Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

Endpoint summary

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 4500 ppm (mean achieved doses of 267 mg/kg bw/day for males, 271 mg/kg bw/day for toxicity phase females, 283 mg/kg bw/day for females during gestation and 699 mg/kg bw/day for females during lactation). The NOAEL of the test item for reproductive/developmental effects was 4500 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:

5 October 2016 - 02 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

- Dose selection rationale: Effects on body weight and food consumption were seen at 12000 ppm at the start of treatment, from which the animals quickly recovered, were considered likely to be related to the palatability of test item Reaction mass of 1,4-cineole and 1,8-cineole. Therefore, a high dietary concentration of 12000 ppm was chosen, and was expected to elicit initial mean body weight loss or stasis in females and initial low food consumption, as observed in the preliminary toxicity study. The lowest dietary concentration of 1500 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 4500 ppm allowed evaluation of any concentration related trends and provided a geometric progression of dietary concentrations.

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 Crl:CD(SD) was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: Approximately 71 days old; Females: Approximately 85 days old.
- Weight at study initiation: Males: 328-389 g; Females: 238-299 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; 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 light : 12 h dark
- Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

IN-LIFE DATES: 5 October 2016 - 02 April 2017

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.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. 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.
- Storage of formulation: Deep-frozen (nominally -30 to -10 °C). Formulations were prepared and used within the documented stability limits and met the following criteria: 4 days at ambient temperature, and 15 days frozen.

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: 1:1 from 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 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 Week 1 and in the final week of treatment were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

Reproductive phase females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation (necropsy on Day 13 of lactation (the diet was available to the animals until the morning of necropsy)).
Toxicity phase males: Three weeks before pairing up to necropsy after minimum of six weeks.
Toxicity phase females: At least six weeks.
Recovery phase males: Three weeks before pairing up to necropsy after minimum of six weeks followed by a minimum 14-day recovery.
Recovery phase females: At least six weeks followed by a minimum 14-day recovery.

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.:

4 500 ppm (analytical)

Remarks:

Group 3 (mid dose)

Dose / conc.:

12 000 ppm (analytical)

Remarks:

Group 4 (high dose)

No. of animals per sex per dose:

Reproductive phase females: 10 animals/dose
Toxicity phase females: 5 females/dose in all groups; 5 males/dose in control and high dose groups; 10 males/dose in low and mid dose groups
Recovery phase animals: 5 animals/sex/dose in control and high dose groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Effects on body weight and food consumption were seen at 12000 ppm at the start of treatment, from which the animals quickly recovered, were considered likely to be related to the palatability of test item Reaction mass of 1,4-cineole and 1,8-cineole. Therefore, a high dietary concentration of 12000 ppm was chosen, and was expected to elicit initial mean body weight loss or stasis in females and initial low food consumption, as observed in the preliminary toxicity study. The lowest dietary concentration of 1500 ppm was expected to be a no effect level for effects on body weight and food consumption. The intermediate dietary concentration of 4500 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.
Estrous cycles were evaluated prior to treatment. After 14 days evaluation, animals that failed to exhibit typical 4-5 day cycles were not allocated to the reproductive phase of the study.
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.

- Post-exposure recovery period in satellite groups: 14 days

- Other: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Animal Replacement: Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation: Abnormal estrus cycle - 7 females; Body weight range extremes - 4 males; Health status - 1 female

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced, during each week of treatment and recovery, on Days 0, 6, 13 and 20 after mating and on Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before feeding of the treated diets on Day 1).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Toxicity and Recovery phase males and females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly thereafter, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm); On the day of necropsy.
F0 Reproductive phase females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly before pairing; Days 0, 7, 14 and 20 after mating; Day 1, 4, 7 and 13 of lactation; On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Daily, including the recovery phase. On Day 5 of recovery the animals were fed diets prepared for the females in the lactation phase in error (recovery control animals received control diet with the corn oil stabiliser, and recovery animals in Group 4 received treated diet (8000 ppm). Food consumption was not recorded for Toxicity phase males and Reproductive phase females during the period when paired for mating (Week 3), but recommenced for males in Week 4. Food consumption was recorded continuously for Toxicity and Recovery phase females. For Reproductive phase females after mating food consumption was performed daily throughout gestation and lactation (until Day 12).
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

OTHER:
NEUROBEHAVIOURAL EXAMINATION:
- Time schedule:
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 during Week 6 of treatment.
Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and on the lowest numbered toxicity phase males and females in Groups 2 and 3 was measured.

