of scheduled necropsy, for hematology, coagulation, clinicl chemistry and thyroid hormone analysis. On PND 4 at culling, blood was collected from two surplus pups per litter, and samples were pooled per litter. On PND 22-24, blood was collected from all Cohort Surplus animals (10/sex/group), if possible. These F1 culled pups and cohort surplus samples were used for thyroid hormone analysis only.
Urine was collected into a specimen vial from the 10 selected animals/sex/group of P-animals and Cohort 1A animals housed in individual metabolism cages overnight (approximately 15-20 hrs) with absence of food, but water was available

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Cohort 2 was not included in this study.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Cohort 3 was not included in this study.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, after successful mating or at the end of the mating period
- Maternal animals: All surviving animals at lactation day 23-25.

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

HISTOPATHOLOGY / ORGAN WEIGHTS
The organs identified in attachment 1 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated. Representative samples of the tissues identified in attachment 1 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.
Postmortem examinations (offspring):
SACRIFICE
- F1 animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

Cohort 1A (fasted), PND 89-95: macroscopy and organ weights (20/sex/dose), histopathology (full list for controls and high dose, gross and target tissues for low and mid dose animals)
Cohort 1B (non-fasted), PND >90: macroscopy and organ weights (19/sex/dose), no histopathology in first instance
Cohort 1C (non-fasted), after positive vaginal patency.balanopreputial separation: macroscopy (19-20/sex/dose)
Cohort Surplus (non-fasted), PND 22-24: macroscopy and organ weights (10/sex/dose)
Non-selected F1 and spare litters (non-fasted), PND 23-25: macroscopy and tissue collection

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The organs identified in attachment 1 were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated. Representative samples of the tissues identified in attachment 1 were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution), unless otherwise indicated.

