Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Oral: sub-chronic, rat, OECD 443, GLP, JRF 2020

- The NOAEL for systemic toxicity is 100 ppm (dietary equivalent of 7.44 (males) and 11.83 (females) mg/kg bw/day for P0 and 10.11 (males) and 11.94 (females) mg/kg bw/day for F1 Cohort 1B), based on lower body weight (gain) and food consumption, higher liver weights, greyish discolouration of the liver and hypertrophic liver and thyroid;

- The NOAEL for fertility/reproductive toxicity is => 300 ppm (highest dose; P0: dietary equivalent of 22.11 and 27.43/25.46 mg/kg bw/day, for males and females, respectively; F1 Cohort 1B: dietary equivalent of 30.05 and 25.14/26.34 mg/kg bw/day, for males and females, respectively), based on the absence of any treatment-related adverse effects on any fertility and reproductive performance parameters;

- The NOAEL for developmental toxicity (Offpring (F1)) is 100 ppm (dietary equivalent of 7.44 and 13.1 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain), lower body temperature, lower liver weight and depleted glycogen in the liver.

- The NOAEL for developmental toxicity (Offspring (F2)) is 100 ppm (dietary equivalent of 10.11 and 13.71 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain).

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Jul 2018 to 12 Apr 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals : The male and female rats of the parental (P) generation were treated for at least ten weeks prior to mating. In this specific case ten weeks exposure duration is supported by the lipophilicity of the substance to ensure that the steady state in parental animals has been reached before mating.
- Basis for dose level selection : The dose levels were selected based on results of a dose range finding study (496-1-04-19024).
- Inclusion of extension of Cohort 1B, because the uses of the registered substance is leading to significant exposure of professionals and consumers, and there are indications that the internal dose for the registered substance will reach a steady state in the test animals only after an extended exposure.
- Termination time for F2 : F2 pups were sacrificed on postnatal day (PND) 21
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B, no triggers for the inclusion were identified.
- Exclusion of developmental immunotoxicity Cohort 3 , no triggers for the inclusion were identified.
- Route of administration : Oral, through diet, Since the substance to be tested is a crystalline solid, ECHA concludes that testing should be performed by the oral route.
- Other considerations, on choice of species: The rat is the preferred test system because it is a readily available laboratory animal. The rat has been historically shown to be an acceptable animal for reproduction toxicity testing and is recommended by the OECD and other regulatory authorities.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000091655
- Expiration date of the lot/batch: 31 August 2018, The test item was expired during the experimental phase of the study as per the declared expiry date on the CoA (Certificate of Analysis) provided by the Sponsor. To establish the stability of the test item, the active ingredient content was analysed concurrently with the dosing period i.e., within one week before declared expiry and within one week after completion of dosing following the validated analytical method. The CoA and comparison of analysed purity are reported in the final report. Analysis of the active ingredient demonstrated that the content of the test item was within experimental variation of previously established purity of 98.00% and that the purity of the test item was unchanged.
- Purity: 98% (analysed purity); 97.83% (Verification of purity before dosing); 97.79% (Verification of purity after dosing)
- Appearance: White to off-white solid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: As per the instruction received from the Sponsor on storage of the test item, the test item was stored: Storage Temperature : Room Temperature; Storage Container : In original container as supplied by the Sponsor
- Stability under test conditions: The test item is stable up to 14 days at room temperature based on the results of the method validation study.
Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jai Research Foundation
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5-6 weeks
- Housing: During the experiment period, rats were housed in groups of 2/3 rats/cage/sex and enrichment material was provided. During the mating period, rats were housed in groups of two rats/cage (one male and one female). Mated female rats were housed individually and nesting material were provided near parturition. During the study, rats were housed in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless-steel top grille having provision for a polypropylene water bottle with stainless steel drinking nozzle. Separate food hoppers were attached to the cages for diet. The bottom of the cages was layered with clean sterilised rice (paddy) husk as the bedding material. Cages were placed on 5 and 6 tier racks. Cages were changed at a minimum of twice a week. Cages were arranged in such way that possible effects due to cage placement are minimised. Contaminant analysis (chemical and microbial) of samples of the bedding material (paddy husk) were performed at six months intervals and the most recent results were included in the final report.
- Diet: The experimental rats were fed ad libitum with standard rodent diet. The standard pellet food was powdered (using a grinder) before offering to animals or mixing with the test item. Every food consignment received was accompanied with a certificate of analysis of nutrient content from the food supplier. The most recent results of microbial contaminant analyses were included in the final report.
- Water: unlimited supply of clean and filtered drinking water (filtered through reverse osmosis water filtration system) in polypropylene bottles. The most recent results of microbial and chemical contaminant analyses were included in the final report.
- Acclimation period: 6 days (prior to commencement of randomisation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 57-67
- Air changes (per hr): 16-17
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: test diet was prepared at least once a week.
- Mixing appropriate amounts with powder diet: The test item was mixed with the 10% of total diet or 500 g of food (whichever was higher from the total quantity of diet prepared) in a blender in order to prepare the pre-mix. To this pre-mix diet, the remaining amount of diet was added and mixed. This whole amount of diet, thus prepared, was mixed in a blender and made available to the animals ad libitum throughout the study.
- Storage temperature of food: The prepared test diet was stored in labelled stainless steel containers covered inside with polythene bags, at the experimental room condition.
Details on mating procedure:
- M/F ratio per cage: 1:1 pairing (unrelated, same dose group)
- Length of cohabitation: until evidence of copulation was observed or 2 weeks had elapsed
- Proof of pregnancy: presence of sperm or copulatory plug referred to as day 0 of pregnancy;
- If mating had not occurred after 2 weeks, the rats were separated without further opportunity for mating and considered as a presumed mated.
- After successful mating each pregnant female was caged: individually
- To produce the F2 generation, 1 male and 1 female rat from the F1 (Cohort 1B) generation were randomly selected and subsequently mated (14-15 weeks of age) in a 1:1 ratio. Care was taken to avoid sibling mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The active ingredient (a.i.) concentration and homogeneity of the test item in the test diet was analysed once before initiation of treatment and at one-month intervals during the treatment period. Duplicate samples of control from middle and triplicate samples from different locations (i.e., top, middle and bottom of the container) of the test diet from each group were taken to determine the homogeneity and concentration of the test item in the treated diet. On each occasion of test diet analysis, the mean concentration was determined and compared to the nominal value. The acceptance criteria were ± 20% deviation from nominal value and %CV < 20. The samples were analysed using a validated analytical method. Following instrumental parameters will be used for analyses: Instrument : HPLC; Column : 250 mm x 4.6 mm (i.d), 5 µm particle size; Wavelength (nm) : 210; Injection volume (µL) : 20; Flow rate (mL/minute) : 1.0; Mobile phase : Solvent (A) - Acetonitrile (80%), Solvent (B) - 0.1 % Orthophosphoric acid in Milli-Q Water (20%)
Duration of treatment / exposure:
- Parental (P) and F1 (selected for Cohort 1A and 1B) rats were offered the control or test diet continuously on a 7-days/week basis, till the scheduled necropsy. Exposure to the test item to both P sexes were commenced ten weeks prior to mating and continued during the two-weeks mating period. After mating, P male rats were further exposed, up to, and including the day before scheduled sacrifice (i.e. till delivery of P females). P female rats were further exposed during gestation and up to weaning of F1 pups. Unless already initiated during the lactation period, direct treatment of the selected F1 male and female pups began at weaning and continue, until the scheduled necropsy.
- Cohort 1B rats was maintained on treatment beyond PND 90 and bred to obtain a F2 generation. Male rats and female rats were 14-15 weeks old at the time of mating. Procedure of mating was the same as described for the P animals. F2 generation pups were evaluated up to PND 21.
Frequency of treatment:
Continuous
Details on study schedule:
- F1 parental animals not mated until Postnatal day 90 after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age (PND 22).
- Age at mating of the mated animals in the study: 14-15 weeks
Dose / conc.:
50 ppm (analytical)
Remarks:
G2: Low Dose; P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
Dose / conc.:
100 ppm (analytical)
Remarks:
G3: Mid Dose; P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
Dose / conc.:
300 ppm (analytical)
Remarks:
G4: High Dose; P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
No. of animals per sex per dose:
P: 25 rats/sex/group
F1 (Cohort 1A/1B): 20 rats/sex/group
Control animals:
yes, plain diet
Details on study design:
- Dose Justification: The dose levels were selected based on results of a dose range finding study (496-1-04-19024)
- Selection of Pups for Post-Weaning Evaluations on Post-natal Day: A) Cohort 1A: One male and one female F1 pup per litter (20 pups/sex/group; wherever possible) were randomly assigned for primary assessment of effects on reproductive systems and general toxicity. , B) Cohort 1B: One male and one female F1 pup per litter (20 pups/sex/group; wherever possible) were randomly assigned for follow-up assessment of reproductive performance by mating F1 rats.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Rats were observed daily, twice, for mortality and morbidity. All visible clinical signs were noted and recorded daily, once, throughout the study.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical examination of all P and F1 rats (after weaning) was performed on a weekly basis, on the day of the body weight recording. Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of the clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards) were also recorded.

BODY WEIGHT:
Body weight of P male rats was recorded on the first day of treatment and weekly thereafter. Body weight of P female rats was recorded on the first day of treatment and weekly thereafter during the pre-mating and mating period. Body weight of P female rats was recorded on gestation day 0, 7, 14 and 20, and lactation days 0, 4, 7, 14, and 21.
On the day of fasting, body weight of respective rats was recorded. Body weight of all rats was also recorded on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The food consumption was determined by weighing of food input and leftover. Food weight of all P male rats was measured weekly during pre-mating and post-mating period. In P female rats, during pre-mating period, food weight was recorded, at weekly intervals. During gestation period, food weight was recorded, on gestation days 0, 7, 14, and 20. During lactation period, food weight was recorded, on lactation days 0, 4, 7, 14, and 21. Additional food was offered as and when required. Food consumption was not measured during mating period. After weaning, the cage wise food weight was recorded at weekly interval, till necropsy.
Compound intake was calculated as time-weighted averages from the consumption and body weight gain data.

BLOOD AND URINE COLLECTION
At the time of the terminal sacrifice, blood (maximum 3.5 mL) was collected from P and F1 (Cohort 1A) rats (except female rats sacrificed on GD 25) under anaesthesia (isoflurane) by orbital plexus puncture. Rats were deprived of food overnight (water provided), prior to the blood collection. Blood samples were collected for haematology (in vials containing 4% EDTA), coagulation parameters PT and APTT (in vials containing 3.2% sodium citrate), clinical chemistry, and thyroid hormone analysis (in vials without anticoagulant). Samples were collected before 13:00 hours per day. Serum samples were preserved at -70 ± 10 °C, till their analysis. Any residual/retained serum samples will be discarded after confirmation for the finalisation of the study report.
Urine samples were collected overnight from 10 rats/sex/group (P and cohort 1A) in rat metabolic cages using graduated collecting tubes.

CLINICAL PATHOLOGY OBSERVATIONS
The haematology, clinical chemistry, and urine parameters as described in Tables 2, 3 and 4 in 'Any other information on materials and methods, incl. tabels' were analysed from 10 rats/sex/group (from P and Cohort 1A).

THYROID HORMONE ANALYSIS
Serum thyroid hormones T3, T4, and TSH were analysed. Level of T3 and T4 in serum was analysed using a validated bioanalytical method. Level of TSH in serum was analysed through ELISA methods provided in the kit literature. Serum thyroid hormones T3, T4, and TSH levels were analysed as specified in Tables 5 and 7 in 'Any other information on materials and methods, incl. tables'.

LYMPHOCYTE SUBPOPULATION ANALYSIS
From randomly selected 10 F1 rats (cohort 1A)/group/sex, spleen was cut half (transverse section) and used for lymphocyte (phenotypic) subpopulation analysis. From the collected spleen, homogenate was prepared and used for T lymphocytes (CD4+ and CD8+), B lymphocyte, and natural killer cell evaluation using flow cytometer. The other half part of the spleen was preserved for the histopathological examination.
Oestrous cyclicity (parental animals):
Oestrous cycle length and pattern was evaluated by the vaginal smears for all P female rats for two weeks period, prior to the mating and throughout the cohabitation/mating period. It was ensured to avoid disturbance of mucosa while obtaining vaginal smear.
Vaginal smear was taken for F1 (Cohort 1A) female pups since the day of vaginal opening till the first oestrus phase was observed. Oestrous cycle length and pattern was evaluated for, two weeks period prior to the terminal sacrifice by vaginal smears for all F1 female rats belonging to the cohort 1A. Vaginal smear was evaluated in cohort 1B since the time of pairing until the mating evidence was observed.
At the time of necropsy, the vaginal smear was examined (from P and F1 rats) to determine the stage of the oestrous cycle.
Sperm parameters (parental animals):
SPERM PARAMETERS
Sperm count was performed in the control and high dose male rats belonging to P and F1 (cohort 1A) generation. Immediately after the sacrifice, right cauda epididymis, and right testis were excised, weighed, and stored at -20 ± 3 °C for enumeration of cauda epididymal sperm reserves and homogenisation - resistant spermatids, respectively. Vas deferens was used for the assessment of sperm motility (performed in all-male rats of P and F1 generation - cohort 1A) and sperm morphology (performed in control and high dose male rats belonging to P and F1 generation – cohort 1A).
Sample of right cauda epididymis and right testis belonging to the low and mid-dose groups will be discarded at the time of report finalisation.

SPERM MOTILITY
Immediately after sacrifice, right vas deferens was placed in a petri dish containing 3 mL of Dulbecco's Phosphate Buffered Saline (maintained at 36-38 °C) on a slide warming table. The vas deferens was pressed from approximately the mid-point with gentle pressure to allow the sperm ‘swim out”. After completion of this step, the mixture of the sperm and medium was pipetted (one drop) directly onto a pre-warmed slide for motility assessment under a light microscope at 40x magnification. Motility assessment of the sperm was determined based on the movement of the sperm and respective motility grades were assigned.

SPERM MORPHOLOGY
After the assessment of sperm motility, about 1 mL of sperm suspension was transferred to a test tube (10 x 75 mm) and diluted to 4 mL with Dulbecco's Phosphate Buffered Saline, followed by an addition of 1 drop of 5% eosin Y. The contents of the test tube were gently agitated by “flicking” the bottom of the test tube with a fingertip. These samples were then incubated at the room temperature for approximately 30 minutes to allow staining. At the end of the staining period, sperm was re-suspended gently with a pasture pipette. Using pasture pipette, 1 or 2 drops of stained solution was spread uniformly on the glass slide. Two slides were prepared for each rat. These slides were placed at an angle of 35 - 40° on a tray whose surface was covered with absorbent tissue paper to remove excess stained suspension. These slides were air-dried and mounted with DPX. 200 sperms/slide were evaluated for morphological abnormalities from the control and high dose rats belonging to P and F1 generation (cohort 1A).

SPERM COUNT
Frozen right cauda epididymis and testis were removed from the freezer and maintained at the room temperature for approximately 10 to 15 minutes for thawing. A known volume of 15 mL saline merthiolate-triton (SMT) for cauda epididymis and 25 mL SMT for testis was used. The tissue was homogenised for 2 to 3 minutes and allowed the froth to dissipate for a few minutes. The samples were loaded on the chamber of the haemocytometer using a micropipette. The sperms were left undisturbed for approximately 5 minutes, to settle down, prior to their count. When the mean count of the five squares in the first chamber varied by more than 15% on either side of the mean value, the second chamber was loaded, and sperm were counted in a similar manner to that of the first chamber. In this case, the final mean value was calculated from all squares counted. The number of sperm head per gram of tissue was calculated.
Litter observations:
STANDARDISATION OF LITTERS
On postnatal day (PND) four, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five male pups and five female pups per litter. Wherever the number of male pups or female pups prevents having five of each sex per litter, partial adjustment (e.g., six male and four female) was performed. Adjustments were not done for litters of ten pups or less.

PUPS OBSERVATIONS
Each litter was examined as soon as possible, after parturition to establish the number and sex of pups, stillbirths, runts, live birth, and the presence of gross anomalies. Found dead pups, on the day of littering, which were not macerated were examined for possible defects and cause of death. Pups which died during study were weighed and subjected to the post-mortem examination. Each pup was observed for the presence of milk band in the stomach from PND 0 to 4, as a part of the nursing care.
In addition, all live pups on day of delivery were observed for the first clinical examination of the pups which included a qualitative assessment of the body temperature, state of activity, and reaction to handling.

BODY WEIGHT
Live pups (F1 and F2) were counted and weighed individually at birth or soon thereafter and on postnatal day 4, 7, 14, 21, and 22.

ANOGENITAL DISTANCE (AGD)
The anogenital distance (AGD) of each pup (F1 and F2) was measured on PND 0.

NIPPLE RETENTION
On PND 13, nipples/areolae in male pups was observed. As no nipple presence was observed, hence counting was not performed.

PHYSICAL AND SEXUAL DEVELOPMENT LANDMARKS
The day for unfolding of pinna, ear-opening, eye-opening, tooth eruption, and hair growth were recorded on their respective day of occurrence.
The age at the vaginal opening or balano-preputial separation was determined for the F1 weanlings selected for the subsequent evaluations. F1 pups were also weighed on the day of the vaginal opening or balano-preputial separation attended.

BLOOD AND URINE COLLECTION
Blood was collected from F1 surplus pups on PND 4 and F1 weanling pups on PND 22, through decapitation and orbital plexus puncture, respectively for the serum thyroid hormone analysis. Blood was collected from the minimum 1 randomly selected male and female pup per litter, wherever feasible. To increase the sample volume, blood from the additional pups was also collected in some dams where pups were available.

THYROID HORMONE ANALYSIS
Serum thyroid hormones T3, T4, and TSH were analysed. Level of T3 and T4 in serum was analysed using a validated bioanalytical method. Level of TSH in serum was analysed through ELISA methods provided in the kit literature. Serum thyroid hormones T3, T4, and TSH levels were analysed as specified in Table 6 in 'Any other information on materials and methods, incl. tables'.
Postmortem examinations (parental animals):
SACRIFICE
Schedule for sacrifice: P male rats were sacrificed after the delivery of P female rats. P female rats were sacrificed on lactation day 22. Cohort 1A rats were sacrificed during the 14-weeks of the treatment period. Cohort 1B male rats were sacrificed, following during the 21 weeks of the treatment period. Cohort 1B female rats were sacrificed on lactation day 22. Females, which have not delivered by post-coitum day 25, were sacrificed on the same day. Female rats with no live pups were kept fasting on the same day when the observation was made and were sacrificed on the next day.

