Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD Guideline 422), the NOAEL for systemic toxicity was 1500 ppm (mean achieved doses of 85.4 mg/kg bw/day for males, 93.6 mg/kg bw/day for toxicity phase females, 106.9 mg/kg bw/day for females during gestation and 176.9 mg/kg bw/day for females during lactation).

The NOAEL of test item for reproductive/developmental effects was 1500 ppm.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 January to 4 September 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD Guideline 422: the high dose group was terminated earlier due to welfare reasons.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
the high dose group was terminated earlier due to welfare reasons
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
- Basis for dose level selection : Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: JM03MQ) following consultation with the Sponsor.
- Route of administration : The dietary route of administration was chosen to simulate a condition of potential human exposure.
- Animal model: The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley (Crl:CD(SD) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of animals at study initiation: Approximately 71 days (males); Approximately 85 days (females)
- Weight at study initiation:: Males: 338-399 g; Females: 241-314 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre pairing - up to five animals of one sex; During pairing - one male and one female; Males after mating - up to five animals; Gestation - one female; Lactation: one female + litter.
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and
during urine collection).
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection).
- Acclimation period: Females: six days prior to the commencement of estrous cycle evaluation; Males: six days prior to the commencement of treatment


ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h dark / 12 h fluorescent light

IN-LIFE DATES: 28 January to 4 September 2016
Route of administration:
oral: feed
Vehicle:
other: feed
Details on exposure:
DIET PREPARATION
- Diet: SDS VRF1 certified powdered diet
- Correction factor: A correction factor was not required.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: On each occasion of the preparation of the premix the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer. For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly; Each formulation was divided into seven daily aliquots and stored in a freezer until required.
- Storage of formulation: Deep-frozen (nominally -20 °C)
Details on mating procedure:
- Animals: Toxicity phase and Recovery phase males with Reproductive phase females (Toxicity and Recovery phase females were not paired for mating).
- M/F ratio per cage: 1: 1from within the same treatment groups
- Pairing commenced: After a minimum of three weeks of treatment
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of gestation.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 500, 1000, 1500 and 20000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix.
- Achieved concentration: Samples of each formulation prepared for administration in Weeks 1, 7 (to coincide with pairing) and in the final week of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
- Toxicity phase males and females of Groups 1 to 3 were treated for at least seven weeks. The males were paired with the reproductive phase females after six weeks of treatment. Males and females were terminated in Week 8 on completion of the Week 8 investigations.
- Reproductive phase females were treated for six weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk.

Frequency of treatment:
Continuously
Dose / conc.:
0 ppm (analytical)
Remarks:
Group 1 (Control)
Dose / conc.:
1 500 ppm (analytical)
Remarks:
Group 2 (Low dose)
Dose / conc.:
4 000 ppm (analytical)
Remarks:
Group 3 (Mid dose). After 25 days of treatment the dietary concentrations for Group 3 was reduced to 3500 ppm as a consequence of effects seen. After 39 days of treatment the dietary concentration for Group 3 was returned to 4000 ppm.
Dose / conc.:
10 000 ppm (analytical)
Remarks:
Group 4 (High dose). After 25 days of treatment the dietary concentrations for Group 4 was reduced to 8000 ppm as a consequence of effects seen. After 43 days of treatment, the Group 4 animals were prematurely sacrificed, due to effects seen.
No. of animals per sex per dose:
- Reproductive phase: 10 females/dose
- Toxicity phase: 5, 10, 10 and 5 males for 0, 1500, 4000 and 10000 ppm, respectively; 5 females/dose for all groups
- Recovery phase: 5 animals/sex/dose for 0 and 10000, respectively (After 43 days of treatment all the Group 4 animals were prematurely sacrificed which resulted in the cancellation of the recovery phase)
Control animals:
other: Untreated diet of the same batch with corn oil (2%)
Details on study design:
- Dose selection rationale: The initial dietary levels used in this study (0, 1500, 4000 and 10000 ppm) were selected in conjunction with the Sponsor.
Dietary levels were selected following the completion of the preliminary toxicity study (Envigo Study number: JM03MQ) following consultation with the Sponsor. Treatment at 15000 ppm resulted in low body weight gain of males during the first week of treatment. Among females receiving 15000 ppm, marked mean body weight loss was recorded during the first week of treatment: this was followed by high weight gain during Week 2 but further mean weight loss during Week 3; weight loss was considered to preclude the use of 15000 ppm on this study. At 15000 ppm, mean food consumption was markedly low during the first day of treatment of males and the first three days of treatment of females. Overall food consumption of both sexes receiving 15000 ppm was low during Weeks 1 and 3 but similar to Controls during Week 2. Females receiving 7500 ppm showed mean body weight stasis during the first week of treatment. Food consumption of females receiving 7500 or 1500 ppm was slightly low during the first day of treatment. The high dietary concentration of 10000 ppm was expected to elicit initial mean body weight loss or stasis in females and initial low food consumption. The lowest dietary concentration was expected to be a no effect level for effects on bodyweight and food consumption. The intermediate dietary concentration of 4000 ppm allowed evaluation of any concentration related trends and provided a geometric progression of dietary concentrations.
- Rationale for animal assignment: On arrival and non-selective allocation to cages. On Day 1 of study all animals were weighed and body weights were reviewed before feeding of the treated diets by Study Management to ensure variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule:
Sensory reactivity and grip strength assessments were performed on the five lowest numbered surviving toxicity phase males and females in Groups 1, 2 and 3 during Week 8 of treatment.
Motor activity: During Week 8 of treatment, the motor activity of the five lowest numbered surviving toxicity phase males and females in Groups 1, 2 and 3 was measured.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males and females: Before feeding of the treated diets on the day that treatment commenced (Day 1) and daily thereafter. On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations:
F0 animals: Daily, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating, but recommenced for males when pairing of all animals was completed.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