OPHTHALMOLOGY
- Time schedule:
Pre-treatment: All Toxicity and Recovery phase animals and spare animals; Week 6: All Toxicity phase females and the first five Toxicity phase males of Groups 1 and 4
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope. 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:
- Time schedule for collection of blood:
Week 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All male Recovery animals
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food; animals were also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein. Sampling was performed on the morning after overnight collection of urine.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute 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 6: Five lowest numbered surviving Toxicity phase males and females in each group; Recovery: All Recovery animals
Metabolism cages used for collection of urine: Yes; animals were placed in an individual metabolism cage, without access to food or water. Urine samples were collected overnight.
- Parameters:
Using manual methods: Clarity and Color/Appearance (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, Bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular Analyzer: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride
A microscopic examination of the urine sediment was performed: Epithelial cells, Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals.

Thyroid Hormone Analysis
- Time schedule for examination
At termination: F0 males, All F0 Reproductive phase females
Day 4 of age - F1 offspring, two females per litter (where possible) - no pups were eliminated when litter size dropped below ten/litter
- one for T4 (serum)#
- one for TSH (plasma - optional analysis)
# priority given to serum sample
Day 13 of age: F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female
# priority given to serum sample

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):

Estrous Cycle
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 the Reproductive phase of the study.
- After pairing until mating.
- For four days before scheduled termination (all Reproductive phase, Toxicity phase and Recovery phase females).

Dry smears:
Reproductive phase females: from beginning of treatment until animals were paired for mating, using cotton swabs (approximately three weeks).

Litter observations:

Clinical observations: Examined at approximately 24 h 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 of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: 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
Time of necropsy
Toxicity phase:
F0 males and females: After Week 6 investigations were completed.
Reproductive phase females:
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 13 of lactation.
Recovery phase
F0 Males and females: After at least 14 days without treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination. (No animal was exposed to carbon dioxide until after completion of thyroid hormone assays).

GROSS NECROPSY
- Necropsy: All adult animals 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.
Main phase females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed.

ORGAN WEIGHTS
- For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

HISTOPATHOLOGY
- 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 F0 animals killed or dying prematurely; Toxicity phase males and females in Groups 1 and 4 at scheduled termination.
Abnormalities: All remaining adult animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Postmortem examinations (offspring):

SACRIFICE
Time of necropsy:
Selected offspring for Day 4 thyroid hormone 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 selected for thyroid hormone sampling: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues retained.
- F1 offspring on Day 4 of age:
Blood sampling required
Externally normal offspring discarded without examination.
Externally abnormal offspring identified on despatch to necropsy, examined externally, and retained pending possible future examination.
- F1 offspring on Day 13 of age
Blood sampling required
All animals (but not including those selected for thyroid hormone analysis) were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved 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.

Statistics:

See "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 and Index: Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.
Gestation index was calculated for each group as:
- 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 of lactation / Number live offspring on Day 4 (after blood sampling)) x 100
Sex ratio: The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 13 of age.
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs considered related to treatment with test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female receiving test item at 12000 ppm (4F 44) was found dead on Day 20 of gestation having shown no clinical signs indicative of a problem during gestation. Macroscopic findings at necropsy included red staining of the fur in the perigenital region, dark mesenteric lymph nodes and stomach contents, an oedematous thymus and bedding material in the buccal cavity. At microscopic evaluation there were no unequivocal signs indicative of the cause of death. All findings were likely to be associated with post-mortem changes, such as erythrocytosis/erythrophagocytosis in the mesenteric lymph node, or associated with a complication of pregnancy, such as foci of hepatic necrosis. This animal was found to have 19 implantations, including one early resorption, one late resorption, and 17 dead fetuses (considered to have died following the death of the dam).
Another female receiving test item at 12000 ppm (4F 48) showed piloerection on Day 20 of gestation; there was evidence of blood in the cage and this animal was found to be bleeding from the vagina at the first parturition check. At the second parturition check piloerection was still apparent and the female was pale in colour but the bleeding appeared to have stopped as there was no fresh evidence. The female showed signs of reduced body temperature, piloerection and pallor at the third scheduled parturition check. This animal was subsequently killed for welfare reasons. Macroscopic examination revealed compacted bedding material in the stomach and two early resorptions and two dead fetuses and seven live fetuses in the uterus, and pale internal organs consistent with the clinical sign. At microscopic evaluation, most findings were likely to be associated with stress, such as hypertrophy of the cortical adrenal and apoptosis in the thymus, or associated with a complication of pregnancy, such as foci of hepatic necrosis and kidney necrosis. In the lungs, minimal, multifocal, microthrombi were seen.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