OTHER – Cohort 1A
The following additional assessments were performed:
Sperm analysis (al males), splenic lymphocytic subpopulation anaylsis (10/sex/group; 1/sex/litter)
In addition to the procedures described above, HE stained step sections of ovaries and corpora lutea at a thickness of 5 micrometers (5 step sections in total, including the routine section) were prepared. One of the ovaries will be quantitatively evaluated for follicles (primordial and small growing follicles counted together), as well as corpora lutea initially.
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.
Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Incidence: 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:
Mating index males (%): (Number of males mated/Number of males paired) x 100
Mating index females (%): (Number of females mated/Number of females paired) x 100
Precoital time: (Number of days between initiation of cohabitation and confirmation of mating
Fertility index males (%): (Number of pregnant females/Number of males mated) x 100
Fertility index males (%): (Number of pregnant females/Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100
Offspring viability indices:
Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Weaning index (%): (Number of live offspring on Day 21 after littering/Number live offspring on Day 4 (after culling)) x 100
Percentage live males at weaning (%): (Number of live male pups on Day 21 after littering/ Number of live pups on Day 21 after littering) x 100
Percentage live females at weaning (%): (Number of live female pups on Day 21 after littering/ Number of live pups on Day 21 after littering) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental findings that were noted included alopecia, wounds and scabs. These findings 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 not to be signs of toxicological relevance.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated animals were affected by treatment at 16, 55 and 160 mg/kg/day.
In males, body weights and body weight gain were statistically significantly lower than controls at 160 mg/kg/day from Week 2 of the pre-mating period onwards. Mean body weight gain was 0.79x and 0.78x of controls at the end of the pre-mating and mating period, respectively, resulting in a terminal body weight (on Day 80-85) of 0.87x of controls.
At 55 mg/kg/day, lower body weights were observed from Week 10 of pre-mating until Week 2 of mating, hereafter body weights appeared to recover for the remaining animals. At 16 mg/kg/day, lower body weights were observed from Week 9 of pre-mating until Day 1 of mating, followed by recovery to normal body weights. At necropsy, terminal body weights were lower with 0.94x and 0.93x compared with controls for treatment groups 16 and 55 mg/kg/day, respectively.
In females treated at 160 mg/kg/day, body weights were statistically significant lower than controls from Week 4 of the pre-mating period onwards. Mean body weight gain was 0.75x, 0.74x and 0.70x of controls at the end of the pre-mating, beginning of mating and end of post-coitum period, respectively, resulting in mean body weights of 0.91x, 0.91x and 0.83x of controls, respectively. Despite the still statistically significantly lower body weights at the end of the lactation period (0.92x), body weight gain increased during the lactation period for these animals, resulting in statistically significantly increased body weight gain at the end of the lactation period (2.1x).
In females treated at 55 mg/kg/day, body weights and body weight gains were lower than controls from Week 8 of treatment until Week 1 of the mating period. Mean body weight gain was 0.91x-0.89x of controls from Week 8 until Week 1 of mating, resulting in mean body weights of 0.96x (not statistically significant)-0.95x, respectively. Despite the still statistically significantly lower body weights on most days during the lactation period (0.93-0.95x), body weight gain increased during the lactation period for these animals, resulting in statistically significantly increased body weight gain at the end of the lactation period (1.5x).
Body weights and body weight gain for females at 16 mg/kg/day were considered to be unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was not affected by treatment up to 160 mg/kg/day in males and up to 55 mg/kg/day in females.
In females at 160 mg/kg/day, absolute food consumption levels were lower compared with controls during the post-coitum and lactation phase (0.86x and 0.88x, respectively). As relative food consumption levels were not affected, the lower food consumption levels were considered to be the result of the lower body weights observed for females during the post coitum period.
Any other statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and duration of treatment during the pre-mating and mating period.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption was affected by treatment up to 160 mg/kg/day in males and females.
In males, water consumption before correction for body weights was considered to be unaffected during the pre-mating and the mating period. The incidental changes observed in the two-daily water consumption measurements were considered to have arisen as a result of naturally occurring day to day variation and to be unrelated to treatment. The water consumption corrected for body weight was increased on multiple days of treatment at 16, 55 and 160 mg/kg/day.
In females, water consumption was considered to be increased, particularly during the first 5 weeks, during the premating and the mating period at treatment with 55 and 160 mg/kg/day. During the post-coitum and the lactation period, water consumption at 160 mg/kg/day was reduced on most days. The water consumption corrected for body weight was increased on multiple days of treatment at 16, 55 and 160 mg/kg/day during the pre-mating period.
Taken into account the direction of the change and in absence of corroborative morphological alterations, the increased (relative) water consumption was considered non-adverse.
Test item concentration was adjusted during the study to account for periodic changes in water consumption and body weight. The adjustments were based on the DRF study and historical control data.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, a decrease in mean corpuscular volume and mean corpuscular haemoglobin at 55 and 160 mg/kg/day and an increase in red blood cells at 160 mg/kg/day were noted. The increase in red blood cells may have been related to the increased incidence and severity in extramedullary hematopoiesis observed in the spleen at 160 mg/kg/day. In females, changes consisted of a decrease in lymphocytes, red blood cell distribution width and platelets at 160 mg/kg/day.
The expression of the haematological changes were mild in comparison with the controls and were in absence of degenerative changes; they were therefore considered non-adverse changes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical biochemistry changes consisted of an increase in total protein in males (55 and 160 mg/kg/day) and a decrease in creatinine in males (160 mg/kg/day) and females (at 55 and 160 mg/kg/day). The observed changes were considered slight and without morphological correlates.
In addition, an increase was noted in total T4 levels in males (55 mg/kg/day) and females (55 and 160 mg/kg/day). This increase in total T4 levels in males may have been related to the increased incidence and/or severity of follicular cell hypertrophy of the thyroid gland in males at 160 mg/kg/day, though this does not explain the increase in total T4 levels in females.
These changes were therefore considered non-adverse.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 160 mg/kg/day, an increase in pH (1.09x control) was noted in females, which was slightly above the upper limit of the historical control range.
In absence of corroborative microscopic findings, the effect on pH was considered non-adverse.
At 16 mg/kg/day, a decrease in specific gravity noted in males, was considered to have been the result of slightly increased control levels which may have been the result of the somewhat lower urine volumes and therefore unrelated to treatment.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related increases in organ weights were noted for the liver (males; +10% and +20% (absolute) at 55 and 160 mg/kg/day, respectively, and +18% and +38% (relative to body weight) at 55 and 160 mg/kg/day, respectively, compared to controls; females; +9% (relative to body weight) at 160 mg/kg/day) and kidney (males; +8% and +18 (relative to body weight) at 55 and 160 mg/kg/day, respectively, compared to controls; females; +7% (absolute) at 160 mg/kg/day, and +7% and +21% (relative to body weight) at 55 and 160 mg/kg/day, respectively, compared to controls). In absence of correlating morphological alterations and in absence of relevant liver enzyme changes (for the liver), these changes in organ weights were considered non-adverse.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related macroscopic findings. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. Watery fluid in the uterus, found in several females in all dose groups, is related to a stage in the estrous cycle and is a normal finding.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Thyroid gland: an increased incidence and/or (mean) severity of follicular cell hypertrophy and colloid alteration was present in males treated at 160 mg/kg/day.
In males at 160 mg/kg bw/day, follicular cell hypertrophy was minimal in 12/25 animals, and slight in 7/25 (controls: 15/25 minimal, 3/25 slight), the colloid alteration was minimal in 12/25 animals, and slight in 1/25 (controls: minimal 5/25, slight 0/25).