WATER CONSUMPTION: No

OPHTHALMIC EXAMINATION: Yes
- Eyes of the animals were examined by means of a binocular indirect ophthalmoscope.
- Time schedule:
Pretreatment: All toxicity and recovery phase animals and spare animals.
Week 8: All toxicity phase females and the first five toxicity phase males of Groups 1 and 3.
Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 8; Five lowest numbered surviving toxicity phase males and females in each group
- Animals fasted: Yes, Animals were deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of the blood sampling procedures.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

URINALYSIS
- Time schedule for collection of urine: Week 8; Five lowest numbered surviving toxicity phase males and females in each group.
- Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight.
- Parameters:
Using manual methods: Appearance (clarity and color) (App) - by visual assessment; Volume (Vol) - using a measuring cylinder; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek®500 instrument: Ketones (Keto); Bile pigments (Bili); Blood pigments (UBld); Urobilinogen (Urob)
Using a Roche P Modular Analyzer: Protein concentration (Prot) and total output (T-Prot); Glucose concentration (U-Gluc) and total output (T-Gluc); Creatinine concentration (U-Creat) and total output (T-Creat); Sodium concentration (U-Na) and total output (T-Na); Potassium concentration (U-K) and total output (T-K); Chloride concentration (U-Cl) and total output (T-Cl)
Microscopic examination of the urine sediment: Epithelial cells (Epi), Leucocytes (WBC), Erythrocytes (RBC), Casts, Other abnormal components (A)

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Oestrous cyclicity (parental animals):
Wet smears: Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
- After pairing until mating.
- For four days before scheduled termination (all reproductive, toxicity phase and recovery phase females).

Dry smears: Reproductive phase females: from beginning of treatment until animals are paired for mating, using cotton swabs.
Sperm parameters (parental animals):
No data
Litter observations:
PARAMETERS EXAMINED
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4, 7 and 13 of age
- Individual offspring bodyweights: Days 1, 4, 7 and 13 of age
- Ano-genital distance: Day 1 - all F1 offspring
- Nipple/areolae count: Day 13 of age - male offspring
Postmortem examinations (parental animals):
SACRIFICE
- All Group 4 males and females: After seven weeks of treatment
Toxicity phase:
- Males and females Groups 1, 2 and 3: After final investigations completed (during Week 8)
Reproductive phase
- F0 females failing to produce a viable litter: Day 25 after mating
- F0 females whose litter died before Day 13: On or after day the last offspring died
- F0 females: Day 14 of lactation

- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
- Adult animals of Group 4 were culled and discarded without further examination. All adult animals of Group 1, 2 and 3 were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
In each uterine horn, number of implantation sites was recorded.
ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The organs weighed, tissue samples fixed and sections examined microscopically are detailed in table 7.8.1/1 and 7.8.1/2 for F0 animals.
- Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid; Eyes: In Davidson’s fluid.
- Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All animals (Groups 1, 2 and 3) killed or dying prematurely. The five lowest toxicity phase males and females in Groups 1 and 3 at scheduled termination.
Kidneys: The five lowest toxicity phase males in Group 2.
Abnormalities only: All remaining adult animals
Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
- Light microscopy: Tissues preserved for examination were examined as follows:
Premature deaths: All F0 animals from all groups
Scheduled kill:
Five lowest numbered toxicity phase males and females in Groups 1 and 3
All F0 animals: Abnormalities only
Five lowest numbered toxicity phase males in Group 2: Kidneys
Postmortem examinations (offspring):
SACRIFICE:
- F1 offspring:
Selected offspring for T3, T4, TSH analysis - Day 4 of age.
Scheduled kill - Day 13 of age
- Method of sacrifice:
Offspring (selected for thyroid hormone sampling on Day 4 or Day 13 of age): Decapitation
Offspring (not subjected to thyroid hormone sampling): Intraperitoneal injection of sodium pentobarbitone

GROSS NECROPSY
- Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
- F1 offspring on Day 4 of age: Externally normal offspring discarded without examination. Externally abnormal offspring examined and where possible tissues retained as specified in Table 7.8.1/3.
F1 offspring on Day 13 of age: All animals (but not including those selected for thyroid hormone analysis) were subject to a complete macroscopic examination; particular attention was paid to the external genitalia. Organs and tissues retained as specified in Table 7.8.1/3 from one male and one female in each litter.
Animals selected for thyroid hormone analysis: externally normal offspring discarded without examination; externally abnormal offspring examined and where possible tissues retained as specified in Table 7.8.1/3.
Statistics:
See section "Any other information on materials and methods incl. tables”.
Reproductive indices:
Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100

Gestation length
- Gestation index (%) = (Number of live litters born / Number pregnant ) x 100
Offspring viability indices:
Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = [(Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering)[ x 100
- Lactation index (%) = [(Number of live offspring on Day 13) / (Number live offspring on Day 4 (after blood sampling))] x 100