One female receiving test item at 12000 ppm (4F 44) was found dead on Day 20 of gestation having shown no clinical signs indicative of a problem during gestation. Macroscopic findings at necropsy included red staining of the fur in the perigenital region, dark mesenteric lymph nodes and stomach contents, an oedematous thymus and bedding material in the buccal cavity. At microscopic evaluation there were no unequivocal signs indicative of the cause of death. All findings were likely to be associated with post-mortem changes, such as erythrocytosis/erythrophagocytosis in the mesenteric lymph node, or associated with a complication of pregnancy, such as foci of hepatic necrosis. This animal was found to have 19 implantations, including one early resorption, one late resorption, and 17 dead fetuses (considered to have died following the death of the dam).
Another female receiving test item at 12000 ppm (4F 48) showed piloerection on Day 20 of gestation; there was evidence of blood in the cage and this animal was found to be bleeding from the vagina at the first parturition check. At the second parturition check piloerection was still apparent and the female was pale in colour but the bleeding appeared to have stopped as there was no fresh evidence. The female showed signs of reduced body temperature, piloerection and pallor at the third scheduled parturition check. This animal was subsequently killed for welfare reasons. Macroscopic examination revealed compacted bedding material in the stomach and two early resorptions and two dead fetuses and seven live fetuses in the uterus, and pale internal organs consistent with the clinical sign. At microscopic evaluation, most findings were likely to be associated with stress, such as hypertrophy of the cortical adrenal and apoptosis in the thymus, or associated with a complication of pregnancy, such as foci of hepatic necrosis and kidney necrosis. In the lungs, minimal, multifocal, microthrombi were seen.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of males and females receiving test item at 12000 ppm was slightly lower than that of the Control during Days 1-7 of treatment and similar to that of the Control thereafter (-12% and -17% for males and females, respectively). The food consumption of males and females receiving test item at 1500 or 4500 ppm was generally similar to that of the Control throughout the treatment period, but slightly low at 1500 and 4500 ppm on the first day of treatment.
Food consumption during Days 1-15 of recovery was similar to that of the Control for males and females which had received test item at 12000 ppm.
Food consumption during Days 1-22 of treatment for reproductive phase females (before pairing) was similar to that of the Control for females receiving test item at 1500 or 4500 ppm, and marginally lower than that of the Control during the first six days for females receiving test item at 12000 ppm (-13% than the Control for Days 1-7 and -5% for Days 1-22).
Food consumption during gestation for reproductive phase females was generally similar to that of the Control for females receiving test item at 1500, 4500 or 12000 ppm. At 12000 ppm, food intake was slightly low on Days 0-2 of gestation (-20% than the Control).
Food consumption during Days 1-13 of lactation for reproductive phase females was variable, but generally similar to that of the Control for females receiving test item at 1500 or 4500 ppm. At 12000 ppm, food intake was slightly low on Days 1-9 of lactation (especially on Days 1-3).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmic lesions that were considered to be related to treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology investigations after 6 weeks of treatment revealed a slightly but statistically significant reduction in haematocrit in female animals receiving 12000 ppm when compared to controls. In all groups of treated females a dose dependent decrease in total white blood cell counts, due to decreases in all white cell populations was apparent. All other haematological differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
Haematology investigations during recovery revealed there remained a suggestion of slightly lower white cell counts (especially lymphocytes, basophils and large unstained cells) in females previously treated at 12000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis after 6 weeks of treatment revealed, when compared to controls:
- high alanine amino-transferase in all groups of males treated with test item.
- high asparate amino-transferase in all groups of males and females treated with test item.
- high bile acid concentration in all groups of males treated with test item; bile acid values for females are highly variable, with no clear dose response apparent.
- high urea and creatine concentrations in all groups of males treated with test item, with males receiving 12000 ppm showing statistical significance; a dose related trend was apparent for both parameters.
- statistically significant higher total protein concentration in both males and females receiving test item at 12000 ppm.
- statistically significant higher albumin concentration in females receiving receiving test item at 12000 ppm.

All other biochemical differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
Blood chemistry investigations during recovery revealed high urea and creatinine values for males which had previously received 12000 ppm when compared with that of the Control (+19% and +37% for urea and creatinine, respectively).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis investigations during Week 6 of treatment revealed glucose concentrations which were higher than Control in groups of males receiving test item at 12000 ppm (+54% than the Control). All other urinary differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
Urinalysis investigations during recovery revealed glucose concentrations which remained higher than Control for males which had previously received 12000 ppm when compared with that of the Control (+35% and +30% than the Control for T-Glucose and U-glucose, respectively).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The sensory reactivity observations conducted during Week 6 of treatment revealed no findings which were considered treatment related in either males or females receiving the test item.
The motor activity assessment conducted during Week 6 of treatment revealed no treatment related effects in male animals at all dose levels. In female animals treated at 12000 ppm a higher than control statistical significance was seen in the high beam breaks, with a similar trend seen in the low beam break during the first 30 minutes of testing resulting in high total values. During recovery, in female animals previously treated at 12000 ppm, there was a trend towards higher than Control high and low beam breaks throughout the testing period. When compared to Historical Control Data (HCD), the total high and low beam breaks seen for Group 4 females are within the HCD range, with high beams breaks for females in Groups 1, 2 and 3 being lower than HCD , so no effect of treatment was inferred.