Spleen: an increased incidence and/or (mean) severity in extramedullary hematopoiesis and pigmentation was seen in males and females at 160 mg/kg/day.
In males at 160 mg/kg bw/day, extramedullary hematopoiesis was minimal in 14/25 animals, slight in 9/25, and moderate in 1/25 (controls: 13/25 minimal, 2/25 slight, 0/25 moderate), the pigmentation was minimal in 1/25 animals, slight in 16/25, and moderate in 8/25 (controls: minimal 13/25, slight 11/25 and moderate 1/25). In females at 160 mg/kg bw/day, extramedullary hematopoiesis was minimal in 7/25 animals, slight in 1/25, and moderate in 1/25 (controls: 2/25 minimal, 0/25 slight, 0/25 moderate), the pigmentation was minimal in 1/25 animals, slight in 15/25, and moderate in 9/25 (controls: minimal 14/25, slight 11/25 and moderate 0/25).

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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm motility, concentration, morphology and spermatogenesis were considered not affected by treatment.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 160 mg/kg/day, the number of non-pregnant females versus mated females was considered increased compared with controls. The number of non-pregnant females was 0/25, 1/25, 0/24 and 5/24 in the control, 16, 55 and 160 mg/kg/day groups, respectively, resulting infertility indices of
100%, 96%, 100% and 79%, respectively. As fertility is affected by body weight, the decreased fertility index may have been the result of the decreased body weights and body weight gains of the females observed at 160 mg/kg/day. No effects on sperm parameters, estrous cycle and microscopic correlates were noted that could have explained the decreased fertility index.


No test item-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, precoital time, number of implantations and histopathological examination of reproductive organs).
Gestation indices were 100%, 96%, 100% and 100% for the control, 16, 55 and 160 mg/kg/day groups, respectively. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 95%, 95%, 94% and 93% for the control, 16, 55 and 160 mg/kg/day groups, respectively. The live birth indices were 100%, 100%, 100% and 99% for the control, 16, 55 and 160 mg/kg/day groups, respectively. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
55 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
16 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No mortality occurred that was considered to be attributable to treatment with the test item.
One male at 55 mg/kg/day was sacrificed in extremis on Day 18 post weaning, based on abnormal posture and/or gait, tilted head, a lean appearance and slightly lower body weight. At macroscopy, a misshapen spinal cord was the only relevant finding, correlating to the observations made in-life for this animal.
One male at 160 mg/kg/day was sacrificed in extremis on Day 32 post weaning, based on overgrowth teeth, causing food intake issues and resulting in lower body weight (gain) and slight body weight loss (-5%) in the week prior to sacrifice. At macroscopy, overgrown teeth were confirmed and in addition, enlarged mandibular lymph nodes were observed.
On Day 9 post weaning, one animal (dosed at 16 mg/kg/day) appeared to be a male animal instead of a female animal and was sacrificed.
These deaths were all considered to be unrelated to treatment.