Sex ratio:
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- There were no clinical signs or arena observations considered to be related to treatment amongst males and females receiving the test item at 1500 or 4000/3500/4000 ppm, or males receiving test item at 10000/8000 ppm.
- Amongst females receiving the test item at 10000/8000 ppm, hunched posture, piloerection and abnormal gait was seen in some animals during the treatment period. There was also an increase in brown staining on the head/muzzle.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females receiving 4000/3500/4000 ppm were killed for welfare reasons due to poor clinical condition on Day 1 of lactation:
- Female 71 had shown piloerection throughout gestation and on Day 1 of lactation. The litter was cold to touch with a small amount of milk apparent in the stomach and was killed for welfare reasons. No macroscopic abnormalities were observed at necropsy of the dam but histopathology of the full range of tissues examined revealed slight multifocal necrosis in the liver.
- Female 74 had dull eyes on Day 1 of lactation. The litter was cold to touch with a small amount of milk apparent in the stomach and was killed for welfare reasons. Macroscopic examination of the dam revealed pale mammary glands, dark caecum contents and a few pale punctate foci on the lungs. Histopathology of the full range of tissues examined revealed minimal focal necrosis in the liver and slight multifocal aggregates of alveolar macrophages in the lungs.
- Since both females showed poor clinical condition on Day 1 of lactation and necrosis in the liver a relationship to treatment cannot be completely ruled out.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- The body weight and body weight gain of males receiving test item at 1500 or 4000/3500/4000 ppm was similar to that of the Control throughout the treatment period.
- The body weight and body weight change of males receiving test item at 10000 ppm was lower than that of the Control during Days 1-26 of treatment. Once the dose was reduced from 10000 to 8000 ppm from Day 26 and until Day 36, the bodyweight gain for these males was similar to that of Control. Thereafter, the body weight gain for these males was lower than that of the Control until termination on Day 43 of treatment.
- The body weight and body weight gain of females allocated to the toxicity phase at 1500 ppm was generally slightly lower than that of the Control throughout the treatment period, with overall body weight gain during Days 1-56 being 41% of Control.
- Females allocated to the toxicity phase and receiving test item at 4000 ppm experienced bodyweight loss in Week 1, bodyweight stasis in Week 2 and during Weeks 3 to six bodyweight gain was similar to that of Control. The dose was lowered to 3500 ppm from Day 26, and increased to 4000 ppm from Day 40 of treatment. Overall body weight gain during Days 1-56 was 21% of Control.
- Females allocated to the toxicity and reproductive phase and receiving test item at 10000 ppm experienced a bodyweight loss of 23g in Week 1 of treatment however during Weeks 2 to 6 the bodyweight change of these females was variable but generally similar to that of Control, but overall body weight gain during Days 1-43 was low.
- The body weight and body weight gain of females allocated to the reproductive phase at 1500 ppm was variable, but overall body weight gain during Days 1-43 was the same as that of the Control.
- Females allocated to the reproductive phase and receiving test item at 4000 ppm experienced bodyweight loss in Week 1, bodyweight stasis in Week 2 and during Weeks 3 to 6 bodyweight gain was similar to that of Control. The dose was lowered to 3500 ppm from Day 26, and increased to 4000 ppm from Day 40 of treatment.
- During gestation, the body weight and body weight gain of females at 1500 ppm was generally similar to that of the Control. At 4000/3500/4000 ppm mean bodyweights during gestation were lower than in Controls. Bodyweight gain during Days 0-13 of gestation was similar to Control but weight gain was low during Days 13-20 of gestation.
- During lactation, the body weight gain of females at 1500 ppm was lower than that of the Control during Days 1-7, and similar to that of the Control from Days 7-13. The females at 4000/3500/4000 ppm showed bodyweight loss during Days 1-7, compared to weight gain in the Controls, and body weight gain was similar to that of the Control from Days 7-13.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- The food consumption of males was generally similar to that of the Controls for males receiving test item at 1500 ppm or 4000/3500/4000 ppm; while food consumption was lower than that of the Controls for males receiving 10000/8000 ppm during Weeks 1 to 6 of treatment.
- The food consumption of females allocated to the toxicity phase receiving test item at 1500 ppm ppm was generally similar to that of the Controls during Weeks 1 to 6 of treatment. The food consumption of females allocated to the toxicity phase receiving test item at 4000/3500 ppm or 10000/8000 ppm was lower than that of the Control in Week 1 of treatment, and generally similar to that of the Controls from Week 2 onwards.
- The food consumption of females allocated to the reproductive phase receiving test item at 1500 ppm was generally similar to that of the Controls during Weeks 1 to 6 of treatment. The food consumption of females allocated to the reproductive phase receiving test item at 4000/3500/4000 ppm or 10000/8000 ppm was lower than that of the Control in Week 1 of treatment, and generally similar to that of the Controls from Week 2 onwards.
- During gestation, the food consumption of females at 1500 ppm was generally similar to that of the Control. The food consumption of females at 4000/3500/4000 ppm was lower than that of the Control throughout gestation.
- At 4000/3500/4000 ppm, mean food consumption during lactation was slightly lower than in Controls. At 1500 ppm, food consumption during Days 3-6, 8-10 and 12-13 of lactation was slightly lower than in Controls.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no findings at ophthalmic examination that were considered related to treatment with test item.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- At haematological examination in Week 8 of treatment, there were no significant changes amongst males receiving test item at 4000/3500/4000 ppm or 1500 ppm.
- At haematological examination in Week 8 of treatment, there were some differences amongst females receiving test item at 4000/3500/4000 ppm or 1500 ppm when compared with that of the Control:
White blood cell counts were statistically significantly low for females in the 4000/3500/4000 ppm and 1500 ppm groups.
Neutrophil counts were low for females in the 4000/3500/4000 ppm and 1500 ppm groups, with statistical significance achieved for females in the 4000/3500/4000 ppm group.
Lymphocyte counts were slightly low for females in the 1500 ppm group and statistically significantly low for females in the 4000/3500/4000 ppm group.
Monocyte counts were statistically significantly low for females in the 4000/3500/4000 ppm and 1500 ppm groups.