GROSS NECROPSY
Surviving rats (including weanling pups) were sacrificed, by using an overdose of carbon dioxide. Gross pathological observations were made for all adult rats (P and F1). Gross necropsy was conducted under the direct supervision of a veterinary pathologist. Rats were examined carefully for the external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. It was ensured to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of the organs was carried out to detect abnormalities. Special attention was paid to the organs of the reproductive systems during the gross examination of all rats. The uteri of all cohabited female rats were examined for the presence and number of implantation sites. Number of corpora lutea were recorded from those female rats in which implants were observed on the gestation day 25.

ORGAN WEIGHTS
On the day of the terminal sacrifice, wet weight of the below-mentioned organs (see Table 1 in 'Any other information on materials and methods incl. tables') from all adult rats (P and F1) were determined as soon as possible after dissection to avoid drying. Paired organs were weighed individually except four rats of GD 25. The organs were weighed and preserved in 10% NBF except testes and eyes in a modified Davidson’s fixatives and Davidson’s fixatives, respectively.

HISTOPATHOLOGY
Histopathology of the organs was performed on all high dose and control rats. Organs (liver – target organ and thyroid) demonstrating treatment-related changes were examined in the low and mid-dose groups from Parent and cohort 1A rats.
Parent animals: Histopathology was performed for the organs listed in Table 1 in 'Any other information on materials and methods incl. tables'. Additionally, histopathology of the reproductive organs of rats suspected of reduced fertility was performed in dams sacrificed on GD 25 and their respective male rats and also in dams in which oestrous cycle was affected. Gross lesions were subjected to histopathological evaluation.

F1 (Cohort 1A) animals: Histopathology was performed for the organs listed in Table 1 in 'Any other information on materials and methods incl. tables'. Quantitative evaluation of primordial and small growing follicles, as well as corpora lutea (from 10 rats of each group G1 and G4), oviduct, uterus, and vagina were examined for appropriate organ-typic development. Detailed testicular histopathology examinations were conducted on the F1 male rats in order to identify treatment-related effects on testes differentiation and development and on spermatogenesis. Periodic Acid Schiff (PAS) and hematoxylin and eosin staining were used for examination of the testes. Caput, corpus, and cauda of the epididymis and the vas deferens were examined for appropriate organ-typic development.

F1 (Cohort 1B) animals: Organs/tissues of cohort 1B rats were processed to the block stage.
Postmortem examinations (offspring):
SACRIFICE
Schedule for sacrifice: Unselected weanlings of F1 were sacrificed on PND 22. F2 pups were sacrificed on the PND 21.

GROSS NECROPSY
Surviving rats (including weanling pups) were sacrificed, by using an overdose of carbon dioxide. Culled pups on PND 4 (not subjected for hormone analysis) were sacrificed, through intraperitoneal administration of thiopentone sodium. Gross pathological observations were made for all adult rats (P and F1) and all pups (F1 and F2). Gross necropsy was conducted under the direct supervision of a veterinary pathologist. Rats were examined carefully for the external abnormalities. After opening the abdominal cavity, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the rat. It was ensured to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened, and a thorough examination of the organs was carried out to detect abnormalities. Special attention was paid to the organs of the reproductive systems during the gross examination of all rats including pups.
Pups, which were found dead on the day of delivery (i.e., PND 0), were subjected to a gross examination and a portion of the lung was immersed in water for confirmation of the status (stillbirth or dead). Pups which were found dead were observed for the presence of milk band.
Pups, which died before scheduled sacrifice (during PND 0 to 21) were examined, for gross lesions and the cause of death or condition, as soon as possible, after the observation is made. In case of a pup with gross lesions (PND 0 to 21), the whole pup was kept in Bouin’s fluid. Pups without gross lesions were discarded, after examination.

ORGAN WEIGTHS
See Table 1 in 'Any other information on materials and methods incl. tables'.

HISTOPATHOLOGY
Microscopic examination of liver (target organ) from F1 and F2 pups was carried out from control and all dose group rats (10 pups/sex/group).
Statistics:
The statistical analysis was carried out for the parameters using validated statistical software. Non-pregnant rats were excluded from analysis. Assumed pregnant rats were excluded from analysis of gestation phase parameters. Pups data (litter size, AGD, pups body weight, and physical development landmark) was evaluated using the litter as the unit for data analysis. All parametric data were analysed to calculate the significance at 95% and 99% whereas non-parametric data was analysed to calculate significance at 95%.
Data such as body weight (gain), food consumption, food efficiency, physical & sexual development landmark, organ weight (ratio), % pre-natal loss, % post-natal loss, male sex ratio, oestrous cycle length, sperm count, % sperm morphology, haematology, clinical chemistry, urinalysis, thyroid hormone, and litter parameters (pups body weight (gain), body temperature, and AGD) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data did not meet the homogeneity of variance, statistical analysis was extended following a decision tree as provided by Gad, S.C., 2007. AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.
Count data (viz., litter size, number of implants, pre-coital interval, duration of gestation, number of oestrous cycles; pre-natal loss, post-natal loss) were subjected to non-parametric test i.e., Kruskal-Wallis test. The non-parametric data (sperm morphology - number and ovarian follicular count) was analysed using the Mann-Whitney test.
Non-parametric indices such as mating, fertility, gestation, parturition, pregnancy rate, survival, mortality, live birth and lactation were analysed using a Chi-Square test.
Flags for significant difference between control and treated groups (single arrow for p≤0.05 and double arrows for p≤0.01) were given in the table along with the footnote.
Reproductive indices:
Mating index = Number of mated animals / Number of paired animals x 100
Male fertility index = Number of males impregnating female / Number of males used for cohabitation x 100
Female fertility index = Number of females with confirmed mating / Number of female cohabited x 100
Gestation index = Number of females with live-born pups / Total N° of female with evidence of mating x 100
Sperm count = (Mean number of sperm head counted x Square factor x Haemocytometer factor x Dilution factor) / Tissue (Testis or Epididymis) weight x 100
Offspring viability indices:
Post-implantation loss = (Number of implants - Number of viable pups) / Number of implants x 100
Post-natal loss = Number of live pups born – pups alive at postnatal day 4
Pup survival index = (N° of live pups on lactation day 0 or 4 or 7 or 14 or 21) / (N° of pups born or N° of pups retained on lactation day 4) x 100
Live birth index = Number of live-born pups / Number of delivered pups x 100
Male sex ratio = Number of male pups on lactation day 0 / (Total number of males + female pups on lactation day 0) x 100
Lactation index = Number of pups alive on lactation day 21 / Number of pups retained on lactation Day 4 x 100
Clinical signs:
no effects observed
Description (incidence and severity):
All rats belonging to the control and the test item treated groups were normal, throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality and morbidity were observed during the study period in the control and the test item treated groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
MALE
The mean body weight of male rats of the test item treated groups was comparable with that of the control group.

FEMALE
- Pre-mating: The mean body weight of female rats of the test item treated groups was comparable with that of the control group.
- Gestation: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during gestation on days 0, 7, 14, and 20 when compared with that of the control group.
- Lactation: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during lactation on days 0, 4, 7, 14, and 21 when compared with that of the control group.
This lower mean body weight during gestation and lactation days could be due to a lower mean food consumption of female rats, belonging to the 300 ppm dose group. This effect could be considered as a treatment-related adverse effect of the test item.

BODY WEIGHT GAIN
MALE
The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 43-50 and 64-71 of treatment when compared with that of the control group.
These occasional lower mean body weight gain without significant effect on food consumption could be considered incidental and without any toxicological relevance.

FEMALE
- Pre-mating: The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 15-22, 43-50, and 50-57 of treatment when compared with that of the control group. The mean body weight gain of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 29-36 of treatment when compared with that of the control group.
- Gestation: The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 0-7 of gestation when compared with that of the control group.
- Lactation: The mean body weight gain of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 4-7 of lactation when compared with that of the control group.
This decrease in the mean body weight gain during the pre-mating and gestation days could be due to a lower mean food consumption of female rats, belonging to the 300 ppm dose group. This effect could be considered as a treatment-related adverse effects of the test item. However, an occasional lower mean body weight gain without a significant effect on the food consumption and dose dependency in the 50 and 100 ppm dose groups, could be considered incidental, and without any toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
FOOD CONSUMPTION
MALE
The mean food consumption of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 85-92 and 92-TS (Terminal Sacrifice) of treatment when compared with that of the control group. These occasional lower mean food consumptions without a significant effect on body weight could be considered incidental, and without any toxicological relevance.

FEMALE
- Pre-mating: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 15-22, 29-36, 43-50, 50-57, 57-64, 64-71, and 1-71 of treatment when compared with that of the control group. The mean food consumption of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 15-22 and 43-50 of treatment when compared with that of the control group. The mean food consumption of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 15-22, 43-50, and 50-57of treatment when compared with that of the control group.
- Gestation: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 0-7, 7-14, 14-20, and 0-20 of gestation when compared with that of the control group. The mean food consumption of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 7-14 of gestation when compared with that of the control group.
- Lactation:
The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 4-7 of lactation when compared with that of the control group. The mean food consumption of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 4-7 of lactation when compared with that of the control group.
The lower mean food consumption during the pre-mating, gestation, and lactation days of female rats belonging to the 300 ppm dose group, could be considered as a treatment-related adverse effect of the test item. However, an occasionally lower food consumption without significant effect on the mean body weight and dose dependency in the 50 and 100 ppm dose groups, could be considered incidental, and without any toxicological relevance.

TEST ITEM INTAKE
Test item intake, with respect to the dose level, was consistent throughout the treatment period (i.e., the test item intake for G3 was approximately twice that of G2, and the same for G4 was approximately thrice of G3). Overall test item intake is given in Table 1 in 'Any other information on results, incl. tables'.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
MALE
The mean food efficiency of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 43-50 and 64-71 of treatment when compared with that of the control group.

FEMALE
- Pre-mating: The mean food efficiency of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 50-57 treatment when compared with that of the control group. The mean food efficiency of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 29-36 of treatment when compared with that of the control group. These occasional lower in the mean food efficiency without a significant effect on the body weight could be considered incidental, and without any toxicological relevance.
- Gestation and Lactation: The mean food efficiency of female rats of the test item treated groups was comparable with that of the control group.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant lower haematocrit value was noted in the males of the 300 ppm dose group. This effect was considered to be related to the test item treatment and considered a mild effect as the reduction was less than 6% when compared to the control group.
A statistically significant higher value was observed in the reticulocyte count of males of the 300 ppm dose group, which was considered related to the lower haematocrit value, hence was considered related to the test item treatment.
A statistically significant higher value in the reticulocyte count of males of the 50 and 100 ppm dose groups was not considered related to the treatment as a relevant effect was not observed in any of the red cell mass parameters (RBC, haemoglobin or haematocrit).
A statistically significant lower MCV was noted in females of the 50 ppm dose group. This alteration was not related to treatment in the absence of dose dependency.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant lower globulin level in males (and apparent decrease in females) of the 300 ppm dose group caused a significantly higher albumin: globulin ratio. It was considered to be related to the test item treatment.
A statistically significant lower value was observed for triglycerides of females of the 300 ppm dose group. Statistically significant higher values were noted for ALT and AST of females of the 300 ppm dose group. These effects were considered to be related to the test item treatment and more specifically to be related to alterations in the liver.
Statistically significant higher values were noted for urea, BUN, and potassium of females of the 300 ppm dose group. These alterations were not considered to be related to test item treatment as it lacked consistency between generation/sexes and support from histopathology. Statistically significant lower values were observed for bile acids of males and total bilirubin and creatinine of females of the 300 ppm dose group which were considered as toxicologically insignificant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Urinalysis did not reveal any treatment-related effect. Count/values of treated groups were comparable with those of the control group.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examination revealed treatment-related lesions in the liver (hepatocyte hypertrophy) and in the thyroid (follicular cell hypertrophy).
Hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid were observed with higher number of incidences in 300 ppm dose rats of both sexes. Both lesions were also present in 100 ppm dose rats of both sexes with marked lower incidence than that of the 300 ppm dose. Incidences noted in 50 ppm in male rats were comparable to that of the control. In female rats at 50 ppm incidences were significantly lower compared to that of 300 ppm. Lesions observed in liver were well comparable with increased weight of the liver.
Histopathology of the reproductive organs of female rats suspected of reduced fertility, their respective male rats and a dam in which the oestrous cycle was affected, did not reveal lesions definitive for the cause.
Microscopic lesions observed in other organs in male and female rats were at a lower rate of occurrence with almost comparable incidence amongst the control and the 300 ppm dose. Further, these lesions were mostly minimal to mild in nature, non-specific, insignificant, and correlated to the respective gross finding. Hence, these lesions were spontaneous or incidental and not related to the test item treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES
Serum T3, T4, and TSH levels of male and female rats were comparable amongst the control and the test item treated groups.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean oestrous cycle length, number of normal oestrous cycle, and pre-coital interval prior to mating were comparable with that of the control group.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm Motility: Sperm motility of male rats was comparable with that of the control group.
Sperm Count: Cauda epididymis sperm and testicular spermatid counts were comparable amongst the control and the high dose groups.
Sperm Morphology: No test item treatment-related change was observed in sperm morphology (number and percent) amongst the control and the high dose groups.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats, and mating index were comparable between the control and the test item treated groups. The duration of the gestation was comparable amongst the control and the test item treated groups.

PRE AND POSTNATAL DATA
No test item related effect on mean number of implants was observed when compared with that of the control group. A statistically significant lower mean percentage prenatal loss was observed in the 300 ppm dose group when compared with that of the control group. The lower mean percentage prenatal loss could be considered as an incident and of no toxicological importance.
Key result
Dose descriptor:
NOAEL
Remarks:
SYSTEMIC
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Dietary equivalent to 7.44 and 11.83 mg/kg bw/day, for males and females, respectively
Key result
Dose descriptor:
NOAEL
Remarks:
FERTILTY
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 22.11 and 27.43 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
REPRODUCTION
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent for 22.11 and 25.46 mg/kg bw/day, for mailes and females, respectively.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
COHORT 1B:
All rats (except the one found dead) belonging to the control and the test item treated groups were normal throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
COHORT 1B:
A female belonging to the 50 ppm dose group was found dead on gestation day 10. All remaining rats belonging to the control and the test item treated groups were normal throughout the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
BODY WEIGHT
MALE
The mean body weight of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, and 85 when compared with that of the control group.  

FEMALE
- Pre-mating: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically significantly lower throughout the treatment period when compared with that of the control group.
- Gestation: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during gestation day, 0, 7, 14, and 20 when compared with that of the control group.
- Lactation: The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during lactation day, 0, 4, 7, 14, and 21, when compared with that of the control group. This lower mean body weight of male and female rats could be due to a decrease in mean food consumption of rats belonging to the 300 ppm dose group. This effect could be considered as a treatment-related adverse effects of the test item.

BODY WEIGHT GAIN
MALE
The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 1-8, 8-15, 15-22, 22-29, 36-43, and 106-113 of treatment, when compared with that of the control group. The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically significantly higher over days 50-57, 71-78, and 92-99 of treatment, when compared with that of the control group. The mean body weight gain of male rats, belonging to the 100 ppm dose group, was statistically significantly higher over days 50-57, 71-78, and 78-85 of treatment, when compared with that of the control group. The mean body weight gain of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 15-22 and 36-43 of treatment when compared with that of the control group. The mean body weight gain of male rats, belonging to the 50 ppm dose group, was statistically significantly higher over days 71-78, 78-85, and 127-134 of treatment, when compared with that of the control group.

FEMALE
- Pre-mating: The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 1-8, 8-15, and 1-78 when compared with that of the control group. The mean body weight gain of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 8-15 when compared with that of the control group.
- Gestation: The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 0-7, 14-20, and 0-20 of gestation when compared with that of the control group.
The mean body weight gain of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 0-7 of gestation when compared with that of the control group.
This lower mean body weight gain during pre-mating and gestation days could be due to a decrease in mean food consumption of female rats, belonging to the 300 ppm dose group. This effect could be considered to be a treatment-related adverse effect of the test item. However, an occasionally lower mean body weight gain without a significant effect on food consumption and dose dependency in the 50 and 100 ppm dose groups, could be considered to be incidental and without any toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
FOOD CONSUMPTION
Male
The mean food consumption of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 1-8, 8-15, 15-22, 22-29, 29-36, 36-43, and 43-50 of treatment when compared with that of the control group.
The mean food consumption of male rats, belonging to the 100 ppm dose group, was statistically significantly higher over days 92-99 of treatment when compared with that of the control group. It could be considered incidental and without any toxicological relevance.
This lower mean food consumption during treatment days of male rats, belonging to the 300 ppm dose group, could be considered as a treatment-related adverse effects of the test item.

FEMALE
- Pre-mating: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 1-8, 8-15, 15-22, 22-29, and 1-78 of treatment when compared with that of the control group.
The mean food consumption of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 8-15 of treatment when compared with that of the control group.
- Gestation: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 7-14 of gestation when compared with that of the control group.
- Lactation: The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 7-14 of lactation when compared with that of the control group.
This lower mean food consumption during pre-mating, gestation and lactation days of female rats, belonging to the 300 ppm dose group, could be considered to be a treatment-related adverse effect of the test item.

TEST ITEM INTAKE
Test item intake, with respect to the dose level, was consistent throughout the treatment period (i.e., the test item intake for G3 was approximately twice that of G2, and the same for G4 was approximately thrice of G3).
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
MALE
The mean food efficiency of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 8-15 and 106-113 when compared with that of the control group. The mean food efficiency of male rats, belonging to the 300 ppm dose group, was statistically significant higher during treatment days 50-57, 71-78, and 92-99, when compared with that of the control group. The mean food efficiency of male rats, belonging to the 100 ppm dose group, was statistically significant higher during treatment days 50-57 when compared with that of the control group. The mean food efficiency of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 8-15 when compared with that of the control group. The mean food efficiency of male rats, belonging to the 50 ppm dose group, was statistically significant higher during treatment days 71-78 when compared with that of the control group.