Viability indices (number of live offspring on Day 4 before culling compared to the number of offspring on Day 1) were thus 99%, 98%, 96% and 98% for the control, 16, 55 and 160 mg/kg/day groups, respectively. The weaning indices (number of live offspring at weaning (PND 21) compared to the number of live offspring on Day 4 (after culling)) were 99%, 100%, 100% and 100%% for the control, 16, 55 and 160 mg/kg/day groups, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 160 mg/kg/day, body weights of male and female pups were statistically significant decreased during the complete lactation period. The decreased body weights were likely the result of the decreased body weights observed for the dam during the post coitum period. As the body weights of pups did not recover to normal levels by the end of the lactation period, this finding was considered adverse.
Post-weaning, test item-related statistically significant decreased body weights were observed in males from 16 mg/kg/day onwards and in females from 55 mg/kg/day onwards and test item-related decreased body weight gains were observed in males at 160 mg/kg/day. The decreased body weights at the start of weaning were considered to have arisen from the slightly decreased body weights at PND 21. As body weight gains did not recover to normal levels for males at 160 mg/kg/day, the decreased body weight and body weight gain for males at 160 mg/kg/day was considered adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 160 mg/kg/day, food intake was lower throughout the treatment period, when compared with controls. Mean food consumption in Week 10 of treatment was 0.88x and 0.78x for males and females, respectively. For males at 55 mg/kg/day, food intake was also statistically significantly lower on several occasions during the treatment period, with the mean food consumption being 0.96x of controls in Week 10.
Food consumption after correction for body weight was statistically significantly increased for males at 55 mg/kg/day (on some occasions) and for both sexes at 160 mg/kg/day.
The lower food consumption levels were considered the result of the lower body weights of these animals and was therefore considered non adverse.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption was lower during the complete treatment period for animals treated at 160 mg/kg/day, reaching statistical significance on most occasions for the males and on approximately half of the occasions for the females. Mean water consumption over the complete treatment period was 0.87x and 0.88x of controls for males and females, respectively.
The lower water consumption levels were considered the result of the lower body weights of these animals and was therefore considered non adverse.
Test item concentration was adjusted during the study to account for periodic changes in water consumption and body weight. The adjustments were based on the DRF study and historical control data.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Similar to the P-generation, non-adverse changes were noted in males of the F1-generation for hematology and clinical chemistry parameters. Test item-related changes in red blood cell distribution width and decreased mean corpuscular haemoglobin and potassium at 160 mg/kg/day, were considered non-adverse, in absence of correlating microscopic alterations and at the low magnitude of the effects.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant increases in reticulocytes observed in males and females at 160 mg/kg/day, were possibly related to the observed extramedullary hematopoiesis and/or pigmentation in the spleen. As the observed slight alterations were in absence of degenerative changes, these effects were considered to be non-adverse.
Serum T4 levels in male and female pups culled at PND 4 were considered not to be affected by treatment. Serum T4 levels in male and female pups of Cohort Surplus at PND 22-24 were statistically significantly higher at 160 mg/kg/day. A statistically significant increase was noted in total T4 levels in males and females (at 55 and 160 mg/kg/day). Also, this increase in total T4 levels in males may have been related to the thyroid gland findings in males observed at 160 mg/kg/day, though this does not explain the increase in total T4 levels in females. As all values were considered within normal range and in absence of degenerative changes, the changes in total T4 levels were considered non-adverse.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinary parameters were unaffected by treatment.
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
The age at which balanopreputial separation (prepuce opening) occurred in males was unaffected by treatment.
The age at which vaginal patency (vaginal opening) was attained in females was unaffected by treatment up to 160 mg/kg/day. A statistically significant increase in the age of reaching vaginal patency was noted at 160 mg/kg/day (1.05x control). This increase was considered the result of the lower body weights of the animals on the day of reaching vaginal patency (0.89x control), and was therefore considered not to be a direct response to treatment.
For animal Nos. 556, 761, 772, 786 and 807, a vaginal thread was observed on the day of vaginal patency . For animal No. 772, a vaginal thread was observed at least until first estrous was reached.
For Cohort 1A, the time between acquisition of vaginal patency and the first estrus was considered to be unaffected by treatment.
Time until first estrous was on average 4, 4, 4 and 5 days for the control, 16, 55 and 160 mg/kg/day groups, respectively. First estrous was observed on average on PND 36, 34, 36 and 38, respectively. As values generally remained within the historical control range , these slight (non-statistical) differences were considered to be of no toxicological relevance. Length and regularity of the estrous cycle were not affected by treatment at 160 mg/kg/day. 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.
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 to be affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 160 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):
Test item-related statistically significant decreased axillary lymph node weights were observed in females of Cohort 1A at 160 mg/kg/day. In absence of correlating morphological alterations, this change in weights was considered non-adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the thyroid gland, an increased incidence and/or (mean) severity of follicular cell hypertrophy and colloid alteration was present in males treated at 160 mg/kg/day.
Follicular cell hypertrophy was minimal in 7/20, 8/20, 9/20 and 9/20 males at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 2/20, 0/20, 4/20 and 6/20 males at 0, 16, 55, and 160 mg/kg bw/day. The colloid alteration was minimal in 1/20, 2/20, 2/20 and 6/20 males at 0, 16, 55, and 160 mg/kg bw/day, respectively.