Large unstained cell counts were low for females in the 1500 ppm and 4000/3500/4000 ppm groups, with statistical significance achieved for females in the 4000/3500/4000 ppm group.
Prothrombin times were statistically significantly shorter than Controls for females in the 4000/3500/4000 ppm group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- At blood chemistry examination in Week 8 of treatment, there were no significant changes amongst males or females receiving test item at 4000/3500 ppm or 1500 ppm. There were some minor differences from Control, however, these occurred in one sex only and are considered unlikely to be related to treatment with test item. The differences were as follows:
Sodium levels were statistically significantly high for males in the 4000/3500/4000 ppm and 1500 ppm groups, there was no relationship to concentration.
Calcium levels were statistically significantly low for females in the 4000/3500/4000 ppm and 15000 ppm groups, although a dose response was not apparent.
Phosphorus levels were statistically significantly low for females in the 4000/3500/4000 ppm group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- At examination of the urine in Week 8 of treatment, there were no significant changes amongst males or females receiving test item at 4000/3500/4000 ppm or 1500 ppm. Protein levels but not total protein output were statistically significantly high for females in the 4000/3500/4000 ppm group, no other changes achieved statistical significance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory Reactivity and Grip Strength: Group mean forelimb grip strength values for all groups of treated males and females (4000/3500/4000 or 1500 ppm) were marginally low compared with Controls. However, these values did not attain statistical significance and the same pattern is not evident in the hindlimbs. Therefore, no definite effect of treatment is inferred. There was no effect of treatment on sensory reactivity.
Motor Activity: There was considered to be no effect of treatment on motor activity.
Group mean activity scores show a few inter-group differences with statistical significances attained at the 18-minute interval for high and low beams for males at 1500 or 4000/3500/4000 ppm and the 30-minute interval for low beams for males at 1500 or 4000/3500/4000 ppm. However, there is no dose relationship and statistical significance has not been attained for the total scores. Group mean activity scores for females at 1500 or 4000/3500/4000 ppm are similar to Controls.
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):
Animals killed at the end of treatment
Treatment related findings
- Changes related to treatment with test item were seen in the kidneys.
- Kidneys: An increased incidence and/or severity of cortical tubule hyaline droplet accumulation was seen in males given 1500 or 4000/3500/4000 ppm. The severity of this finding was dose dependent.
Incidental Findings
- All other histological changes were considered to be unrelated to treatment.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis: there was no clear effect of treatment with Reaction mass of beta-phellandrene and d-limonene and l-limonene at 4000/3500/4000 ppm or 1500 ppm on the thyroid hormone levels of adult males at termination or F1 offspring on Day 13 of age.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
- The oestrous cycle data show that there was a much higher percentage of the reproductive females showing an irregular cycle or extended oestrus at 10000/8000 ppm during the treatment period, compared with Controls. This has achieved statistical significance. Because of the effects seen, we have presented individual cycle lengths as well as cycle categories. So, during the treatment period, 50 per cent of the females at 10000/8000 ppm showed at least one extended cycle of 6-10 days and 40 per cent showed at least one extended cycle of 11-20 days compared with the Control group that showed only 20 per cent of females with at least one extended cycle of 6-10 days. 20% of females at 10000/8000 ppm also showed a cycle length of 3 days compared with none in the Control group. When looking at the incidence of cycle length, the number of cycles at 10000/8000 ppm were much reduced compared to Controls (and the other treated groups) with a total of 78 cycles throughout the treatment period compared with 107 in the Control group making it evident that there were fewer cycles due to the extended length. Statistical significance has again been achieved at 10000/8000 ppm.
- There was no effect of treatment at 4000/3500/4000 or 1500 ppm on estrous cycles during treatment or at termination.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance, pre coital interval, fertility, gestation length and index were unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Remarks on result:
other: mean achieved doses of 85.4 mg/kg bw/day for males, 93.6 mg/kg bw/day for toxicity phase females, 106.9 mg/kg/day for females during gestation and 176.9 mg/kg bw/day for females during lactation
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive effects
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs seen in some litters at 4000/3500/4000 ppm group included small amount of milk in stomach and pups cold to touch.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Amongst the offspring of females in the 4000/3500/4000 ppm group, there were a high number of offspring found dead or missing up until Day 4 of age. Clinical signs seen in some litters included small amount of milk in stomach and pups cold to touch.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 4000/3500/4000 ppm, mean body weights of male and female offspring on Day 1 of age were marginally lower than in Controls. Subsequent mean body weight gains of male and female offspring between Days 1 and 13 of age were slightly lower than in Controls.
- At 1500 ppm, there was no conclusive effect of treatment on mean offspring body weights on Day 1 of age. Subsequent mean body weight gains of male and female offspring were marginally lower than in Controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- Amongst offspring killed or dying before scheduled termination, the most common finding was no milk in stomach. There were no macroscopic findings at necropsy that could be associated with the maternal administration of test item.
- Amongst offspring killed at scheduled termination on Day 13 of age, there were no macroscopic findings at necropsy that could be associated with the maternal administration of test item.
Other effects:
no effects observed
Description (incidence and severity):
Litter Size, Sex Ratio and Survival Indices
- There was considered to be no effect of treatment on litter size, offspring survival or the sex ratio following maternal treatment at 1500 ppm.
The litter size, post implantation survival index and viability index in the 4000/3500/4000 ppm group was lower than in Controls. Sex ratio was unaffected in this group.
Ano-genital distance in male and female offspring was unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