FEMALE
- Pre-mating: The mean food efficiency of female rats, belonging to the 300 ppm dose group, was statistically significant higher during treatment days 15-22 when compared with that of the control group. The mean food efficiency of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 8-15 when compared with that of the control group.
- Gestation: The mean food efficiency of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during gestation days 0-7 when compared with that of the control group. The mean food efficiency of female rats, belonging to the 100 ppm dose group, was statistically and significantly lower during gestation days 0-7 when compared with that of the control group.
- Lactation: The mean food efficiency of female rats was comparable with that of the control group. This lower mean food efficiency during pre-mating and gestation days of rats, belonging to the 300 ppm dose group, is correlated with lower food consumption and considered as a treatment-related adverse effects of the test item. However, an occasional decrease in mean food efficiency without significant effect on body weight gain and dose dependency in the 50 and 100 ppm dose groups, could be considered incidental and without any toxicological relevance.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
Statistically significant lower terminal body weight was noted in female rats. Statistically significant higher relative liver weight was noted in male and female rats of the 300 ppm dose, while higher absolute liver weight was also observed in the 300 ppm dose males. Moreover, three female rats of the 300 ppm dose group had values above historical range for relative weight of liver, while none of the control rat had values above historical range (Female: 3.382 to 5.097%, central 95 percentile, n = 138). These findings were considered related to the test item treatment as similar effects were observed in parental and cohort 1A rats.
Statistically significant higher relative weights were noted in the thyroid of female rats of the 300 ppm dose which was not considered related to the test item treatment as values of female rats of the 300 ppm dose group were below upper limit of historical range (0.0051 to 0.0110%, central 95 percentile, n = 138).
Statistically significant lower values were observed in absolute weights of left epididymis (males) and pituitary (females) of rats of the 300 ppm dose, while lower values were noted in absolute and relative weights of uterus of females of the 300 ppm dose. These changes were considered unrelated to the test item treatment due to lack of consistency between sexes and lack of similar effect in other generation (parent and cohort 1A).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
COHORT 1B:
Macroscopic examination revealed treatment-related lesions in liver (greyish discolouration).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
COHORT 1B:
PRE-COITAL INTERVAL
The pre-coital interval was comparable with that of the control group.

FERTILITY
Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats, and mating index were comparable between the control and the test item treated groups. The duration of the gestation days was comparable among all the test item treated groups with that of the control group.

PRE AND POSTNATAL DATA:
A statistically significant lower mean number of implants was observed in the 50 and 300 ppm dose groups when compared with that of the control group. However, pre- and postnatal data of cohort 1B female rats were comparable with that of the control group. The lower mean number of implants did not show a dose dependency. Histopathology of uterus and ovarian follicular counts of cohort 1A females, belonging to 50 and 300 ppm dose groups, were also comparable with that of the control group. Therefore, it could be considered as incidental and without toxicological relevance.
Key result
Dose descriptor:
NOAEL
Remarks:
COHORT 1B (SYSTEMIC)
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
other: Dietary equivalent to 10.11 and 11.94 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
COHORT 1B (FERTILITY)
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 30.05 and 25.14 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
COHORT 1B (REPRODUCTION)
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 30.05 and 26.34 mg/kg bw/day, for males and females, respectively.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, non-treatment-related: No treatment-related clinical signs were observed in all pups. However, weakness was occasionally observed in following F1 pups.

COHORT 1A: No effects observed: All rats belonging to the control and the test item treated groups were normal throughout the study period.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Mortality observed, non-treatment-related: During the lactation period, no treatment-related mortality was observed in the test item treated groups.
The mortality index of male pups, belonging to the 100 ppm dose group, was statistically and significantly lower on the postnatal day 7, when compared with that of the control group. It was reflected as significantly higher survival of male pups on the postnatal day 7. This effect could be considered incidental to treatment and without any toxicological relevance.

COHORT 1A: No mortality observed: No mortality and morbidity were observed during the study period in the control and the test item treated groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related:
BODY WEIGHT
MALE PUPS
The mean body weight of male pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal day 0 when compared with that of the control group.

FEMALE PUPS
The mean body weight of female pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal days 14 and 21 when compared with that of the control group.

COMPOSITE OF MALE AND FEMALE PUPS
The mean body weight of pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal day 0 when compared with that of the control group. These lower body weights could be due to the lower gestation body weight of pregnant rats and considered as a growth retardation effect of the test item on developing pups.

BODY WEIGHT GAIN
MALE PUPS
The mean body weight gain of male pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 7-14 when compared with that of the control group.

FEMALE PUPS
The mean body weight gain of female pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 7-14 and 14-21 when compared with that of the control group.

COMPOSITE OF MALE AND FEMALE PUPS
The mean body weight gain of pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 7-14 and 14-21 (without statistical significance) when compared with that of the control group. This lower mean body weight gain of pups, belonging to the 300 ppm dose group toward the end of the lactation period could be due to decreased milk production of lactating females and direct exposure of pups to the test item through diet. Therefore, it could be considered as an adverse effect of the test item on developing pups.

COHORT 1A: Effects observed, treatment-related:
BODY WEIGHT
MALE
The mean body weight of male rats, belonging to the 300 ppm dose group, was statistically significant lower throughout the treatment period when compared with that of the control group. The mean body weight of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 71 and TS when compared with that of the control group.

FEMALE
The mean body weight of female rats, belonging to the 300 ppm dose group, was statistically significant lower throughout the treatment period when compared with that of the control group.

This lower mean body weight of male and female rats during the treatment period could be due to a lower mean food consumption of male and female rats, belonging to the 300 ppm dose group. This effect could be considered to be a treatment-related adverse effect of the test item. However, an occasionally lower mean body weight of male rats without significant effect on food consumption and dose dependency in the 50 ppm dose group, could be considered incidental and without any toxicological relevance.

BODY WEIGHT GAIN
MALE
The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 1-8, 8-15, 22-29, 50-57, 64-71, and 1-TS of treatment when compared with that of the control group. The mean body weight gain of male rats, belonging to the 300 ppm dose group, was statistically significant higher over days 71-TS of treatment, when compared with that of the control group. The mean body weight gain of male rats, belonging to the 100 ppm dose group, was statistically and significantly lower over days 29-36 and 57-64 of treatment when compared with that of the control group. The mean body weight gain of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 22-29, 36-43, 57-64, and 1-TS of treatment when compared with that of the control group.

FEMALE
The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower over days 8-15, 64-TS, and 1-TS of treatment when compared with that of the control group. The mean body weight gain of female rats, belonging to the 300 ppm dose group, was statistically significantly higher over days 50-57 of treatment, when compared with that of the control group. The mean body weight gain of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower over days 43-50 of treatment when compared with that of the control group.

These lower mean body weight gains of male and female rats during the treatment period could be due to a lower mean food consumption of male and female rats, belonging to the 300 ppm dose group. This effect could be considered to be a treatment-related adverse effect of the test item. However, an occasionally lower mean body weight of rats without a significant effect on food consumption and dose dependency in the 50 and 100 ppm dose groups, could be considered incidental and without any toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: Effects observed, treatment-related
FOOD CONSUMPTION
MALE
The mean food consumption of male rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 1-8, 8-15, 15-22, and 22-29 when compared with that of the control group.

FEMALE
The mean food consumption of female rats, belonging to the 300 ppm dose group, was statistically and significantly lower during treatment days 1-8, 8-15, and 1-TS when compared with that of the control group. This lower mean food consumption during treatment days of male and female rats could be considered a treatment-related adverse effect of the test item.

TEST ITEM INTAKE
Test item intake, with respect to the dose level, was consistent throughout the treatment period (i.e., the test item intake for G3 was approximately twice that of G2, and the same for G4 was approximately thrice of G3).
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: Effects observed, non-treatment-related
MALE
The mean food efficiency of male rats, belonging to the 300 ppm dose group, was statistically significantly higher during treatment days 50-57 when compared with that of the control group.
The mean food efficiency of male rats, belonging to the 100 ppm dose group, was statistically significant higher during treatment days 50-57 and lower during treatment days 29-36, 57-64, when compared with that of the control group.
The mean food efficiency of male rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days 15-22, 36-43, and 64-71 when compared with that of the control group.

FEMALE
The mean food efficiency of female rats, belonging to the 300 ppm dose group, was statistically significant higher during treatment days 50-57 and lower during treatment days 64-TS when compared with that of the control group. The mean food efficiency of female rats, belonging to the 50 ppm dose group, was statistically and significantly lower during treatment days, 8-15 and 43-50 when compared with that of the control group.

These occasional higher and lower mean food efficiencies without dose dependency could be considered incidental and without any toxicological relevance.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: Effects observed, treatment-related: Statistically significant lower values were noted for haematocrit, RBC, and haemoglobin of males of the 300 ppm dose. These effects were considered to be related to the test item treatment and considered a mild effect as the decrease compared to control was below 6%. A statistically significant higher value was observed for PT of males of the 100 and 300 ppm doses, and WBC and lymphocyte of females of the 300 ppm dose which were not considered to be an effect of the test item treatment due to inconsistencies between sexes and/or generations. A statistically significant lower MCV was noted in females of the 50 and 300 ppm dose groups, and APTT of females of the 50 and 100 ppm dose groups. These alterations were not related to treatment in the absence of dose dependency.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: Effects observed, treatment-related: Statistically significant lower globulin was noted in male and female rats of the 300 ppm dose. This effect lead to a statistically significant higher albumin: globulin ratio of male and female rats of the 300 ppm dose group. The effect was also observed in males of the 100 ppm dose. It was considered to be related to the test item treatment. A statistically significant higher albumin: globulin ratio of female rats of the 100 ppm group was observed whereas no changes were observed in globulin or albumin level. Statistically significant lower total cholesterol was observed in male and female rats of the 300 ppm dose group. These effects were considered to be related to the test item treatment and more specifically to be related to alterations in the liver.
Urinalysis findings:
no effects observed
Description (incidence and severity):
F1 PUPS: Not examined

COHORT 1A: No effects observed: Urinalysis did not reveal any treatment-related effect. Count/values of treated groups were comparable with that of the control group.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related
MALE
The mean balano-preputial separation (BPS) of male pups (cohort 1A and 1B), belonging to the 300 ppm dose group, was statistically and significantly increased when compared with that of the control group. The mean balano-preputial separation of male pups (cohort 1B), belonging to the 50 and 100 ppm dose groups, was statistically and significantly increased when compared with that of the control group. The mean body weight of male pups (cohort 1A and 1B) on the day of balano-preputial separation was comparable between the control and the test item treated groups. This increase in balano-preputial separation in the 300 ppm dose group could be due to a lower preweaning body weight. This effect could be considered a treatment-related growth retardation effect of the test item on developing pups. The mean balano-preputial separation of male pups (cohort 1B), belonging to the 50 and 100 ppm dose groups, were within historical control data range (44.27 to 49.05) of the performing laboratory. Therefore, it was considered to be a biological variation without any toxicological importance.

FEMALE
Vaginal opening of female pups was comparable between the control and the test item treated groups. The mean body weight on the day vaginal opening of female pups (cohort 1A and 1B), belonging to the 300 ppm dose groups, was statistically and significantly decreased when compared with that of the control group. It could be considered a growth retardation effect of the test item on developing pups.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, non-treatment--related: The mean anogenital distance of female pups, belonging to the 300 ppm dose group, was statistically and significantly increased on postnatal day 0 when compared with that of the control group. However, anogenital distance of F2 pups was comparable with that of the control group. Therefore, this effect could be considered incidental to treatment and without any toxicological relevance.
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related: Statistically significant lower values were noted for terminal body weight of male and female pups. A statistically significant lower value was noted for the absolute weight of the liver in 300 ppm dose females. This effect was also noted in 300 ppm dosed males and although it did not reach statistical significance in males, it was considered related to treatment as it was supported by histopathology. Statistically significant higher values were noted in relative weights of the brain in 300 ppm dosed male and female pups, which was caused by a lower body weight. The lower value noted for the absolute weight of the brain in 300 ppm dosed females was considered not related to treatment.

COHORT 1A: Effects observed, treatment-related: Statistically significant lower terminal body weights were noted in male and female rats. Statistically significant higher relative liver weight was noted in the male and female rats of the 300 ppm dose. Moreover, six male rats of the 300 ppm dose group had values above historical range for relative weight of liver, while none of the control male rat had values above historical range (Male: 2.409 to 3.541 %, central 95 percentile, n = 155; Female: 3.382 to 5.097%, central 95 percentile, n = 138). The effect was considered related to the test item treatment and was supported by hypertrophy observed with histopathology. Statistically significant higher relative weights were also noted in the thyroid of males of the all three dose levels. Values of male rats of the 300 ppm dose group were below upper limit of historical range, while only 1 male rat each of low dose and mid dose level had values above historical range (0.0033 to 0.0077 %, central 95 percentile, n = 155). Hence, effect was not related to the test item treatment. Higher relative weight of the kidneys was noted in males of the 300 ppm dose. Effects on relative kidney weights were likely to be due to a decrease in body weight.
A statistically significant reduction was observed in the absolute weight of the heart and spleen of females of the 300 ppm dose, which could be owed to lower body weight. A statistically significant lower value was noted in the absolute weight of the brain of males and an increase was observed in the relative weight of the thymus of females of the 300 ppm. These effects were not related to the test item treatment as it lacked histopathological lesions. Statistically significant lower values were noted in terminal body weight, absolute weights of liver, heart, epididymis (left), and epididymis cauda (right) of males of the 50 ppm dose, which was unrelated to treatment in the absence of a dose dependency. Similarly, statistically significant lower values noted in whole epididymis and cauda (left) of males of the 300 ppm dose were not related to treatment in the absence of consistency between the two sides. Statistically significant lower values observed in absolute weight of the adrenals of male rats of the 50 ppm dose (left and right), 300 ppm dose (left) and 50 ppm dose (right) were not associated with the test item treatment as it lacked a dose-dependency/consistency between sexes and histopathological examination did not reveal any changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, non-treatment-related: Macroscopic examination of F1 weanling pups did not reveal any pathologic lesions. One of the pups which was found dead in the 100 ppm dose group revealed neck oedema upon gross examination.

COHORT 1A: Effects observed, treatment-related: Macroscopic examination revealed treatment-related lesions in the liver (greyish discolouration).
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related: Glycogen depletion in the liver was observed in the 300 ppm dose group pups. It could be due to nutritional insufficiency and it was considered as an effect of the test item.

COHORT 1A: Effects observed, treatment-related: Histopathological examination revealed treatment-related lesions in the liver (hepatocyte hypertrophy) and in the thyroid (follicular cell hypertrophy). Hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid were observed with higher number of incidences in the 300 ppm dose rats of both sexes. Incidences noted in the 50 and 100 ppm dose were almost comparable to that of the control except for a higher number of incidences of liver hypertrophy found in male rats of the 100 ppm dose group. Lesions observed in liver were well comparable with increased weight of the liver.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
F1 PUPS: Effects observed, treatment-related

LIVE BIRTH, SURVIVAL, AND LACTATION INDICES
Live birth, survival, and lactation indices were comparable amongst the control group and the test item treated groups. Survival index of male pups, belonging to the 100 ppm dose group, was statistically significant higher on the postnatal day 7, when compared with that of the control group. It was reflected as significantly lower mortality of male pups on the postnatal day 7. This effect could be considered incidental and without any toxicological relevance.

LITTER SIZE AND MALE SEX RATIO
Litter size and male sex ratio of F1 pups were comparable with that of the control group.

BODY TEMPERATURE
The mean body temperature of male, female, and a combination of both sexes’ pups, belonging to the 300 ppm dose group, was statistically significantly lower on postnatal day 0 when compared with that of the control group. This lower body temperature could be due to a decrease in mean body weight of the pups on postnatal day 0. This effect could be considered a treatment-related adverse effect of the test item on developing pups.

PHYSICAL DEVELOPMENT LANDMARK
Physical development landmark of male and female pups was comparable between the control and the test item treated groups.

THYROID HORMONES
Serum T3 level of female rat pups (F1) on PND 22, belonging to the 100 ppm dose group, was statistically and significantly higher when compared with that of the control group. However, serum T3 level of male rat pups (F1) on PND 22 was comparable with that of the control group. The changes observed at 100 ppm lack dose dependency, hence, they are considered as incidental in nature. Serum T4 and TSH levels of male and female pups (F1) on PND 22 were comparable between the control and the test item treated groups. Serum T4 and T3 levels of pups (F1) on PND 4 were comparable between the control and the test item treated groups.

COHORT 1A: Effects observed, treatment-related
OESTROUS CYCLE
The mean oestrous cycle length of female rats was comparable between the control and the test item treated groups.

SPERM PARAMETERS
- Sperm motility: Sperm motility of male rats was comparable with that of the control group.
- Sperm Count: Cauda epididymis sperm and testicular spermatid counts were comparable between the control and the high dose groups.
- Sperm Morphology: No test item treatment-related changes were observed in sperm morphology (number and percentage) between the control and the high dose groups.

SPLENIC LYMPHOCYTE SUBPOPULATION
Splenic lymphocyte subpopulation of male and female rats was comparable with that of the control group.

THYROID HORMONES
Serum TSH and T4 levels of cohort 1A female rats, belonging to the 300 ppm dose group, were statistically significantly higher when compared with that of the control group. Increased serum thyroid levels of female rats could be due to hypertrophy of the thyroid.

OVARIAN FOLLICULAR QUANTITATION
The total number and ovarian follicles of different types i.e. primordial, growing and antral follicles in the 300 ppm dose group of F1 generation females were found comparable with that of the control group.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
FETAL TOXICITY
Generation:
F1
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
other: lower body temperature
Remarks on result:
other: Dietary equivalent to 7.44 and 13.1 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
SYSTEMIC
Generation:
F1 (cohort 1A)
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Dietary equivalent to 11.13 and 13.42 mg/kg bw/day, for males and females, respectively.
Key result
Dose descriptor:
NOAEL
Remarks:
REPRODUCTION
Generation:
F1 (cohort 1A)
Effect level:
>= 300 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Dietary equivalent to 34.63 and 40.86 mg/kg bw/day, for males and females, respectively.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed in all pups. However, weakness was occasionally observed in some F2 pups( G1, 2; G2, 1; G3, 2; G4, 2).
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The mortality index of F2 pups was comparable between the control and the test item treated groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT
Male Pups
The mean body weight of male pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal days 14 and 21 when compared with that of the control group.

Female Pups
The mean body weight of female pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal days 14 and 21 when compared with that of the control group.

Composite of Male and Female Pups
The mean body weight of pups, belonging to the 300 ppm dose group, was statistically and significantly lower on postnatal day 14 and 21 when compared with that of the control group. This lower mean body weight of pups, belonging to the 300 ppm dose group toward the end of the lactation period could be due to decreased milk production of lactating females and direct exposure of pups to the test item through diet during 3rd week of lactation (i.e., approximately from postnatal day 14 onwards). Therefore, it could be considered as an adverse effect of the test item on developing pups.

BODY WEIGHT GAIN
Male Pups
The mean body weight gain of male pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 4-7, 7-14, and 14-21 when compared with that of the control group.
The mean body weight gain of male pups, belonging to the 100 ppm dose group, was statistically and significantly lower during postnatal days 7-14 when compared with that of the control group.

Female Pups
The mean body weight gain of female pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 4-7, 7-14, and 14-21 when compared with that of the control group. The mean body weight gain of female pups, belonging to the 100 ppm dose group, was statistically and significantly lower during postnatal days 7-14 when compared with that of the control group.