In the spleen, an increased incidence and/or (mean) severity of extra medullary hematopoiesis was seen in males and females at 55 and 160 mg/kg/day. Pigmentation was increased in incidence and/or (mean) severity in males at all doses and in females at 160 mg/kg/day.
In males, extramedullary hematopoiesis was minimal in 11/20, 11/20, 12/20 and 12/20 animals at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 2/20, 3/20, 6/20 and 8/20 animals at 0, 16, 55, and 160 mg/kg bw/day. In females, extramedullary hematopoiesis was minimal in 8/20, 5/20, 11/20 and 11/20 animals at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 1/20, 1/20, 4/20 and 7/20 animals at 0, 16, 55, and 160 mg/kg bw/day,
In males, pigmentation was minimal in 12/20, 18/20, 18/20 and 15/20 animals at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 0/20, 1/20, 1/20 and 4/20 animals at 0, 16, 55, and 160 mg/kg bw/day. In females, pigmentation was minimal in 13/20, 13/20, 12/20 and 5/20 animals at 0, 16, 55, and 160 mg/kg bw/day, respectively, and slight in 7/20, 6/20, 8/20 and 14/20 animals at 0, 16, 55, and 160 mg/kg bw/day, Moderate pigmentation was seen in 1 female each at 16 and 160 mg/kg bw/day.

A test item-related statistically significant decrease in ovarian follicle counts (primordial and primary oocytes) was noted at 55 and 160 mg/kg/day. However, the variation in follicle counts in control females was rather high and the number of ovulating follicles were likely the same as the number of corpora lutea was not decreased by treatment with 160 mg/kg/day. In addition, no histological changes or organ weight changes of reproductive organs were noted, and therefore, there was no indication that the reproductive function was adversely affected by treatment up to 160 mg/kg/day. Furthermore, the use of ovarian follicle counts is commonly used only as a second-tier screening method as the initial reproductive toxicity will more commonly be detected by qualitative microscopic assessment or other standard methods.



Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
Splenic lymphocyte subpopulation were considered to be unaffected by treatment.
Statistically significant shifts in T-cell, T-helper cell, T-cytotoxic cell and B-cell splenic lymphocyte subpopulations in males at 55 mg/kg/day when compared to splenic lymphocyte subpopulations in control males were minor in magnitude and occurred in the absence of a dose-related trend. In addition, histopathology findings in spleen of F1-animals were unrelated to lymphocytes. Therefore, these shifts were considered to represent biological variability and considered to be unrelated to treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
55 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
160 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this extended one generation reproductive toxicity study (Cohort 1) performed according to OECD guideline 443 and following GLP principles, the following no-observed-adverse-effect level (NOAEL) of NHDT-solution (4,6-dichloro-1,3,5-triazin-2(1H)-one, sodium salt) were established (the presented NOAELs are target dose levels):
General toxicity (P and F1): 16 mg/kg/day (based on body weights and body weight gain in females)
Reproduction NOAEL (P): 55 mg/kg/day (based on a lower fertility index; possibly secondary to lower body weights)
Developmental NOAEL (P and F1): 55 mg/kg/day (based on lower body weight gains in males; possibly secondary to lower maternal body weights)