None

Conclusions:
The No-observed-adverse-effect-level (NOAEL) of the test item for systemic toxicity was 1500 ppm (mean achieved doses of 85 mg/kg bw/day for males, 94 mg/kg bw/day for toxicity phase females, 107 mg/kg bw/day for females during gestation and 177 mg/kg bw/day for females during lactation).
The NOAEL of the test item for reproductive/developmental effects was 1500 ppm.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, groups of Crl:CD(SD) rats received the test item, Reaction mass of beta-phellandrene and D-limonene and L-limonene orally, via the diet at concentrations of 0,1500, 4000/3500/4000, 10000 ppm (Group 1, 2, 3 and 4, respectively). After 43 days of treatment all the Group 4 animals were prematurely sacrificed, due to effects seen. Toxicity phase males and females of Groups 1 to 3 were treated for at least seven weeks. The males were paired with the reproductive phase females after six weeks of treatment. Males and females were terminated in Week 8 on completion of the Week 8 investigations. Reproductive phase females were treated for six weeks before pairing,throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 certified diet with corn oil throughout the same relative period.

During the study, clinical condition, detailed physical examination and arena observations, body weight, food consumption, ophthalmic examination, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, sensory reactivity, grip strength, motor activity, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, nipple count (males only), ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. 

Achieved dose generally maintained the intervals between dietary concentrations. During lactation achieved doses were higher reflecting the increased physiological demand on the dams.

Two females at 4000/3500/4000 ppm and their litters were killed for welfare reasons due to poor clinical condition on Day 1 of lactation. Since both females showed poor clinical condition on Day 1 of lactation and necrosis in the liver a relationship to treatment cannot be completely ruled out. There were no clinical signs or arena observations considered to be related to treatment amongst males and females at 1500 or 4000/3500/4000 ppm.

The body weight, body weight gain and food consumption of males receiving test item at 4000/3500/4000 ppm was similar to that of the Control throughout the treatment period. Females allocated to the toxicity phase and receiving test item at 4000 ppm experienced bodyweight loss in Week 1, bodyweight stasis in Week 2 and during Weeks 3 to six bodyweight gain was similar to that of Control. The dose was lowered to 3500 ppm from Day 26, and increased to 4000 ppm from Day 40 of treatment. Overall body weight gain during Days 1-56 was 21% of Control. Females allocated to the reproductive phase and receiving test item at 4000 ppm experienced bodyweight loss in Week 1 with food consumption also lower than Control in this week, bodyweight stasis in Week 2 and during Weeks 3 to 6 bodyweight gain was similar to that of Control. Food consumption of these females was generally similar to that of the Controls from Week 2 onwards. The dose was lowered to 3500 ppm from Day 26, and increased to 4000 ppm from Day 40 of treatment. At 4000/3500/4000 ppm mean bodyweights and food consumption during gestation were lower than in Controls. Bodyweight gain during Days 0-13 of gestation was similar to Control but weight gain was low during Days 13-20 of gestation. During lactation, the females at 4000/3500/4000 ppm showed bodyweight loss during Days 1-7, compared to weight gain in the Controls, and body weight gain was similar to that of the Control from Days 7-13. Mean food consumption during lactation was slightly lower than in Controls.