Composite of Male and Female Pups
The mean body weight gain of pups, belonging to the 300 ppm dose group, was statistically and significantly lower during postnatal days 4-7, 7-14, and 14-21 when compared with that of the control group. The mean body weight gain of pups, belonging to the 100 ppm dose group, was statistically and significantly lower during postnatal days 7-14 when compared with that of the control group.
This lower mean body weight gain of pups, belonging to the 100 and 300 ppm dose groups toward the end of the lactation period could be due to decreased milk production of lactating females and direct exposure of pups to the test item through diet. Therefore, it could be considered as an adverse effect of the test item on developing pups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The mean anogenital distance of F2 pups was comparable between the control and the test item treated groups.
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant lower terminal body weight was noted in male and female pups. In F2 weanling pups, statistically significant lower values were noted for absolute weight of spleen and thymus in the 300 ppm dose of both sexes and also of the thymus in the 100 ppm dose males, while higher values were noted for relative weights of brain in the 300 ppm dose of both sexes. Effects were considered related to a lower body weight. A statistically significant higher relative liver weight was noted in the 300 ppm dosed females which was not related to treatment due to the absence of consistency between sexes and the lack of histopathological support.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of F2 weanling pups did not reveal any pathologic lesions.
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopic findings in the liver were comparable between the control and the test item treated groups of F2 pups.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
LIVE BIRTH, SURVIVAL AND LACTATION INDICES
The live birth index of pups, belonging to the 50 ppm dose group, was statistically and significantly lower, when compared with that of the control group. The lower live birth index without any dose dependency could be considered as incidental and without any toxicological relevance. Survival and lactation indices of F2 pups were comparable between the control and the test item treated groups.

LITTER SIZE AND MALE SEX RATIO
Litter size and male sex ratio of F2 pups was comparable with that of the control group.

BODY TEMPERATURE
The mean body temperature of male and female pups, belonging to the 100 ppm dose group, was statistically and significantly lower on postnatal day 0 when compared with that of the control group. This lower body temperature without any dose dependency could be considered as incidental.

PHYSICAL DEVELOPMENT LANDMARK
Physical development landmark of male and female pups was comparable amongst the control and the test item treated groups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
FETAL TOXICITY
Generation:
F2 (cohort 1B)
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Dietary equivalent to 10.11 and 13.71 mg/kg bw/day, for males and females, respectively.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Verification of homogeneity and concentration of the test item in the test diet

The mean percent recovery obtained for the test diet was within the acceptance level of ±20% of the nominal concentration demonstrating that the exposure concentrations were as intended by the study plan and the %CV was less than 20, suggesting that the test diets were homogeneously mixed.

TABLE 1a. Test item intake during treatment period (P0)

Group N°

Dose Levels (ppm)

Test Item Intake (mg/kg body weight/day)

Male Rats

Female Rats

Treatment Period

Pre-mating Period

Gestation Period

Lactation Period

G1

0

0.00

0.00

0.00

0.00

G2

50

3.74

4.66

3.99

9.03

G3

100

7.44

9.30

7.95

18.25

G4

300

22.11

27.43

23.48

56.19

TABLE 1b. Test item intake during the treatment period (F1 Cohort 1A)

Group N°

Dose Levels (ppm)

Test Item Intake (mg/kg body weight/day)

Male Rats

Female Rats

G1

0

0.00

0.00

G2

50

5.74

6.98

G3

100

11.13

13.42

G4

300

34.63

40.86

 

TABLE 1c. Test item intake during the treatment period (F1 Cohort 1B)

Group N°

Dose Levels (ppm)

Test Item Intake (mg/kg body weight/day)

Male Rats

Female Rats

Treatment Period

Pre-mating Period

Gestation Period

Lactation Period

G1

0

0.00

0.00

0.00

0.00

G2

50

5.93

4.22

4.47

9.34

G3

100

10.11

8.40

8.84

18.58

G4

300

30.05

25.14

27.54

62.23

 

TABLE 2: Summary of Effect on Fertility, Reproduction, and Development – Parent Female (P)

Observations

Values

Group & Dose (ppm)

G1 (0)

G2 (50)

G3 (100)

G4 (300)

Pairs Started (N)

25

25

25

25

Females Achieving Pregnancy (N)

22

24

23

18

Conceiving Days 1 - 5 (N)

24

25

25

25

Conceiving Days 6 - 10 (N)

0

0

0

0

Conceiving Days³11 (N)

0

0

0

0

Pregnancy = 21 days (N)

0

0

0

0

Pregnancy = 22 days (N)

7

16

14

12

Pregnancy = 23 days (N)

12

7

8

6

Pregnancy³24 days (N)

2

1

0

0

Female Sacrificed on Gestation Day 25 (N)

3

1

3

7

Dams with Live Pups at Day 4 (N)

21

24

22

18

N° of Female Rats Standardized on Lactation Day 4

8

12

12

11

Dams with Live Pups at Weaning (N)

20

24

22

18

N° of Female Rats (along with Pups) Sacrificed on Lactation Day 22

20

24

22

18

N° of Female Rats Sacrificed (Without Pups)

2

0

0

0

Implants (Mean±SD)

10.18±4.43

11.17±2.76

10.96±3.40

11.33±2.74

ABNORMAL PUPS (excluding dead/cannibalised pups)

Dams with 0

20

22

19

17

Dams with 1 (Weakness)

2

2

2

1

Dams with ≥2 (Weakness)

0

0

1

0

LOSS OF OFFSPRING

Pre-natal (implantations minus live births)

Females with 0

6

10

10

10

Females with 1

8

7

9

5

Females with 2

6

7

1

2

Females with ≥ 3

2

0

3

1

Post-natal (live births minus alive at post-natal day 4)

Females with 0

16

18

17

16

Females with 1

5

5

5

1

Females with 2

1

1

0

1

Females with ≥ 3

0

0

0

0

FERTILITY DATA

Number of Males Housed with Female

25

25

25

25

Number of Males Impregnating Female

25

25

25

25

Number of Females Housed with Male

25

25

25

25

Number of Females with Sperm Positive Vaginal Smear

25

25

25

25

Number of Females Confirmed Pregnant

22

24

23

18

Number of Females Giving Birth

22

24

22

18

Number of Females Giving Birth to at least a Viable Pup

22

24

22

18

Number of Females Giving Birth to All Viable Pups

21

23

21

17

Duration of Gestation (days) (Mean±SD)

22.76±0.62

22.38±0.58

22.36±0.49

22.33±0.49

FERTILITY INDEX

Male Fertility Index

100.00

100.00

100.00

100.00

Female Fertility Index

88.00

96.00

92.00

72.00

Gestation Index

88.00

96.00

88.00

72.00

Parturition Index

88.00

96.00

88.00

72.00

Percentage of Pregnant Rats

88.00

96.00

92.00

72.00

Percentage of Non-pregnant Rats

12.00

4.00

8.00

28.00

Mating Index

100.00

100.00

100.00

100.00

Prenatal Loss (Mean±SD)

1.23±1.07

0.88±0.85

0.96±1.26

0.67±0.91

Postnatal Loss (Mean±SD)

0.32±0.57

0.29±0.55

0.23±0.43

0.17±0.51

Prenatal Loss (%) (Mean±SD)

15.52±18.87

7.72±7.52

13.25±22.94

5.42±7.09¯*

Postnatal Loss (%) (Mean±SD)

11.03±26.27

2.72±4.97

1.66±3.15

1.35±4.23

Key: N = Number of rats, SD = Standard deviation,* =Student’s t-test

Key:  ¯=Significantly lower than control (p=<0.05)

Note: Non-pregnant rats were excluded from statistical analysis of implants and pre- and postnatal losses.

Presumed mated female was not considered for calculation of conceiving and pregnancy days

TABLE 3. Summary of Effect on Fertility, Reproduction, and Development - Pup data

Number of Pups

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

Total N° of Pups on Day of Littering

94

104

198

121

127

248

117

114

231

93

100

194

Cannibalized pups on day of littering

0

0

0

0

0

0

0

0

0

0

0

1

Pups Still birth on Day of Littering

1

0

1

1

0

1

1

0

1

1

0

1

Total N° of Live Pups on Day of Littering

93

104

197

120

127

247

116

114

230

92

100

192

Litter Size on PND 4

89

101

190

116

124

240

114

111

225

90

99

189

Litter Size on PND 4 (R)

83

87

170

103

112

215

97

99

196

82

85

167

Litter Size on PND 7

78

85

163

102

110

212

97

98

195

79

82

161

Litter Size on PND 14

77

82

159

100

108

208

93

96

189

77

79

156

Litter Size on PND 21

75

80

155

99

106

205

93

95

188

74

78

152

Pups Mortality Index

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

1-4 (N°)

4

3

7

4

3

7

2

3

5

2

1

3

Mortality Indexon PND 4

4.30

2.88

3.55

3.33

2.36

2.83

1.72

2.63

2.17

2.17

1.00

1.56

4R -7 (N°)

5

2

7

1

2

3

0

1

1

3

3

6

Mortality Indexon PND 7

6.02

2.30

4.12

0.97

1.79

1.40

0.00¯©

1.01

0.51

3.66

3.53

3.59

4R -14 (N°)

6

5

11

3

4

7

4

3

7

5

6

11

Mortality Indexon PND 14

7.23

5.75

6.47

2.91

3.57

3.26

4.12

3.03

3.57

6.10

7.06

6.59

14R -21 (N°)

8

7

15

4

6

10

4

4

8

8

7

15

Mortality Indexon PND 21

9.64

8.05

8.82

3.88

5.36

4.65

4.12

4.04

4.08

9.76

8.24

8.98

Key:  M = Male, F = Female, T = Total, R = Retained, N° = Number,a= Cannibalized (sex not identified),© = Chi-square test

¯=Significantly lower than control (p=<0.05)


Pups survival Index

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

Survival Index on PND 4

95.70

97.12

96.45

96.67

97.64

97.17

98.28

97.37

97.83

97.83

99.00

98.44

Survival Index on PND 7

93.98

97.70

95.88

99.03

98.21

98.60

100.00­©

98.99

99.49

96.34

96.47

96.41

Survival Index on PND 14

92.77

94.25

93.53

97.09

96.43

96.74

95.88

96.97

96.43

93.90

92.94

93.41

Survival Index on PND 21

90.36

91.95

91.18

96.12

94.64

95.35

95.88

95.96

95.92

90.24

91.76

91.02

 

Lactation Index, Live Birth Index and Postnatal Loss

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

Lactation Index (%)

90.36

91.95

91.18

96.12

94.64

95.35

95.88

95.96

95.92

90.24

91.76

91.02

Live Birth Index (%)

98.94

100.00

99.49

99.17

100.00

99.60

99.15

100.00

99.57

98.92

100.00

98.97

Postnatal Loss (PND 0-4)

4

3

7

4

3

7

2

3

5

2

1

3

Key:    M = Male, F = Female, T = Total, R = Retained, N° = Number, PND = Postnatal Day, © = Chi-square test

­ = Significantly higher than control (p=<0.05)

TABLE 4. Organ weight percent change compared to control

Generation

Parameters

Group

Percent Change

G1

G2

G3

G4

G2

G3

G4

Parent

B. wt. (TS)-Female

247.345

235.104

237.327

218.806¯¯

-4.9

-4.1

-11.5

Liver (Male)-R

2.614

2.641

2.669

3.026­­

1.0

2.1

15.8

Liver (Female)-R

3.894

4.095

4.083

4.696­­

5.2

4.9

20.6

Thyroid (Female)-R

0.0066

0.0072

0.0072

0.0080­­

9.1

9.1

21.2

Cohort 1A

B. wt. (TS)- Male

322.685

297.62

315.23

295.965¯¯

-7.8

-2.3

-8.3

B. wt. (TS)-Female

191.63

183.54

194.27

176.300¯

-4.2

1.4

-8.0

Liver (Male)-R

3.069

2.974

3.069

3.435­­

-3.1

0.0

11.9

Liver (Female)-R

3.174

3.154

3.178

3.558­­

-0.6

0.1

12.1

Thyroid (Male)-R

0.0047

0.0053­

0.0053­

0.0058­­

12.8

12.8

23.4

Cohort 1B

B. wt. (TS)-Female

237.415

228.653

233.494

212.606¯¯

-3.7

-1.7

-10.4

Liver (Male)-R

2.593

2.570

2.628

2.946­­

-0.9

1.3

13.6

Liver (Female)-R

4.226

4.397

4.444

4.847­­

4.0

5.2

14.7

Thyroid (Female)-R

0.0054

0.0052

0.0053

0.0060­

-3.7

-1.9

11.1

F1-Weanling Pups

B. wt. (TS)-Male

42.569

40.443

38.764

35.340¯

-5.0

-8.9

-17.0

Liver (Male)-A

1.971

1.865

1.734

1.564

-5.4

-12.0

-20.6

B. wt. (TS)-Female

39.833

39.890

37.471

32.515¯

0.1

-5.9

-18.4

Liver (Female)-A

1.786

1.853

1.689

1.438¯$

3.8

-5.4

-19.5

F2-Weanling Pups

B. wt. (TS)-Male

40.780

42.018

37.589

34.871¯¯

3.0

-7.8

-14.5

B. wt. (TS)-Female

40.495

40.900

36.718

33.900¯¯

1.0

-9.3

-16.3

Key: M-Male, F-Female, TS-Terminal sacrifice

TABLE 5. Microscopic Findings of Parents, Cohort 1A and F1& F2 Pups

Lesion

Generation

Sex

Group

G1

G2

G3

G4

Liver: Hypertrophy, hepatocytes

Parent

M

1

1

3

14

F

0

2

5

14

Cohort 1A

M

0

1

3

9

F

0

0

1

8

Thyroid: Hypertrophy, follicular cell

Parent

M

3

3

6

15

F

2

6

6

18

Cohort 1A

M

4

3

4

12

F

0

0

0

8

Liver: Glycogen depletion, hepatocytes

F1 Pups

M

0

0

0

8

F

1

0

0

6

F2 Pups

M

0

1

0

1

F

1

1

1

2

Key: M-male, F-Female

TABLE 6. Summary ofSerum T3 and T4 Level in Parent Male Rats

Groups and Dose: G1 - 0; G2 - 50; G3 - 100; G4 - 300 ppm

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

406.896

85.522

10

379.680

66.411

10

354.102

47.875

10

430.092

93.733

10

T4

162884.407

25143.440

10

159581.646

22284.244

10

164727.417

30623.771

10

161425.613

35243.036

10

 

TABLE 7. Summary ofSerum T3 and T4 Level in Parent Female Rats

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

634.485

77.520

10

669.095

89.733

10

666.089

78.733

10

542.548

107.996

10

T4

52170.941

12929.265

10

59036.481

6080.748

10

59296.682

12923.489

10

49277.953

9673.531

10

 

TABLE 8. Summary of Serum T4 Level in PND 4 Rat Pups (F1)

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T4

166263.378

36641.300

8

151498.847

31071.914

12

183356.165

39221.749

11

175831.884

15736.875

12

T3

173.989

28.964

8

194.562

26.907

12

183.808

32.597

11

164.592

21.045

12

 

TABLE 9. Summary of Serum T3 and T4 Level in PND 22 Male Rat Pups (F1)

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

947.624

149.214

10

893.210

128.097

10

885.531

99.916

10

828.537

155.784

10

T4

32088.317

5505.246

10

30624.640

3437.663

10

32546.858

4931.690

10

38553.791

21088.103

10

Key: N = Number of animals, SD = Standard deviation, N = Number


TABLE 10. Summary of Serum T3 and T4 Level in PND 22 Female Rat Pups (F1)

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

1166.706

98.492

10

1130.596

152.894

10

1300.428­*

171.315

10

1371.200

395.309

10

T4

75886.896

12449.540

10

74471.364

8805.684

10

78445.631

13454.459

10

91869.842

25523.427

10

 

TABLE 11. Summary of Serum T3 and T4 Level i nCohort 1A Male Rats

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

608.631

105.624

10

634.192

114.392

10

673.808

92.116

10

631.297

97.648

10

T4

46283.928

4749.377

10

46933.934

4885.995

10

52575.119

5758.954

10

44885.595

6853.688

10

 

TABLE 12. Summary of Serum T3 and T4 Level in Cohort 1A Female Rats

Hormone

Group N°

G1

G2

G3

G4

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

T3

657.453

131.922

10

690.129

108.068

10

691.313

68.543

10

792.604

184.617

10

T4

30867.390

6085.017

10

34932.867

7235.657

10

32105.700

5251.861

10

49568.749­­#

10927.196

10

Keys: N = Number of animals, SD = Standard deviation, N = Number

­= Significantly higher than control (p=<0.05); ­­= Significantly higher than control (p=<0.01)

* =Student’s t-test, # = Dunnett’s t-test

 

TABLE 13: Pup body temperature

Postnatal
Day 0

Group N°

G1

G2

G3

G4

 

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

F1 Male

24.95

0.48

20

24.9

0.41

24

24.82

0.52

22

24.43↓↓$

0.37

18

F1 Female

25.01

0.62

20

24.97

0.45

24

24.79

0.47

22

24.40↓↓$

0.3

18

F1 Male+ female

24.97

0.58

22

24.93

0.42

24

24.8

0.47

22

24.40↓↓$

0.31

18

F2 Male

24.94

0.57

20

24.77

0.6

17

24.39↓$

0.54

18

24.62

0.42

17

F2 Female

25.02

0.49

20

24.71

0.6

17

24.45↓↓$

0.52

17

24.65

0.43

17

F2 Male+ female

24.99

0.49

20

24.75

0.6

17

24.42↓↓$

0.53

18

24.65

0.41

17

N = Number of observations, SD = Standard deviation, N° Number, $ t-test with Bonferroni’s adjustment, ↓ = Significantly lower than control (p≤0.05), ↓↓ = Significantly lower than control (p≤0.01)

 

 

TABLE 14: Summary of Effect on Fertility, Reproduction, and Development – Parent Female (P1)

Observations

Values

Group & Dose (ppm)

G1 (0)

G2 (50)

G3 (100)

G4 (300)

Pairs Started (N)

20

20

20

20

Females Achieving Pregnancy (N)#

20

20

20

20

Conceiving Days 1 - 5 (N)

19

16

17

19

Conceiving Days 6 - 10 (N)

0

0

0

1

Conceiving Days ³ 11 (N)

0

3

2

0

Pregnancy = 21 Days (N)

0

0

2

1

Pregnancy = 22 Days (N)

16

14

15

16

Pregnancy = 23 Days (N)

3

2

0

0

Pregnancy ³ 24 Days (N)

0

1

1

0

Female Sacrificed on Gestation Day 25 (N)

0

2

2

3

Dams with Live Pups at Day 4 (N)

20

17

18

17

N° of Female Rats Standardized on Lactation day 4

6

4

6

0

Dams with Live Pups at Weaning (N)

20

17

18

17

N° of Female Rats (along with pups) Sacrificed on Lactation Day 22

20

17

18

17

N° of Female Rats Sacrificed (without pups)

0

0

0

0

Implants (Mean ± SD)

11.05 ± 1.93

9.22 ± 2.88¯β

9.47 ± 3.29

9.59 ± 1.37¯β

ABNORMAL PUPS (excluding dead/cannibalised pups)

Dams with 0

18

16

16

15

Dams with 1 (Weakness)

2

1

2

2

Dams with ≥2 (Weakness)

0

0

0

0

LOSS OF OFFSPRING

Pre-natal (implantations minus live births)

Females with 0

7

8

9

8

Females with 1

7

4

9

7

Females with 2

6

4

1

1

Females with ≥ 3

0

2

0

1

Post-natal (live births minus alive at post-natal day 4)

Females with 0

16

15

17

14

Females with 1

2

1

1

3

Females with 2

1

0

0

0

Females with ≥ 3

1

1

0

0

FERTILITY DATA

Number of Males Housed with Female

20

20

20

20

Number of Males Impregnating Female

20

20

20

20

Number of Females Housed with Male

20

20

20

20

Number of Females with Sperm Positive Vaginal Smear

19

19

19

20

Number of Females Confirmed Pregnant

20

20

20

20

Number of Females Giving Birth

20

17

18

17

Number of Females Giving Birth to at least a Viable Pup

20

17

18

17

Number of Females Giving Birth to All Viable Pups

20

15

18

17

Duration of Gestation (days) (Mean ± SD)

22.16 ± 0.37

22.24 ± 0.56

22.00 ± 0.59

21.94 ± 0.24

FERTILITY INDEX

Male Fertility Index

100

100

100

100

Female Fertility Index

100

90

95

85

Gestation Index

100

85

90

85

Parturition Index

100

85

90

85

Percentage of Pregnant Rats

100

90

95

85

Percentage of Non-pregnant Rats

0

10

5

15

Mating Index

100

100

100

100

Prenatal Loss (Mean ± SD)

0.95 ± 0.83

1.00 ± 1.08

0.58 ± 0.61

0.71 ± 0.85

Postnatal Loss (Mean ± SD)

0.45 ± 1.19

0.24 ± 0.75

0.06 ± 0.24

0.18 ± 0.39

Prenatal Loss (%) (Mean ± SD)

8.75 ±7.97

14.59 ± 24.15

12.20 ± 24.27

6.70 ± 7.33

Postnatal Loss (%) (Mean ± SD)

4.15 ± 9.44

2.38 ± 8.16

0.79 ± 3.37

1.91 ± 4.29

Key: N = Number of rats, SD = Standard deviation,* =Student’s t-test

Key:  ¯=Significantly lower than control (p=<0.05)

Note: Non-pregnant rats were excluded from statistical analysis of implants and pre- and postnatal losses.

Presumed mated female was not considered for calculation of conceiving and pregnancy days

 

 

TABLE 15. Summary of Effect on Fertility, Reproduction, and Development - Pup data

Number of Pups (F2)

Group N°

G1

G2

G3

G4

Sex

M

F

T

M

F

T

M

F

T

M

F

T

Number of Pups

Total N° of Pups on Day of Littering

93

109

202

72

77

151a

88

81

169

76

75

151

Cannibalized Pups on Day of Littering

0

0

0

0

0

2

0

0

0

0

0

0

Pups Still birth on Day of Littering

0

0

0

1

0

1

0

0

0

0

0

0

Total N° of Live Pups on Day of Littering

93

109

202

71

77

148

88

81

169

76

75

151

Litter Size on

91

102

193

70

74

144

88

80

168

74

74

148

PND 4

Litter Size on

86

95

181

65

72

137

84

75

159

74

74

148

PND 4 (R)

Litter Size on

84

94

178

65

71

136

83

74

157

73

73

146

PND 7

Litter Size on

81

93

174

65

71

136

83

73

156

73

72

145

PND 14

Litter Size on

80

93

173

64

71

135

81

73

154

73

71

144

PND 21

Pups Mortality Index

1-4 (N°)

2

7

9

1

3

4

0

1

1

2

1

3

Mortality Index on PND 4

2.15

6.42

4.46

1.41

3.9

2.7

0

1.23

0.59

2.63

1.33

1.99

4R -7 (N°)

2

1

3

0

1

1

1

1

2

1

1

2

Mortality Index on PND 7

2.33

1.05

1.66

0

1.39

0.73

1.19

1.33

1.26

1.35

1.35

1.35

4R -14 (N°)

5

2

7

0

1

1

1

2

3

1

2

3

Mortality Index on PND 14

5.81

2.11

3.87

0

1.39

0.73

1.19

2.67

1.89

1.35

2.7

2.03

14R -21 (N°)

6

2

8

1

1

2

3

2

5

1

3

4

Mortality Index on PND 21

6.98

2.11

4.42

1.54

1.39

1.46

3.57

2.67

3.14

1.35

4.05

2.7

Pups Survival Index

Survival Index on PND 4

97.85

93.58

95.54

98.59

96.1

97.3

100

98.77

99.41

97.37

98.67

98.01

Survival Index on PND 7

97.67

98.95

98.34

100

98.61

99.27

98.81

98.67

98.74

98.65

98.65

98.65

Survival Index on PND 14

94.19

97.89

96.13

100

98.61

99.27

98.81

97.33

98.11

98.65

97.3

97.97

Survival Index on PND 21

93.02

97.89

95.58

98.46

98.61

98.54

96.43

97.33

96.86

98.65

95.95

97.3

Lactation Index, Live Birth Index and Postnatal Loss

Lactation

93.02

97.89

95.58

98.46

98.61

98.54

96.43

97.33

96.86

98.65

95.95

97.3

Index (%)

Live Birth

100

100

100

98.61

100

98.01¯©

100

100

100

100

100

100

Index (%)

Postnatal

2

7

9

1

3

4

0

1

1

2

1

3

Loss (PND 0-4)

Key:    M = Male, F = Female, T = Total, R = Retained, N° = Number, PND = Postnatal Day, © = Chi-square test

­ = Significantly higher than control (p=<0.05)

 

 

 

Conclusions:
In this GLP-compliant Extended One Generation Reproductive Toxicity Test, performed according to OECD 443, with the inclusion of Cohorts 1A and 1B (including extension to F2), the following NOAELs were found:
- Parental - systemic toxicity: 100 ppm (dietary equivalent of 7.44 (males) and 11.83 (females) mg/kg bw/day for P0 and 10.11 (males) and 11.94 (females) mg/kg bw/day for F1 Cohort 1B), based on lower body weight (gain) and food consumption, higher liver weights, greyish discolouration of the liver and hypertrophic liver and thyroid;
- Parental - fertility/reproductive toxicity: => 300 ppm (highest dose; P0: dietary equivalent of 22.11 and 27.43/25.46 mg/kg bw/day, for males and females, respectively; F1 Cohort 1B: dietary equivalent of 30.05 and 25.14/26.34 mg/kg bw/day, for males and females, respectively), based on the absence of any treatment-related adverse effects on any fertility and reproductive performance parameters;
- Offspring (F1) - developmental toxicity: 100 ppm (dietary equivalent of 7.44 and 13.1 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain), lower body temperature, lower liver weight adn depleted glycogen in the liver.
- Offspring (F2) - developmental toxicity: 100 ppm (dietary equivalent of 10.11 and 13.71 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain).
Executive summary:

The objective of this GLP-compliant study, performed according to OECD 443, was to provide data on the possible effects of the test item on reproductive performance of Wistar rats and the development of pups consequent to daily dietary exposure to various concentrations of the test item (intended target concentrations: 0 ppm, 50 ppm, 100 ppm and 300 ppm) to male and female rats during a premating period of 10 weeks and during mating (max. 2 weeks), gestation and lactation until postnatal day (PN) 21. At weaning on postnatal day 21 (PN 21), pups were distributed to Cohorts 1A and 1B and were exposed to the test item (intended target concentrations: 0 ppm, 50 ppm, 100 ppm and 300 ppm). To produce the F2 generation, 1 male and 1 female rat from the F1(cohort 1B) generation were randomly selected and subsequently mated (14 - 15 weeks of age). 

The mean per cent recovery obtained for diet was within the acceptance level of ± 20% of the nominal concentration demonstrating that the exposure concentrations were as intended in the study plan and the %CV was less than 20, suggesting that the diet was homogeneously mixed.

Body weights for all generations were recorded weekly and at termination. Food consumption was measured weekly. Sperm parameters (count and morphology) were assessed for all P and F1A males rates of the control and high dose group, whereas sperm motility was assessed for all groups. Oestrous cycle length and pattern was assessed for all P females and F1A female pups (from the day of vaginal opening, until first oestrus phase). Vaginal smear was examined (from P and F1 rats) at the time of necropsy to determine the stage of the oestrous cycle. Lymphonic subpopulation analysis was performed on 10 rats/sex/group belonging to cohort 1A. Necropsy of the parent generation was scheduled as follows: male rats were sacrificed after delivery of P female rats and female rates on lactation day 22. F1 weanlings were sacrificed on the lactation day 22.Cohort 1A rats were sacrificed during 13-14 weeks of the treatment period. For Cohort F1B, males were sacrificed after sacrifice of F1 female rats on lactation day 22. F2 weanlings were sacrificed on lactation day 21. Clinical pathology included: Haematology, clinical chemistry, and urine parameters were analysed from 10 rats/sex/group (from P and cohort 1A).Thyroid hormones (T3, T4, and TSH) were analysed from parent, weanling pups, and cohort 1A rats.Organs, as defined in the study plan, were excised, weighed, preserved, and subjected to the histopathological examination.From 10 F1 female rats of control and high dose groups Primordial Follicle Quantitative Evaluation was performed.

P Generation

No morality, morbidity, and clinical signs of toxicity were observed in male and female rats up to the 300 ppm dose level.

The mean body weight, body weight gain, food consumption, and food efficiency of male rats were comparable with those of the control group. The mean body weight (gestation and lactation), body weight gain (pre-mating and gestation), and food consumption (pre-mating,gestation, and lactation) of female rats, belonging to the 300 ppmdose group,werestatistically and significantly lower when compared with those of the control group.

A statistically significant lower haematocrit (HCT) value was noted in males of the 300 ppm dose group. This effect was considered to be a treatment-related adverse effect of the test item. A statistically significant higher value was observed for the reticulocyte count of males of the 300 ppm dose group, which was considered to be related to the decrease in HCT and was considered to be related to the test item treatment.

The statistically, significant lower globulin level in males (and apparent decrease in females) of the 300 ppm dose group caused a significantly higher albumin: globulin ratio.A statistically significant lower value was observed for triglycerides of females of the 300 ppm dose group. Statistically significant higher values were noted for ALT and AST of females of the 300 ppm dose group. These effects were considered to be related to the test item treatment and more specifically to be related to alterations in the liver.

Statistically significant higher values were noted for the relative weight of the liver (males and females) of rats of the 300 ppm dose group. Effects were related to the treatment, as these were supported by the histopathology.

Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose. A higher incidence of hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid were observed in male and female rats of the 300 ppm dose. Lesions observed in liver and thyroid were well comparable with the increased weight of the liver and thyroid.

Oestrous cycle and sperm parameters were comparable between the control and the test item treated groups.

F1 Pups

The mean body weight, body weight gain, and temperature of pups, belonging to the 300 ppm dose group,were statistically and significantly lower during the postnatal period when compared with those of the control group. These lower values could be due to a decreased gestation body weight of pregnant rats and considered as growth retardation effect of the test item on the developing pups.

A statistically significant lower value was noted for the absolute weight of liver of females of the 300 ppm dose. this effect was also noted in males of the 300 ppm dose group and although it did not reach statistical significance in males, it was considered to the related to treatment, as it was supported by histopathology. Glycogen depletion was observed upon the histopathological examination of the liver of pups of the 300 ppm dose group. It could be considered an adverse effect of the test item.

No treatment-related clinical signs were observed in pups up to the 300 ppm dose level. Mortality, survival, live birth, and lactation indices were comparable amongst the control group and the test item treated groups.Litter size, male sex ratio, thyroid hormones, and physical development landmarks of F1 pups were comparable with that of the control group. The increase in the mean balano-preputial separation of F1 pups of the 300 ppm dose group could be due to a lower preweaning body weight.

F1 Adults (Cohort 1A)

No morality, morbidity, and clinical signs of toxicity were observed either in male or female rats up to the 300 ppm dose level.

The mean body weight, body weight gain, and food consumption of male and female rats, belonging to the 300 ppm dose group, were statistically and significantly lower, throughout the treatment period, when compared with those of the control group.

Statistically significant lower haematocrit, RBC, and haemoglobin values were noted in males of the 300 ppm dose. Statistically significant lower globulin levels were noted in male and female rats of the 300 ppm dose. This effect led to a statistically significant higher albumin: globulin ratio of male and female rats of the 300 ppm dose groups.

Statistically significant higher values were noted for the relative weight of liver (male and female) of rats of the 300 ppm dose which were considered related to the test item treatment and which were corroborated by the hypertrophy observed with histopathology.

Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose group. A higher incidence of hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid was observed in male and female rats of the 300 ppm dose rats.

Oestrous cycle, ovarian follicular counts, sperm parameters, lymphocyte subpopulation, and thyroid hormones were comparable amongst the control and the test item treated groups.

F1 Adults (Cohort 1B)

A female belonging to the 50 ppm dose group was found dead on gestation day 10. Other remaining rats were normal, throughout the study period.

The mean body weight, body weight gain, food consumption, and food efficiency of male rats, belonging to the 300 ppm dose group, were statistically and significantly lower, throughout the treatment period, when compared with those of the control group.

The mean body weight (pre-mating, gestation, and lactation), body weight gain and food efficiency (pre-mating and gestation), and food consumption (pre-mating, gestation, and lactation) of female rats, belonging to the 300 ppm dose group, were statistically and significantly lower when compared with that of the control group.

Statistically significant higher values were noted for the relative weight of the liver (males and females) of rats of the 300 ppm dose, whereas increases in absolute weight of the liver was also observed in the 300 ppm dosed males. Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose.

F2 Pups

No treatment-related clinical signs were observed in pups up to the 300 ppm dose level. Mortality, survival, live birth, and lactation indices were comparable amongst the control group and the test item treated groups. Litter size, male sex ratio, anogenital distance, body temperature, and physical development landmarks of F2 pups, were comparable with those of the control group.

The mean body weight and body weight gain of pups, belonging to the 300 ppm dose group, were statistically and significantly lower during the postnatal period when compared with that of the control group. These lower values for the mean body weight and the body weight gain of pups, belonging to the 300 ppm dose group towards the end of the lactation period could be due to the decreased milk production of the lactating females and direct exposure of pups to the test item during the last week of the lactation period (i.e., approximately from postnatal day 14 onwards), through their diet.

Macroscopic and microscopic findings of the liver were comparable amongst the control and the test item treated groups.

Conclusion

In conclusion, the following NOAELs were found:

- Parental - systemic toxicity: 100 ppm (dietary equivalent of 7.44 (males) and 11.83 (females) mg/kg bw/day for P0 and 10.11 (males) and 11.94 (females) mg/kg bw/day for F1 Cohort 1B), based on lower body weight (gain) and food consumption, higher liver weights, greyish discolouration of the liver and hypertrophic liver and thyroid;

- Parental - fertility/reproductive toxicity: => 300 ppm (highest dose; P0: dietary equivalent of 22.11 and 27.43/25.46 mg/kg bw/day, for males and females, respectively; F1 Cohort 1B: dietary equivalent of 30.05 and 25.14/26.34 mg/kg bw/day, for males and females, respectively), based on the absence of any treatment-related adverse effects on any fertility and reproductive performance parameters;

- Offspring (F1) - developmental toxicity: 100 ppm (dietary equivalent of 7.44 and 13.1 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain), lower body temperature, lower liver weight adn depleted glycogen in the liver.

- Offspring (F2) - developmental toxicity: 100 ppm (dietary equivalent of 10.11 and 13.71 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
22.11 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD 443, in compliance with GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

OECD 443 (EOGRTS)

The objective of this GLP-compliant study, performed according to OECD 443, was to provide data on the possible effects of the test item on reproductive performance of Wistar rats and the development of pups consequent to daily dietary exposure to various concentrations of the test item (intended target concentrations: 0 ppm, 50 ppm, 100 ppm and 300 ppm) to male and female rats during a premating period of 10 weeks and during mating (max. 2 weeks), gestation and lactation until postnatal day (PN) 21. At weaning on postnatal day 21 (PN 21), pups were distributed to Cohorts 1A and 1B and were exposed to the test item (intended target concentrations: 0 ppm, 50 ppm, 100 ppm and 300 ppm). To produce the F2 generation, 1 male and 1 female rat from the F1(cohort 1B) generation were randomly selected and subsequently mated (14 - 15 weeks of age). 

The mean per cent recovery obtained for diet was within the acceptance level of ± 20% of the nominal concentration demonstrating that the exposure concentrations were as intended in the study plan and the %CV was less than 20, suggesting that the diet was homogeneously mixed.

Body weights for all generations were recorded weekly and at termination. Food consumption was measured weekly. Sperm parameters (count and morphology) were assessed for all P and F1A males rates of the control and high dose group, whereas sperm motility was assessed for all groups. Oestrous cycle length and pattern was assessed for all P females and F1A female pups (from the day of vaginal opening, until first oestrus phase). Vaginal smear was examined (from P and F1 rats) at the time of necropsy to determine the stage of the oestrous cycle. Lymphonic subpopulation analysis was performed on 10 rats/sex/group belonging to cohort 1A. Necropsy of the parent generation was scheduled as follows: male rats were sacrificed after delivery of P female rats and female rates on lactation day 22. F1 weanlings were sacrificed on the lactation day 22.Cohort 1A rats were sacrificed during 13-14 weeks of the treatment period. For Cohort F1B, males were sacrificed after sacrifice of F1 female rats on lactation day 22. F2 weanlings were sacrificed on lactation day 21. Clinical pathology included: Haematology, clinical chemistry, and urine parameters were analysed from 10 rats/sex/group (from P and cohort 1A).Thyroid hormones (T3, T4, and TSH) were analysed from parent, weanling pups, and cohort 1A rats.Organs, as defined in the study plan, were excised, weighed, preserved, and subjected to the histopathological examination.From 10 F1 female rats of control and high dose groups Primordial Follicle Quantitative Evaluation was performed.

P Generation

No morality, morbidity, and clinical signs of toxicity were observed in male and female rats up to the 300 ppm dose level.

The mean body weight, body weight gain, food consumption, and food efficiency of male rats were comparable with those of the control group. The mean body weight (gestation and lactation), body weight gain (pre-mating and gestation), and food consumption (pre-mating,gestation, and lactation) of female rats, belonging to the 300 ppmdose group,werestatistically and significantly lower when compared with those of the control group.

A statistically significant lower haematocrit (HCT) value was noted in males of the 300 ppm dose group. This effect was considered to be a treatment-related adverse effect of the test item. A statistically significant higher value was observed for the reticulocyte count of males of the 300 ppm dose group, which was considered to be related to the decrease in HCT and was considered to be related to the test item treatment.