There were no findings at ophthalmic examination that were considered related to treatment with the test item. There was no effect of treatment on grip strength,motor activity and sensory reactivity.

At haematological examination in Week 8 of treatment, there were no significant changes amongst males at 4000/3500/4000 ppm or 1500 ppm. In females, white blood cell, neutrophil, lymphocyte, monocyte and large unstained cell counts were lower at 4000/3500/4000 ppm or 1500 ppm when compared with that of the Control.

At blood chemistry examination in Week 8 of treatment, there were no significant changes amongst males or females at 4000/3500 ppm or 1500 ppm. There were some minor differences from Control, however, these occurred in one sex only and are considered unlikely to be related to treatment with test item.

At examination of the urine in Week 8 of treatment, there were no significant changes amongst males or females at 4000/3500/4000 ppm or 1500 ppm. Protein levels but not total protein output were statistically significantly high for females in the 4000/3500/4000 ppm group, no other changes achieved statistical significance.

There was no effect of treatment at 4000/3500/4000 or 1500 ppm on estrous cycles during treatment or at termination. Mating performance, pre coital interval, fertility, gestation length and index were unaffected by treatment.

After 8 weeks of treatment, the mean adjusted and unadjusted liver weights among males and females at 4000/3500/4000 ppm was marginally high compared with Controls.

The macroscopic examination performed after eight weeks of treatment or reproductive phase revealed no test-item related lesions.

At pathological examination, administration of the test item to rats by diet for at least 5 weeks resulted in treatment related effects in the kidneys (increased incidence and/or severity of cortical tubule hyaline droplet accumulation) in males treated with 1500 or 4000/3500/4000 ppm.

Amongst the offspring of females in the 4000/3500/4000 ppm group, there were a high number of offspring found dead or missing up until Day 4 of age. Clinical signs seen in some litters included small amount of milk in stomach and pups cold to touch. There was considered to be no effect of treatment on litter size, offspring survival or the sex ratio following maternal treatment at 1500 ppm.

The litter size, post implantation survival index and viability index in the 4000/3500/4000 ppm group was lower than in Controls. Sex ratio was unaffected in this group.

At 4000/3500/4000 ppm, mean body weights of male and female offspring on Day 1 of age were marginally lower than in Controls. Subsequent mean body weight gains of male and female offspring between Days 1 and 13 of age were slightly lower than in Controls. At 1500 ppm, there was no conclusive effect of treatment on mean offspring body weights on Day 1 of age. Subsequent mean body weight gains of male and female offspring were marginally lower than in Controls. Ano-genital distance in male and female offspring was unaffected by treatment.

No macroscopic findings were observed in offsprings at necropsy that could be associated with the maternal administration of the test item.

 

Therefore, it is concluded that the No-observed-adverse-effect-level (NOAEL) of the test item for systemic toxicity was 1500 ppm (mean achieved doses of 85 mg/kg bw/day for males, 94 mg/kg bw/day for toxicity phase females, 107 mg/kg bw/day for females during gestation and 177 mg/kg bw/day for females during lactation).

The NOAEL of the test item for reproductive/developmental effects was 1500 ppm.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
89.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP study conducted acording to OECD guideline 422 with one dose missing (high dose group was terminated earlier due to welfare reasons).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a recent GLP repeated dose toxicity study (OECD Guideline 422), systemic toxicity changes were observed in male and female rats exposed to the registered substance mainly due to a decrease in food consumption because of the lack of palatability of the test item mixed in the diet.

Furthermore, no effects on fertility and post-natal effects due to the lack of food consumption of dams were observed.

In this OECD Guideline 422 study, the NOAEL was set at 1500 ppm.

Therefore, the registered substance does not need to be classified for reproductive toxicity according to CLP Regulation (EC) No 1272/2008.