The statistically, significant lower globulin level in males (and apparent decrease in females) of the 300 ppm dose group caused a significantly higher albumin: globulin ratio.A statistically significant lower value was observed for triglycerides of females of the 300 ppm dose group. Statistically significant higher values were noted for ALT and AST of females of the 300 ppm dose group. These effects were considered to be related to the test item treatment and more specifically to be related to alterations in the liver.

Statistically significant higher values were noted for the relative weight of the liver (males and females) of rats of the 300 ppm dose group. Effects were related to the treatment, as these were supported by the histopathology.

Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose. A higher incidence of hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid were observed in male and female rats of the 300 ppm dose. Lesions observed in liver and thyroid were well comparable with the increased weight of the liver and thyroid.

Oestrous cycleandsperm parameters were comparable between the control and the test item treated groups.

F1 Pups

The mean body weight, body weight gain, and temperature of pups, belonging to the 300 ppm dose group,were statistically and significantly lower during the postnatal period when compared with those of the control group. These lower values could be due to a decreased gestation body weight of pregnant rats and considered as growth retardation effect of the test item on the developing pups.

A statistically significant lower value was noted for the absolute weight of liver of females of the 300 ppm dose. this effect was also noted in males of the 300 ppm dose group and although it did not reach statistical significance in males, it was considered to the related to treatment, as it was supported by histopathology. Glycogen depletion was observed upon the histopathological examination of the liver of pups of the 300 ppm dose group. It could be considered an adverse effect of the test item.

No treatment-related clinical signs were observed in pupsup to the 300 ppm dose level.Mortality, survival, live birth, and lactation indices were comparable amongst the control group and the test item treated groups.Litter size, male sex ratio, thyroid hormones, andphysical development landmarksof F1pups were comparable with that of the control group.The increase in the meanbalano-preputial separation of F1 pups of the 300 ppm dose groupcould be due to a lower preweaning body weight.

F1 Adults (Cohort 1A)

No morality, morbidity, and clinical signs of toxicity were observed either in male or female rats up to the 300 ppm dose level.

The mean body weight, body weight gain, and food consumption of male and female rats, belonging to the 300 ppm dose group, were statistically and significantly lower, throughout the treatment period, when compared with those of the control group.

Statistically significant lower haematocrit, RBC, and haemoglobin values were noted in males of the 300 ppm dose. Statistically significant lower globulin levels were noted in male and female rats of the 300 ppm dose. This effect led to a statistically significant higher albumin: globulin ratio of male and female rats of the 300 ppm dose groups.

Statistically significant higher values were noted for the relative weight of liver (male and female) of rats of the 300 ppm dose which were considered related to the test item treatment and which were corroborated by the hypertrophy observed with histopathology.

Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose group. A higher incidence of hepatocyte hypertrophy in liver and follicular cell hypertrophy in thyroid was observed in male and female rats of the 300 ppm dose rats.

Oestrous cycle, ovarian follicular counts, sperm parameters, lymphocyte subpopulation, and thyroid hormones were comparable amongst the control and the test item treated groups.

F1 Adults (Cohort 1B)

A female belonging to the 50 ppm dose group was found dead on gestation day 10. Other remaining rats were normal, throughout the study period.

The mean body weight, body weight gain, food consumption, and food efficiency of male rats, belonging to the 300 ppm dose group, were statistically and significantly lower, throughout the treatment period, when compared with those of the control group.

The mean body weight (pre-mating, gestation, and lactation), body weight gain and food efficiency (pre-mating and gestation), and food consumption (pre-mating, gestation, and lactation) of female rats, belonging to the 300 ppm dose group, were statistically and significantly lower when compared with that of the control group.

Statistically significant higher values were noted for the relative weight of the liver (males and females) of rats of the 300 ppm dose, whereas increases in absolute weight of the liver was also observed in the 300 ppm dosed males. Treatment-related greyish discolouration of the liver was observed in male and female rats of the 300 ppm dose.

F2 Pups

No treatment-related clinical signs were observed in pupsup to the 300 ppm dose level.Mortality, survival, live birth, and lactation indices were comparable amongst the control group and the test item treated groups.Litter size, male sex ratio, anogenital distance, body temperature, andphysical development landmarksof F2pups, were comparable with those of the control group.

The mean body weight and body weight gain of pups, belonging to the 300 ppm dose group, were statistically and significantly lower during the postnatal period when compared with that of the control group. These lower values for the mean body weight and the body weight gain of pups, belonging to the 300 ppm dose group towards the end of the lactation period could be due to the decreased milk production of the lactating females and direct exposure of pups to the test item during the last week of the lactation period (i.e., approximately from postnatal day 14 onwards), through their diet.

Macroscopic andmicroscopicfindings of the liver were comparable amongst the control and the test item treated groups.

Conclusion

In conclusion,the following NOAELs were found:

- Parental - systemic toxicity: 100 ppm (dietary equivalent of 7.44 (males) and 11.83 (females) mg/kg bw/day for P0 and 10.11 (males) and 11.94 (females) mg/kg bw/day for F1 Cohort 1B), based on lower body weight (gain) and food consumption, higher liver weights, greyish discolouration of the liver and hypertrophic liver and thyroid;

- Parental - fertility/reproductive toxicity: => 300 ppm (highest dose; P0: dietary equivalent of 22.11 and 27.43/25.46 mg/kg bw/day, for males and females, respectively; F1 Cohort 1B: dietary equivalent of 30.05 and 25.14/26.34 mg/kg bw/day, for males and females, respectively), based on the absence of any treatment-related adverse effects on any fertility and reproductive performance parameters;

- Offspring (F1) - developmental toxicity: 100 ppm (dietary equivalent of 7.44 and 13.1 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain), lower body temperature, lower liver weight adn depleted glycogen in the liver.

- Offspring (F2) - developmental toxicity: 100 ppm (dietary equivalent of 10.11 and 13.71 mg/kg bw/day, for males and females, respectively), based on lower body weight (gain).

OECD 422 (Reproduction / Developmental Toxicity Screening Test)

The study was conducted under GLP conditions to determine the initial information on toxic characteristic, systemic and reproduction/developmental toxicity, occurring as a result of repeated daily dietary administration of the test item during pre-mating, mating, gestation, and lactation period of rats to select the dose levels for definitive study. A total of 36 male and 36 female Wistar rats were randomly divided into four groups (8 rats/sex/group). Test item was given through diet, in graduated doses of 150, 300, and 600 ppm, to three groups of male and female rats. The control group received basal diet. Male rats were treated for 43 days. Female rats were treated for two weeks prior to mating, variable time to conception, the duration of pregnancy and 14 days after delivery. The active ingredient concentration and homogeneity of the test item in diet were analysed once before the initiation of treatment and twice during the treatment period. Oestrous cycle length and pattern of female rats were evaluated by vaginal smears during pre-treatment of two weeks and daily from the beginning of the treatment period until evidence of mating. Rats were observed daily, twice, for mortality and morbidity, and once for visible clinical signs throughout the study period. Body weight of rats was recorded on the first day of treatment and the day of sacrifice. Body weight of male rats were recorded weekly throughout the treatment period. Body weights of female rats were recorded weekly during pre-mating and mating periods. Pregnant females were weighed on gestation days (GDs) 0, 7, 14, 20, within 24 hours of parturition, and post-coitum on day 25. Body weights of dams and pups were recorded on lactation days 0, 4, 7, and 14. Food consumption and test item intake was calculated during the treatment period. Pups were observed for sex, stillbirths, live birth, runts, gross anomalies, and measured for ano-genital distance (AGD) on post-natal day (PND) 0. Each litter was standardised on PND 4 to obtain nearly 4 male and 4 female pups. Rats were terminally sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination. Absolute organ weights were recorded and relative organ weights were calculated.

The results of diet formulation analyses were within the acceptable range of ±20% of the nominal concentration and the %CV was less than 20 which suggests that the prepared diet had an acceptable a.i. concentration and it was homogeneously prepared. Parents: No morality, morbidity, and clinical signs of toxicity were observed in male and female rats up to the 600 ppm dose level. The mean body weight gain, food consumption, and food efficiency of male rats, belonging to the 600 ppm dose group, was statistically significant decreased during treatment, when compared with that of the control group. The mean terminal body weight of female rats, belonging to the 600 ppm dose group, was statistically significant decreased, when compared with that of the control group. The mean body weight gain, food consumption, and food efficiency of female rats, belonging to the 600 ppm dose group, was statistically significant decreased during the pre-mating and lactation period, when compared with that of the control group. These decreases in mean body weight gain, food consumption, and food efficiency of male and female rats, belonging to the 600 ppm dose groups, could be due to an adverse effect of the test item on the liver. Occational decreases in mean food consumption and food efficiency without significant effect on body weight and body weight gain of male and female rats, belonging to the 150 and 300 ppm dose groups, could be considered as an incidental incidence without any toxicological relevance. External examination of terminally sacrificed male and female rats, belonging to the 0, 150, 300, and 600 ppm dose groups did not reveal any abnormality. Greyish discolouration of liver of male and female rats, belonging to the 300 and 600 ppm dose groups, was observed during necropsy. Treatment related increases in mean absolute and relative liver weights of male rats, belonging to the 300 and 600 ppm dose groups was observed. Pups: Pup mortality was observed in litters, belonging to the 300 and 600 ppm dose groups. However, the body weight and body weight gain of pups, belonging to the 600 ppm dose group was comparable. Therefore, this could be considered as an incidental change. The mortality of pups, belonging to the 300 ppm dose group, was attributed to two litters in which all pups were cannibalised and was not considered as treatment related. Pups mortality was also reflected in live birth and survival indices. The mean body weight and body weight gain of pups, belonging to the 600 ppm dose group, was statistically significant decreased during PNDs 7-14, when compared with that of the control group. This could be due to a decrease in mean food consumption and food efficiency of female rats of the 600 ppm dose group which might not produce sufficient milk for the pups. No treatment related differences were observed in litter size, sex ratio, AGD, and nipple retention of the pups. External and internal examination of the pups did not reveal any abnormality. A statistically significant increase in relative weights of thyroid with parathyroid was observed in male and female pups, belonging to the 600 ppm dose group.

Based on the results of the present study, the following dose levels are suggested for the extended one-generation reproductive toxicity study (cohort-1) of the test item in Wistar rats: 75, 150, and 300 ppm. The following NOAELs were determined based on the study:

- NOAEL systemic toxicity: 150 ppm, based on the gross pathology abd greyish discoloration of the liver, both sexes (Dietary equivalent to 8.59 and 18.78 mg/kg bw/day for males and females, respectively)

- NOAEL reproductive toxicity: >600ppm, based on no adverse toxic effect observed, both sexes (Dietary equivalent to 33.53 and 66.91 mg/kg bw/day for males and females, respectively)

- NOAEL developmental toxicity: 300 ppm, based on the effect on the thyroid and parathyroid gland weight and the body weight ratios,  both sexes (Dietary equivalent to 17.59 and 33.92 mg/kg bw/day for males and females, respectively)

Effects on developmental toxicity

Description of key information

Jones 1996 - rat study - subchronic

- The NOAEL for parental toxicity is set 50 mg/kg body weight, highest dose tested.

- The NOAEL for developmental toxicity is 50mg/kg body weight, highest dose tested.

Christian 1999 - rat study - subacute

- The NOAEL for parental toxicity is set 50 mg/kg body weight, highest dose tested.

- The NOAEL for developmental toxicity is 50 mg/kg body weight/day, highest dose tested.

Latha 2018 - rabbit study - subacute

- The NOAEL for parental toxicity is set on 15 mg/kg body weight.

- The NOAEL for developmental toxicity is 15 mg/kg body weight/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04 Sep 2018 to 13 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 0000145096
- Expiration date of the lot/batch: 03-01-2020
- Purity test date: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25ºC)
- Solubility and stability of the test substance in the solvent/vehicle: Based on results of validation study, the test material formulations prepared at 1.0 and 250 mg/mL in the vehicle (Corn oil) are stable and resuspendable in the vehicle for 3 days when stored at room temperature at both the concentrations.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd., No.93, Solur, Thally Road, Anekal, Bengaluru-562106, India
- Age at study initiation: 7 months young adult, nulliparous and non-pregnant females at the initiation of the study and adult males (for mating purpose only).
- Weight at study initiation: G1: 2.975 ± 0.20, G2: 2.938 ± 0.21, G3: 2.984 ± 0.20, G4: 2.995 ± 0.20
- Housing: The rabbits were housed individually, except during cohabitation, when the females rabbits were cohabited with the males in 1:1 ratio in rabbit cages (approximate size: Length 65 cm x BWidth 65 cm x Height 45 cm) with shallow cage body and facilities for providing pelleted food (Stainless steel feed hopper) and drinking water in bottle fitted with sipper tube. The waste collection tray was changed daily (except on Sundays and public holidays). - Diet: Rabbit feed manufactured by Special Diets Services, Essex, England (Batch No.3439) was provided ad libitum in stainless steel feed hoppers to rabbits.
- Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard water filter-cum-purifier was provided ad libitum in polycarbonate bottles with stainless steel sipper tubes to rabbits.
- Acclimation period: After clinical examination for good health and suitability for the study, the rabbits were acclimatized for five days before initiation of mating. During the acclimatization period, all rabbits were observed at least once daily.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 64-65, The relative humidity in the experimental rooms was calculated daily from dry and wet bulb temperature recordings.
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material dose formulations were prepared prior to treatment and used within the stability period. Required quantities of the test item were weighed separately for each dose group and transferred into separate mortars. The test item was triturated gently using a pestle to obtain a fine powder. A small portion of the vehicle (Corn oil) was added and the contents was triturated well to obtain a suspension. This suspension was transferred into a pre-marked beaker (Previously calibrated to the desired volume). The mortar and pestle was rinsed with the vehicle and the contents were transferred to the same pre-marked beaker. The suspension was made up to the intended volume with the vehicle. The homogeneity of the the test material dosing suspension during oral gavage was maintained by constant stirring using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was selected as vehicle for preparing the test material formulations in this study, as the same vehicle was used in pilot rabbit study without any effects.
- Concentration in vehicle: 0 mg/mL, 2.5 mg/mL, 7.5 mg/mL, 25 mg/mL
- Amount of vehicle: 2 mL/kg bw
-The dosing was performed using a suction catheter attached to a plastic disposable syringe from GD 6 to GD 28 of presumed gestation. The actual volume administered was calculated based on the most recent body weight on first day of treatment (Gestation Day 6) and was adjusted according to the most recent body weights recorded till Gestation Day 28. Dose formulations were continuously stirred during dose administrations. The animals in the vehicle control group were handled and administered vehicle in an identical manner to the treatment groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and concentration analysis of test material, the prepared formulations were sampled at the initiation of treatment (GD 6) and at termination of treatment period (GD 28). Samples were drawn at each dose level (two samples from the top, middle and bottom layers) in duplicate sets. Similarly, samples only from middle layer was collected from vehicle control. The analyses was done using a validated analytical method. One set of samples was analyzed for concentration and other set of samples was kept as a backup at room temperature for reanalysis. These samples were discarded as the results of first set of samples were within the acceptable limits. Formulations were considered acceptable as the mean result for all layers was within ± 15% of the nominal concentration.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: During the mating period, females were cohabited randomly with males in a 1:1 ratio
- Proof of pregnancy: After confirmation of mating by visual examination, the day was referred to as day 0 of pregnancy



Duration of treatment / exposure:
The dosing was performed using a suction catheter attached to a plastic disposable syringe from GD 6 to GD 28 of presumed gestation.
Frequency of treatment:
Once per day
Duration of test:
All animals surviving until scheduled euthanasia were euthanised on day 29 post-coitum.
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
Mid dose
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
Total of 92 female rabbits were mated and allocated to a control group and 3 treatment groups (23 female rabbits/ group). Male rabbits were used only for mating.
Control animals:
yes, concurrent vehicle
Details on study design:
Selection of Dose Levels and Dose Justification: A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out using 6 rabbits per group with test material dosed at 5, 15 and 50 mg/kg bw/day along with the concurrent vehicle control group. The rabbits were treated once daily with test item formulations by oral gavage at a dose volume of 2 mL/kg body weight from GD 6 to 28 and observed for clinical signs and mortality. The test material at 5 mg/kg bw/day did not affect body weight, food consumption, and maternal and litter data parameters. At 15 mg/kg bw/day, body weight and food consumption were not affected, but there was reduction in mean fetal weights observed. Treatment at 50 mg/kg bw/day resulted in reduction in food consumption and there was reduction in uterine weight observed further causing reduction in the mean litter weights. These findings were considered test material treatment related. Gross evaluation of the placenta revealed no findings. No abnormality was noticed on external observations of fetuses at any of the doses tested. Based on the results of the dose range finding study and in consultation 0, 5, 15 and 50 mg/kg/day dose levels were selected for the definitive study.
Maternal examinations:
CLINICAL SIGNS AND MORTALITY:
- Clinical Signs and Mortality: All rabbits were observed for morbidity and mortality twice daily, i.e. once in the morning and once in the afternoon. Rabbits were observed for clinical signs during the treatment period of the study: pre dose and post dose (within 1- 2 hours of administration). On 20 September 2018, post dose observation was delayed by approximately 4.00 hrs. Aborted rabbits were necropsied, and gross observations observed were recorded.

BODY WEIGHT:
- All the females included in the study (G1 to G4) were weighed on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 27 and 29. The intermittent body weight gain was calculated for GD 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 - 24, 24 - 27 and 27 - 29. Further the body weight gain for pre-treatment period (GD 0 to 6), treatment period (GD 6 to 28), and for entire gestation period (GD 0 to 29) was derived and statistically analyzed only for rabbits pregnant at caesarean section. The corrected body weight (carcass weight) was obtained by subtracting the unopened uterine weight from terminal body weight (body weight on GD 29). The corrected body weight gain was calculated by subtracting the body weight on GD 6 from corrected body weight.

FOOD CONSUMPTION:
- About 600 g of food (food input) was provided on GD 0. The left over food was recorded and replenished to about 600 g on GD 3, 6, 9, 12, 15, 18, 21, 24 and 27. Prior to terminal sacrifice, left over food was recorded on GD 29 of presumed gestation. There was no spillage of food noticed during the entire study period. Intermittent food consumption i.e GD 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18, 18 - 21, 21 -24, 24 - 27 and 27 - 29 was calculated. Further, food intake for pre-treatment (GD 0 to 6), treatment (GD 6 to 29), and for entire gestation period (GD 0 to 29) was derived and statistically analyzed only for rabbits pregnant at caesarean section.

NECROPSY:
- Prior to caesarean section, random numbers were generated for coding to avoid bias during caesarean section and subsequent fetal evaluations. The animal code was written on the ear and the permanent accession number was striked out. On GD 29, all the rabbits were sacrificed by intravenous injection of sodium thiopentone. Gross pathological changes of all external and visceral organs of dams, including uterine contents were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: gross evaluation of placenta
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
The data on maternal body weight and food consumption, interval body weight changes, gravid uterine weight, body weight change corrected to gravid uterine weight were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant. Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Mann-Whitney/Wilcoxon test pair wise comparisons of the treated groups with the control group was performed, when the group differences were significant. The incidence of dams with and without resorptions was tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.
Indices:
All the indices can be found in the field 'Any other information on materials and methods, incl. tables'.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed at any of the doses tested during the study period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no change in mean maternal (corrected) body weights and (corrected) body weight gains during the different periods of gestation (GD 0-6; GD 6-29; GD 0-29) of the rabbits treated at 5 and 15 mg/kg/day compared to the vehicle control group. At 50 mg/kg/day there was statistically significant decrease in body weight gain during GD 6-29 (116%) and GD 0 – 29 (79%). In addition, there was a trend towards a decrease in mean maternal (corrected) body weight and corrected body weight gain, which was not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Compared to the vehicle control group, there was no change in food consumption of rabbits dosed at 5 and 15 mg/kg/day. Though not significant, there was a trend for a decrease in food consumption during different periods of gestation with stastistical significance during GD 21-24 at 50 mg/kg/day. The reduction in body weight and food consumption at 50 mg/kg/day is considered a treatment-related effect.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was a decrease in uterine weight (25%).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross pathological findings in any of the treated groups except for a single occurrence of pale liver at 15 and 50 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
There were incidental occurrences of abortion in each of the low mid and high dose groups. One animal per group.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was an increase in post implantation loss.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Two pregnant dams at the high dose of 50 mg/kg/day had complete resorptions.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day compared with the vehicle control, there was an increase in dams with resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were found in any of the dose groups
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
The number of rabbits sacrificed at term were 23, 22, 22 and 22, respectively, of which 2, 2, 2 and 3 rabbits were non pregnant. The number of pregnant rabbits in the vehicle control, low, mid and high dose groups were 21, 20, 20 and 19, respectively.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
maternal general toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Remarks:
maternal developmental toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
pre and post implantation loss
total litter losses by resorption
necropsy findings
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day, there was a significant reduction in mean fetal weights (-8.3%) at both the sexes (males and females) as compared to the vehicle control group.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The total number of fetuses were 135, 122, 118 and 94 at 0, 5, 15 and 50 mg/kg/day, respectively.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Information can be found in Table 1 in the field 'Any other information on results, incl. tables'.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related major abnormality was observed during external observations of the fetuses at any of the doses. Anomaly of fore limb flexed at wrist (moderate) was observed in one fetus of a litter with 5 fetuses at 50 mg/kg bw/day and in one fetus of a litter with 8 fetuses in the vehicle control group. These minor changes were considered incidental and not adverse as these observations commonly occur in animals of this test model. The incidence is attributed to random occurrence and not treatment related.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related skeletal malformations observed in fetuses of dams treated up to 50 mg/kg/day. Variants and anomalies observed across vehicle and treated groups were comparable and are consistent with concurrent and historical data. The variants and anomalies were consistent with concurrent and historical data.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related major visceral malformations observed in fetuses of dams treated up to 50 mg/kg/day. Anomalies such as gall bladder hypoplastic in vehicle and test material treated groups, and bilobed gall bladder in the vehicle control group were observed. These findings were not considered adverse as these observations commonly occur in animals of this test model and the incidence of occurrence were consistent with concurrent or historical controls. At 50 mg/kg bw/day, visceral evaluation showed incidences of unilateral absence of kidney and ureter in one dam and unilateral dilated ureter with hydroneprosis in another dam. These findings (2/94 fetuses and 2/17 litters) were considered developmental anomalies.
Other effects:
no effects observed
Description (incidence and severity):
Gross evaluation of the placenta revealed no findings. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental Toxicity
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
visceral malformations
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

For homogeneity and concentration analysis of test material it was found that the relative standard deviation of assay of top, middle and bottom layer (total 6 samples) was less than or equal to 10%.

Table 1. Summary of Litter data

Sex: female

G1

0

mg/kg/day

G2

5

mg/kg/day

G3

15

mg/kg/day

G4

50

mg/kg/day

Pregnant

N

21

20

21

19

Total number of fetuses

Mean 

N

Sum

6.4

21

135.0

6.0

20

119.0

5.9

20

118.0

5.5

17

94.0

% Male Fetuses

 

60.0

45.4

48.3

43.6

Dead

Sum

0

0

0

0

Live Male

Sum

81

55

57

41

Mean fetal [c] weight (M) (g)

Mean

SD

N

39.61

5.20

21

39.82

4.77

20

38.02

5.59

20

35.94

7.38

16

Live Female

Sum

54

67

61

53

Mean fetal [c1] weight (F) (g)

Mean

SD

N

40.22

6.24

21

39.83

4.78

20

37.13

4.22

20

36.03

7.26

17

Live fetuses

Sum

135

122

118

94

Mean fetal [c2] weight (both) (g)

Mean

SD

N

39.81

5.32

21

39.79

4.47

20

37.50

4.38

20

36.51*

7.21

17

[c] – Anova/Anova&Dunnett

{Covariate(s): Number of Live Male Fetuses}

[c1] - Anova/Anova&Dunnett

{Covariate(s): Number of Live Female Fetuses}

[c2] - Anova/Anova&Dunnett

{Covariate(s): Number of both Fetuses}; *=p<0.05

Table 2.            Summarized Details of Necropsy 

Parameters 

Group No. &

G1

G2

G3

G4

Dose (mg/kg/day)

0

5

15

50

Total No. of rabbits / group

23

23

23

23

Duration of treatment 

GD 6 to 28 (total 23 days)

Caesarean section

(day of presumed gestation)

29

Number of rabbits sacrificed at caesarean section

23

22

22

22

Number of rabbits aborted

0

1

1

1

Number of rabbits non-pregnant at caesarean section

2

2

2

3

Number of rabbits pregnant at caesarean section

Number of rabbits with complete resorption

21

0

20

0

20

0

19

2

Number of litters examined

21

20

20

17

Total number of fetuses

135

122

118

94

 

 

 

 

 

Number of fetuses evaluated

 

 

 

 

External, visceral and skeletal

135

122

118

94

Number of fetus for decapitation

61

54

55

44

Number of intact fetuses

74

68

63

50

 

  Table 3. Summary of Maternal Body Weight of Pregnant Rabbits

Sex: Female

 

Day(s)

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Body Weight (kg)

0 [a]

Mean

SD

N

2.9731

0.2061

21

3.0035

0.2150

20

2.9701

0.2116

20

2.9880

0.1671

19

3 [a]

Mean

SD

N

3.0049

0.2171

21

3.0608

0.2312

20

2.9842

0.2261

20

3.0354

0.2011

19

6 [a]

Mean

SD

N

3.0279

0.2159

21

3.0947

0.2496

20

3.0148

0.2489

20

3.0541

0.1908

19

9[a]

Mean

SD

N

2.9963

0.2104

21

3.0166

0.2102

20

2.9551

0.2423

20

2.9587

0.1932

19

12 [a]

Mean

SD

N

3.0370

0.2183

21

3.0246

0.2512

20

2.9803

0.2381

20

2.9600

0.2331

19

15 [a]

Mean

SD

N

3.0925

0.2588

21

3.0756

0.2707

20

2.9968

0.2844

20

2.9672

0.1995

19

18 [a]

Mean

SD

N

3.1110

0.2546

21

3.1034

0.2673

20

2.9996

0.2799

20

2.9928

0.2294

19

21 [a]

Mean

SD

N

3.1415

0.2842

21

3.1024

0.2706

20

3.0086

0.2724

20

2.9965

0.2373

19

24 [a]

Mean

SD

N

3.1610

0.2899

21

3.1264

0.2659

20

3.0384

0.2709

20

3.0274

0.2524

19

27 [a]

Mean

SD

N

3.1636

0.3142

21

3.1256

0.2557

20

3.0443

0.2493

20

3.0205

0.2254

19

29 [a]

Mean

SD

N

3.1781

0.3041

21

3.1374

0.2504

20

3.0505

0.2179

20

3.0307

0.2454

19

 [a] – Anova & Dunnett(Log)

  

Table 4. Summary of Maternal Body Weight Gain of Pregnant Rabbits

Sex: Female

 

Day(s)

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Absolute Weight Gain (kg)

0 -> 3 [a]

Mean

SD

N

0.0318

0.0641

21

0.0573

0.0374

20

0.0140

0.0830

20

0.0475

0.0734

19

 

3 -> 6 [a1]

Mean

SD

N

0.0230

0.0527

21

0.0340

0.0395

20

0.0307

0.0494

20

0.0186

0.0431

19

 

6 -> 9 [a1]

Mean

SD

N

-0.0316

0.0778

21

-0.0781

0.0876

20

-0.0598

0.0743

20

-0.0953

0.0759

19

 

9 -> 12 [a1]

Mean

SD

N

0.0407

0.0855

21

0.0080

0.0967

20

0.0252

0.0856

20

0.0012

0.0840

19

 

12 -> 15 [a]

Mean

SD

N

0.0554

0.0740

21

0.0510

0.0720

20

0.0165

0.0925

20

0.0072

0.1001

19

 

15 -> 18 [a1]

Mean

SD

N

0.0185

0.0664

21

0.0279

0.0513

20

0.0028

0.0775

20

0.0256

0.0610

19

 

18 -> 21 [a]

Mean

SD

N

0.0306

0.0493

21

-0.0010

0.0447

20

0.0090

0.0757

20

0.0036

0.0461

19

 

21 -> 24 [a1]

Mean

SD

N

0.0194

0.0457

21

0.0240

0.0361

20

0.0298

0.0584

20

0.0309

0.0598

19

 

24 -> 27 [a1]

Mean

SD

N

0.0026

0.0701

21

-0.0007

0.0483

20

0.0059

0.0570

20

-0.0069

0.0486

19

 

27 -> 29 [a1]

Mean

SD

N

0.0145

0.0401

21

0.0118

0.0612

20

0.0062

0.0560

20

0.0102

0.0465

19

 

0 -> 6 [a]

Mean

SD

N

0.0548

0.0832

21

0.0912

0.0583

20

0.0447

0.0950

20

0.0661

0.0705

19

 

6 -> 29 [a1]

Mean

SD

N

0.1502

0.2006

21

0.0427

0.1361

20

0.0357

0.1844

20

-0.0234*

0.1614

19

 

0 -> 29 [a]

Mean

SD

N

0.2050

0.2326

21

0.1339

0.1331

20

0.0804

0.1645

20

0.0427*

0.1894

19

 

Corrected/adjusted Body weight (kg)

GD29 - gravid uterine. Wt [a2]

Mean

SD

N

2.8132

0.2704

21

2.7880

0.2443

20

2.7093

0.1993

20

2.7586

0.2638

19

 

Corrected/adjusted Body weight Gain

Corrected Bwt-GD6 [a1]

Mean

SD

N

-0.2147

0.1931

21

-0.3067

0.1271

20

-0.3055

0.1611

20

-0.2954

0.1820

19

 

 

[a] – Anova & Dunnett: *= p<0.05

[a1] – Anova & Dunnett(Rank): *= p<0.05

[a2] – Anova & Dunnett(Log)

  

Table 5. Summary of Maternal Data

Sex: Female

 

Day(s) Relative to mating

G1

0

mg/kg/day

G1

5

mg/kg/day

G1

15

mg/kg/day

G1

50

mg/kg/day

Group size

GD 29

N

23

23

23

23

Pregnant

GD 29

N

21

20

20

19

Gravid Uterus (g)

GD 29 [a]

Mean

SD

N

364.9

79.9

21

349.4

62.5

20

341.3

64.4

20

286.1**

88.2

17

Number of CorporaLutea

GD 29 [k]

Mean

SD

N

8.2

1.4

21

8.1

1.4

20

8.4

2.0

20

8.2

1.9

17

Number of Implantation

GD 29 [k]

Mean

SD

N

7.0

1.7

21

6.6

1.2

20

6.6

1.8

20

6.4

2.0

17

With Early resorption

GD 29

Total

N

21

4

20

5

20

6

17

6

Number of Early Deaths

GD 29 [k]

Mean

SD

N

Sum

0.2

0.5

21

5.0

0.3

0.6

20

6.0

0.4

0.6

20

7.0

0.4

0.5

17

6.0

With Late Resorptions

GD 29

Total

N

21

4

20

4

20

6

17

7

Number of Late deaths

GD 29 [k]

Mean

SD

N

Sum

0.3

0.7

21

6.0

0.2

0.4

20

4.0

0.3

0.5

20

6.0

0.5

0.6

17

8.0

With Resorptions

GD 29

N

N-ve

N+ve

21

13

8

20

11

9

20

9

11

17

5

12

Total number of resorptions

GD 29 [f]

Mean

SD

N

Sum

Trend

0.5

0.8

21

11.0

   .

0.4

0.6

20

10.0

   .

0.7

0.7

20

13.0

0.1969.

0.8

0.6

17

14.0

0.0454*

Pre-imp Loss / Animal

GD 29 [k]

Mean

SD

N

1.24

0.94

21

1.50

1.05

20

1.85

1.25

20

1.82

1.63

17

[a] – Anova & Dunnett(Log)

[k] – Kruskal-Wallis & Wilcoxon 

[f] – Cochran Armitage, Chi-Squared & Chi squared - Pearson

 

 

Conclusions:
In this study, which conducted based on OECD TG 414 and under GLP conditions, prenatal developmental toxicity of the test material was evaluated in New Zealand White rabbits following daily administration by oral gavage at 0, 5, 15 and 50 mg/kg/day during gestation days 6 to 28. External and visceral and skeletal examination of fetuses revealed no signs of teratogenicity. Based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) are as follows:
- NOAEL maternal general toxicity is 15 mg/kg/day
- NOAEL maternal developmental toxicity is 15 mg/kg/day
- NOAEL developmental toxicity is 15 mg/kg/day
Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of the test material when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits.This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. Mated female rabbits were assigned to four groups with 23 animals in each group. The day on which mating had occurred was considered as gestation Day 0 (GD 0) for each individual female rabbit. Test material in vehicle (Corn oil) was administered at 0, 5, 15 and 50 mg/kg/day. The control group received the vehicle only. Dose formulation analysison initiation and termination of treatment periodindicated all samples were within 15% of the nominal concentrations. All rabbits were observed for clinical signs, morbidity and mortality, body weight changes and food consumption.Caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses.The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations. All the live fetuses were examined forvisceral and skeletal variations and malformations. The main findings of the study are presented below:

There were no mortalities, clinical signs or gross necropsy findings at any of the doses tested except for incidental abortions in each of the, low, mid and high dose groups. Mean body weight gain at 50 mg/kg/day was lower compared to the vehicle control animals. Food consumption at 50 mg/kg/day was reduced during treatment period (GD 6 to 28). At 50 mg/kg/day there was a reduction in uterine weight, increase in post implantation loss and two dams with complete resorptions. At 50 mg/kg/day a significant reduction in litter weight was observed. Visceral evaluation at 50 mg/kg/day showed incidences (2/94 fetus) of unilateral absence of kidney and ureter and ureter dilated with hydronephrosis.

In conclusion, based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) for maternal and fetal developmental toxicity is 15 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 June - 30 November 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline on Detection of Toxicity to Reproduction for Medicinal Products, endorsed by the ICH Steering Committee
Principles of method if other than guideline:
The International Conference on Harmonisation (ICFI) Guideline on Detection of Toxicity to Reproduction for Medicinal Products, endorsed by the ICH Steering Committee Step 4 of the ICH process 24 June 1993. Section 4.1.2.was used, since there was no accepted protocol for such a study at that time. This guideline has also been issued as: JMOHW (Japanese Ministry of Health and Welfare): (Yakushin No. 470 of 7 July 1994).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CDBR VAF/Plus
Details on test animals and environmental conditions:
118 sexually mature female rats (Cr1 :CD®BR VAF/Plus strain), approximately 8 - 10 weeks, which were time-mated to males of the same strain, were received from Charles River UK Ltd, Manston Road, Margate, Kent. An additional 10 animals were supplied with batch A for health check purposes and subjected to routine macroscopic examination.
Initial group mean bodyweights within each batch (weight range 244 - 357 g).
Animal room temperature and relative humidity controls were set at 21 ± 2°C and 55 ± 10% respectively. Artificial lighting was controlled to give 12 hours continuous light and 12 hours continuous dark per 24 hours.
All animals had free access to tap water and pelleted SDS Laboratoiy Animal Diet No. 1.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test substance, AHTN, was administered as a solution in corn oil. The highest concentration formulation was prepared by mixing the test substance with vehicle. Lower concentrations were prepared by serial dilution. Control aniinals received the vehicle alone.
The formulations were prepared daily and used within 24 hours of preparation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Acetone extraction followed by High Performance Liquid Chromatography with UV detection.
The mean concentrations of test formulations analysed during the toxicity study were within 5% of the nominal concentration confirming the accuracy of preparation. The results also confirm that test formulations were true solutions and stable during ambient temperature storage for 24 hours, a period representing the maximum time from preparation to the completion of dosing.
Details on mating procedure:
The day of mating, as judged by the appearance of sperm in the vaginal smear or by the presence of a vaginal plug, was considered to be Day 0 of pregnancy.
F1: During the pre-mating period, F1 males and females animals were housed seperately and during the mating period, they were housed on the basis of one male to one female.
Duration of treatment / exposure:
The test substance, AHTN, was administered by gavage; only the F0 females received treatment. Treatment commenced on Day 14 of pregnancy and continued daily up to sacrifice on, or shortly after, Day 22 postpartum. The dosage volume was calculated for individual females on Days 14 and 16 of pregnancy according to bodyweight. Dose volumes were also adjusted according to bodyweight on Days 1, 7, 14 and 21 post partum.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
2, 6 and 20 mg/kg/day
Basis:

No. of animals per sex per dose:
112 animals with the highest weight gains were assigned to four groups i.e. 28 animals per dose group.
Control animals:
yes
Details on study design:
Duration of test: from day 14 of pregnancy up to sacrifice at weaning
Maternal examinations:
Occasional and isolated instances of salivation were noted after dosing amongst some treated animals but not controls.
There were no other clinical signs noted that were considered to be related to treatment.

There were no treatment-related effects on bodyweight gain or food intake following the start of treatment on Day 14 of pregnancy prior to parturition or on the pattern of bodyweight change during lactation.

The mean duration of pregnancy was not affected by treatment. There were no indications of any intergroup differences in the length of parturition.

There were no treatment-related changes in the incidence of minor abnormalities noted at post mortem examination of the females following weaning of their offspring.
Ovaries and uterine content:
Following the start of treatment on Day 14 of pregnancy, there were no treatment-related effects on the incidence of in utero losses.
Statistics:
Significance tests, employing analysis of variance followed by an intergroup comparison with the control, were performed on food consumption, bodyweight change, litter data, sex ratios, pre-weaning development, Post weaning behavioural tests, sexual maturation.

Dependent on the heterogeneity of variance between treatment groups, parametric tests were used to analyse the data, as appropriate. For litter data and pre-weaning development, the basic sampling unit was the litter and, due to the preponderance of non-normal distributions non parametric analyses were routinely used.

All significant (p ≤0.05) intergroup differences from the control are reported.
Where 75% or more of the values for a given variable were the same, a Fishers’ exact test was used, when considered necessary.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No treatment-related effects on mortality, body weight and food intake. The mean duration of pregnancy was also not affected. There were no treatment-related changes in the incidence of minor abnormalities noted at post mortem examination of the females following weaning of their offspring.
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effects in birth weight. Mean values for litter size at birth were comparable for all groups. No evidence of any selective effect of treatment on survival of either sex.
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day
Basis for effect level:
other: No adverse toxic effect observed to the highest tested dose
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Abnormalities:
no effects observed
Developmental effects observed:
no

There were no clinical signs noted amongst the F1 adults that were considered to be related to treatment of the F0 parent female.

Mean weekly bodyweight gain of F1 males and females from nominal Week 4 was unaffected by treatment of the F0 parent females.

There were no intergroup differences in the mean age of attainment or bodyweight on sexual maturation.

Treatment of parent females did not appear to impair the performance of their offspring on the Rotarod, Actimat or Passive Avoidance tests.

 

Treatment of the parent females had no adverse effect upon the mating performance of their offspring.

 

The incidence of macroscopic changes noted at autopsy of F1 adult males and females was low in all groups and considered to be unrelated to maternal treatment.

 

There were no macroscopic changes noted at post mortem examination of F2 pups that died or were sacrificed at weaning that were considered to be attributable to treatment of the F0 parent females.

Conclusions:
Treatment of the pregnant rat during the peri- and post natal period at dosages of AHTN up to (>)20 mg/kg bw/day was without adverse toxic effect. There were no effects on development of the foetus during the peri-natal phase of post natal growth, development, performance in specific behavioural tests and reproductive capacity of the offspring; exposure of the offspring was limited to possible in-utero transfer across the placenta during late pregnancy or via indirect transfer via the mothers milk.
Under the conditions of this study, the no-observable adverse effect level (NOAEL) on the pregnant and lactating rat and peri- and post natal development of the offspring was considered to be 20 mg/kg bw/day.
Executive summary:

The effect of AHTN on the development of the conceptus and offspring following exposure of the pregnant/lactating female during the peri-natal phase (from completion of organogenesis) to weaning. The primary intention of this study was to investigate whether exposure of the newborn to the test material via maternal milk resulted in any changes in Post natal development.

AHTN was administered at dosages of 0 (Control), 2, 6 or 20 mg/kg/day once daily by gavage to groups of 28 time-mated animals from Day 14 of pregnancy (end of organogenesis) through to weaning on Day 21 Post partum. The females were allowed to litter and rear their young to weaning (litters were standardised to 8 pups on Day 4 post partum). From these litters, selected offspring were retained (24 males and females per group) to maturity and assessed for behavioural changes and reproductive capability. The only exposure the F1 generation had to the test substance was in utero during the peri-natal phase or through any transfer in the milk of the lactating dams.

 

Apart from occasional, but not dosage-related, occurrences of salivation after dosing there were no apparent effects of treatment in any of the treated parent females during pregnancy or lactation. No effects were apparent on development of the foetus during the late pre-natal phase on post natal growth, development, performance in specific behavioural tests and reproductive capacity of the offspring.

Since no adverse effects were detected, no LOAEL can be established, and the NOAEL is higher than or equal to the highest dose tested, thus ≥20 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
6 - 30 january 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Equivalent to OECD guideline 414 (confirmed by Research Institute for Fragrance Materials). Study conducted by GLP accredited laboratory.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
AHTN in corn oil was daily administered through the gavage of rats during gestation. The dams were observed for toxicity and sacrificed for gross necropsy.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD BR VAF/Plus
Details on test animals and environmental conditions:
Groups of 25 Crl:CD®BR VAF/Plus® (Sprague-Dawley) female rats, supplied by Charles River Laboratories, Inc., Kingston, NY.
Female rats were individually housed except during the 5-day cohabitation period. All cage sizes and housing conditions were in compliance with the ‘Guide for the Care and Use of Labora tory Animals’ (Institute of Laboratory Animal Resources, 1996). Light cycles were automatically maintained at 12-h light:12-h dark. Rats were given ad libitum access to feed and water.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
AHTN was administered on days 7-17 of presumed gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis to check concentration and stability of AHTN in solution.
Details on mating procedure:
Dosage-range finding studies in rats were conducted at 100, 50, 25, 10 mg/kg per day. One animal from the 100 mg AHTN/kg per day dosage group was sacrificed in a moribund condition. Rats in the two highest AHTN dosage groups had reduced body weight gains.
Based on the results of these studies, dosages tested were 5, 15, 50 mg/kg per day.
Duration of treatment / exposure:
10 days (day 7-day 17 of gestation)
Frequency of treatment:
once daily
Duration of test:
20 days (of gestation)
Remarks:
Doses / Concentrations:
5, 15, 50 mg/kg/day
Basis:
other: The treatment period included days 7-17 of gestation (GD 7—17), and control and test articles were administered once daily by gavage at a dosage volume of 5 ml/kg, adjusted daily according to individual body weights recorded immediately before intubation.
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
Sex: female
Maternal examinations:
The dams were observed for signs of toxicity and body weights and feed intake were recorded. On day 20 of gestation, gross necropsy was performed.
Ovaries and uterine content:
The number of corpora lutea in the ovaries were recorded and the uteri were examined for pregnancy, number and distribution of implantations, live and dead foetuses and early and late resorptions. The placenta was also examined.
Fetal examinations:
All foetuses were weighed and examined for sex and gross external abnormalities. One half of the foetuses in each litter were examined for soft tissue alterations. The remaining foetuses were examined for skeletal alterations.
Statistics:
Statistical evaluations were performed using SAS programs. Clinical observations and other proportional data were analyzed by the Variance Test for Homogeneity of Binomial Distribution. Continuous data were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance or Dunnett’s Test. The Kruskal-Wallis Test along with Dunn’s Method of Multiple Comparisons or Fisher’s Exact Test was used if the Analysis of Variance was not appropriate.
Indices:
Gross necropsy parameters included: gross lesions and numbers of corpora lutea, implantations, live and dead foetuses and resorptions.
Foetus parameter: body weighed, sex and gross external alterations.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnancy rates were unaffected by the fragrances and ranged between 72 and 96% in the fragrance-treated groups, versus 84 and 100% in the two control groups. No abortions, premature deliveries or fragrance-related deaths occurred during the study.
Clinical signs, including urine-stained abdominal fur, excess salivation, red or brown substances on the forepaws, localized alopecia, tremors, chromodacryorrhea and dry faeces, were frequently increased in incidence in mid-dosage groups, and were significantly increased in the high-dosage groups, as compared with controls.
In the high-dosage group rats (50 mg/kg per day) localized alopecia and green or mottled livers were observed.
Dose-dependent, statistically significant reductions in maternal body weight gains and feed consumption occurred in all dosage groups with a significant rebound in body weight gains and feed consumption values in the high group.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The decreases in bodyweight were well within historical ranges and because of no clear dose relationship they were not considered relevant. No effects were observed on numbers of implantations, dead or live foetuses, resorptions or foetal sex ratios. There were no skeletal changes and all gross external, soft tissue or skeletal malformations in the foetuses were incidental and considered unrelated to the test article.
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: not selectively toxic to development; no adverse effects on development occurred
Abnormalities:
no effects observed
Developmental effects observed:
no

No abortions or premature deliveries occurred during the study. One non-pregnant 5 mg/kg/day dosage group rat was moribund sacrificed on presumed gestation day (DG) 11, because it had an injury of the snout/palate on or about DG 9, an event unrelated to the test article.

 

Significantly increased (p0.01) numbers of 50 mg/kg/day dosage group rats had localized alopecia, generally on the underside and back, but sometimes on the limbs. The 50 mg/kg/day dosage group also had a significant increase (p0.01) in the number of rats with colour changes in the liver (green or mottled green and dark red), findings attributable to the test article and not observed in any other dosage group.

 

The 5 mg/kg/day dosage group had a transient, small (p>0.05) reduction in maternal body weight gain, the 15 mg/kg/day dosage group had significantly reduced (p0.05) maternal body weight gain and the 50 mg/kg/day dosage group had significant maternal body weight loss (p0.01) on DGs 7 to 10. During the remainder of the dosage period, the 5 and 15 mg/kg/day dosage groups had comparable or slightly increased weight gains (DGs 12 to 15, p0.05, 15 mg/kg/day dosage group), and the 50 mg/kg/day dosage group had reduced weight gains (p0.01 on DGs 10 to 12 and 15 to 18), as compared with the control group values. As a result, the 50 mg/kg/day dosage group had significantly reduced (p0.01) body weight gain for the entire dosage period (calculated as DGs 7 to 18). After completion of the dosage period (DG5 18 to 20), groups administered 5 mg/kg/day and higher dosages of AHTN had dosage dependent increases in weight gains, as compared with the control group value (p0.05 to p0.01 in the 15 and 50 mg/kg/day dosage groups), rebound phenomena commonly observed in these types of studies after completion of the dosage period. These effects resulted in only the 50 mg/kg/day dosage group demonstrating significant reductions (p0.01) in weight gains for the entire period after the first dosage was administered (DGs 7 to 20) and for the entire pregnancy (DG5 0 to 20).

 

DG 0 maternal body weights were comparable following randomization and assignment to four dosage groups (Groups 1 through IV However, Groups II through IV gained significantly less weight (p0.01) during the pre-treatment period (DGs 0 to 7) than Group 1. These events were unrelated to the test article. On DG 7 (first treatment day), rats assigned to Groups II, III and IV had biologically comparable mean body weights (means ranged from 269.0 g to 265.2 g) that were reduced (Group II) or significantly reduced (Group III, p0.05; Group IV, p0.01), as compared with the control group (Group 1) value (275.2 g). The reduction in maternal body weight on DG 7 was considered unrelated to the test article. Maternal body weights were significantly reduced (p0.05) in Group II on DGs 8 through 10 and DG 12, as compared with the control group (Group 1) value. Maternal body weights were significantly reduced (p0.05 to p0.01) in Group III on DGs 7 through 14 and DG 16. Maternal body weights were significantly reduced (p0.0l) in Group IV on DGs 7 through 20.

 

Groups administered 5 mg/kg/day and higher dosages of AHTN had dosage dependent, significant (p0.05 to p≤0.01) reductions in absolute (g/day) feed consumption values for the entire dosage period (calculated as DGs 7 to 17). Relative (g/kg/day) feed consumption values tended to be reduced in the 5 mg/kg/day dosage group and were significantly reduced (p0.01) in the 15 and 50 mg/kg/day dosage groups on DG5 7 to 17. These effects of the test article reflected the cumulative effect of small reductions in absolute and relative feed consumption values in the 5 mg/kg/day dosage group and a reduced or significantly reduced (p≤0.05 to p≤0.01) absolute and relative feed consumption values throughout the dosage period in the 15 and 50 mg/kg/day dosage groups. As observed for body weight gain, the effects were most severe on DGs 7 to 10. Only the 50 mg/kg/day dosage group had a post-dosage (DGs 18 to 20) increase in relative feed consumption value, a rebound phenomenon. Absolute and relative feed consumption values were significantly reduced (p0.05 to p0.0l) in the 15 and 50 mg/kg/day dosage groups for the entire interval after the first dosage (DGs 7 to 17) and for the entire pregnancy (DGs 0 to 20).

 

The smaller, statistically significant (p0.05) reductions in foetal body weight parameters in the 5 and 15 mg/kg/day dosage groups were considered unrelated to the test article because they did not demonstrate dosage-dependency and were within historical ranges of the Testing Facility. The 50 mg/kg/day dosage group had statistically significant (p0.01) reductions in foetal body weight. These observations were not considered effects of the test article because, while the values were lower than in any other dosage group, the values were within ranges observed historically at the Testing Facility.

All other Caesarean-sectioning and litter parameters were unaffected by dosages of the test article as high as 50 mg/kg/day. No dosage-dependent, statistically significant or biologically important differences occurred in the litter averages for corpora lutea, implantations, litter sizes, live/dead foetuses, early/late resorptions, percent resorbed conceptuses, or percent male foetuses. No dam had a litter consisting of only resorbed conceptuses, and there were no dead foetuses. All gross external, soft tissue or skeletal malformations or variations in the foetuses were considered unrelated to the test article.

Conclusions:
The maternal no-observable-adverse-effect-level (NOAEL) for AHTN is 5 mg/kg/day (minimal reductions in maternal body weight gain and feed consumption values occurred at this dosage). Maternal toxicity was evident in the 15 and 50 mg/kg/day dosage groups as adverse clinical and necropsy observations (50 mg/kg/day dosage group only) and biologically important, significant reductions in maternal body weight gains and absolute and relative feed consumption values.
The developmental NOAEL for AHTN is 50 mg/kg/day. Based on these data, the test article was not selectively toxic to development; no adverse effects on development occurred.
Executive summary:

AHTN was tested for developmental toxicity in rats (25/group, 3 groups/fragrance, 2 fragrances/corn oil control). Dosages tested was 5, 15, 50 mg/kg per day. These dosages exceeded multiples of the estimated maximal daily human dermal exposure. Treatment (gavage, 5 ml/kg) occurred on days 7-17 of gestation and Caesarean-sectioning on day 20 of gestation. Based on the results of these studies, none of the four fragrances tested were more toxic in the conceptuses than in the dams. Maternal NOAEL was 5 mg/kg per day (50 mg/kg per day caused clinical signs and reduced weight gain and feed consumption). Developmental NOAEL was 50mg/kg per day. No adverse effects on embryo-foetal viability, growth or morphology occurred at the highest dosages of AHTN (50 mg/kg per day). The results of this study indicate that under conditions of normal use, the test fragrances do not pose a risk to human conceptuses.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
OECD 414, in compliance with GLP.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The objective of this study was to evaluate the prenatal developmental toxicity of the test material when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits.This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. Mated female rabbits were assigned to four groups with 23 animals in each group. The day on which mating had occurred was considered as gestation Day 0 (GD 0) for each individual female rabbit. Test material in vehicle (Corn oil) was administered at 0, 5, 15 and 50 mg/kg/day. The control group received the vehicle only. Dose formulation analysison initiation and termination of treatment periodindicated all samples were within 15% of the nominal concentrations. All rabbits were observed for clinical signs, morbidity and mortality, body weight changes and food consumption.Caesarean section was performed for all the surviving rabbits on GD 29 and dams were examined for gross pathological changes. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses.The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations. All the live fetuses were examined for visceral and skeletal variations and malformations. The main findings of the study are presented below:

There were no mortalities, clinical signs or gross necropsy findings at any of the doses tested except for incidental abortions in each of the, low, mid and high dose groups. Mean body weight gain at 50 mg/kg/day was lower compared to the vehicle control animals. Food consumption at 50 mg/kg/day was reduced during treatment period (GD 6 to 28). At 50 mg/kg/day there was a reduction in uterine weight, increase in post implantation loss and two dams with complete resorptions. At 50 mg/kg/day a significant reduction in litter weight was observed. Visceral evaluation at 50 mg/kg/day showed incidences (2/94 fetus) of unilateral absence of kidney and ureter and ureter dilated with hydronephrosis.

In conclusion, based on the above findings, the No- Observed- Adverse- Effect Level (NOAEL) for maternal and fetal developmental toxicity is 15 mg/kg/day.

  

The effect of AHTN on the development of the conceptus and offspring following exposure of the pregnant/lactating female during the peri-natal phase (from completion of organogenesis) to weaning. The primary intention of this study was to investigate whether exposure of the newborn to the test material via maternal milk resulted in any changes in Post natal development.

AHTN was administered at dosages of 0 (Control), 2, 6 or 20 mg/kg/day once daily by gavage to groups of 28 time-mated animals from Day 14 of pregnancy (end of organogenesis) through to weaning on Day 21 Post partum. The females were allowed to litter and rear their young to weaning (litters were standardised to 8 pups on Day 4 post partum). From these litters, selected offspring were retained (24 males and females per group) to maturity and assessed for behavioural changes and reproductive capability. The only exposure the F1 generation had to the test substance was in utero during the peri-natal phase or through any transfer in the milk of the lactating dams.

Apart from occasional, but not dosage-related, occurrences of salivation after dosing there were no apparent effects of treatment in any of the treated parent females during pregnancy or lactation. No effects were apparent on development of the foetus during the late pre-natal phase on post natal growth, development, performance in specific behavioural tests and reproductive capacity of the offspring.

Since no adverse effects were detected, no LOAEL can be established, and the NOAEL is higher than or equal to the highest dose tested, thus ≥20 mg/kg bw/day.

 

AHTN was tested for developmental toxicity in rats (25/group, 3 groups/fragrance, 2 fragrances/corn oil control). Dosages tested was 5, 15, 50 mg/kg per day. These dosages exceeded multiples of the estimated maximal daily human dermal exposure. Treatment (gavage, 5 ml/kg) occurred on days 7-17 of gestation and Caesarean-sectioning on day 20 of gestation. Based on the results of these studies, none of the four fragrances tested were more toxic in the conceptuses than in the dams. Maternal NOAEL was 5 mg/kg per day (50 mg/kg per day caused clinical signs and reduced weight gain and feed consumption). Developmental NOAEL was 50mg/kg per day. No adverse effects on embryo-foetal viability, growth or morphology occurred at the highest dosages of AHTN (50 mg/kg per day). The results of this study indicate that under conditions of normal use, the test fragrances do not pose a risk to human conceptuses.

Mode of Action Analysis / Human Relevance Framework

In the OECD TG 443, potential effects of the test item on T3, T4 and TSH levels were investigated. In two occasions, a statistically significant difference was found for the test item treated groups. A statistically significant increase of T4 and TSH was noted in high dose Cohort 1A Female. Statistical analysis of TSH did not reveal any effect related to the test item treatment in parent male and female, Cohort 1A Male and in PND 22 male and female pups. In addition, a statistically significant increase was observed for T3 in the mid dose F1 pups. Due to the limited historical control data available at the test facility, these values should be interpreted with care. Due to the low frequency of these findings in the absence of an apparent dose response, these effects should be considered as chance findings without any toxicological relevance.

Justification for classification or non-classification

Based on the available information classification for toxicity to reproduction is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.