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 After 6 Weeks of Treatment:
Changes related to treatment with tets item were seen in the liver of both males and females and kidneys of males.
Centrilobular hypertrophy was seen in most of the males and all females receiving test item at 12000 ppm, and in one female receiving 4500 ppm. After 2 weeks of recovery, full recovery from test item related findings in the livers was considered to have occurred.
In all males receiving test item at all dose levels there was an accumulation of hyaline droplets, and this was associated with several findings such as cortical tubular basophilia, granular casts, tubular dilatation and inflammatory cell infiltrates.

- Animals Killed After 2 Weeks of Recovery:
In all males receiving Reaction mass of 1,4-cineole and 1,8-cineole at 12000 ppm there was an accumulation of hyaline droplets, and this was associated with several findings such as cortical tubular basophilia, granular casts, tubular dilatation and inflammatory cell infiltrates.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis:
There was no effect of treatment on mean serum T4 concentrations in F0 males.

There was considered to be no effect of treatment on mean serum T4 concentrations in F1 male and female offspring at Day 13 of age: although the mean serum levels of T4 in both sexes were slightly lower than in Controls; review of the Historical control data (HCD) showed that mean T4 levels in 2/3 studies were noticeably lower than in the Controls on this study and only marginally higher than the levels in the 12000 ppm group; therefore, no effect of treatment was inferred.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to the study showed regular 4 or 5 day estrus cycles prior to the start of treatment.
There was no effect of treatment on the estrus cycles of the reproductive phase females. At termination, all reproductive phase females showed diestrous.
All toxicity and recovery phase females showed estrus before termination. There was no effect of treatment on the stage of the estrus cycle at termination.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the pre coital interval: all animals mated within 4 days of pairing at the first estrus.
Mating performance and fertility was 100% in all groups.
There was no effect of treatment on gestation length. The gestation index at 12000 ppm was lower due to the 2 decedent pregnant females on Day 20 of gestation.

Key result

Dose descriptor:

NOAEL

Effect level:

4 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

12 000 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):

There were no clinical signs that were considered to be related to treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was considered to be no effect of treatment on litter size, offspring survival or the sex ratio.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment on mean body weights of male and female offspring in the 1500 or 4500 ppm groups on Day 1 of age, or on subsequent bodyweight gain until termination on Day 13 of age.

Mean bodyweight of male and female offspring in the 12000 ppm group on Day 1 of age was slightly lower than that of the Control. In addition, mean body weight gain of male and female offspring in the 12000 ppm group were marginally lower than in Controls until termination on Day 13 of age.

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:

no effects observed

Description (incidence and severity):

Ano-genital distance in male and female offspring was unaffected by treatment.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no findings in decedent offspring, or offspring at termination on Day 13 of age, that were considered to be related to treatment.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

4 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Formulation Analysis:

The mean concentrations of in test formulations analyzed for the study were within ±11% of nominal concentrations, confirming accurate formulation.

Achieved Dose:

Mean achieved doses for toxicity and recovery phase males were 90, 267 or 702 mg/kg bw/day at 1500, 4500 or 12000 ppm, respectively.

Mean achieved doses for toxicity and recovery phase females were 92, 271 or 709 mg/kg bw/day at 1500, 4500 or 12000 ppm, respectively.

Mean Achieved doses for reproductive phase females at 1500, 4500 or 12000 ppm were respectively 93, 276 or 678 mg/kg bw/day before pairing, 91, 283 or 763 mg/kg bw/day during gestation and 221, 699 or 1664 mg/kg bw/day during Days 1-12 of lactation. The higher achieved intakes during lactation reflect the higher food consumption due to the increased physiological demand on the dams during lactation.

Conclusions:

The No-Observed-Adverse-Effect-Level (NOAEL) of the test item for systemic toxicity was 4500 ppm (mean achieved doses of 267 mg/kg bw/day for males, 271 mg/kg bw/day for toxicity phase females, 283 mg/kg bw/day for females during gestation and 699 mg/kg bw/day for females during lactation). The NOAEL of the test item for reproductive/developmental effects was concluded to be 4500 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, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations of 1500, 4500 and 12000 ppm.

 

Reproductive phase females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation. The F1 generation was killed on Day 4 or Day 13 of age, but received no direct administration of the test item; any exposure was in utero or via the milk. Toxicity phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks. Toxicity phase females were treated daily for a minimum of six consecutive weeks. Recovery phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks followed by a minimum of 14 days recovery. Recovery phase females were treated daily for a minimum of six consecutive weeks followed by a minimum of 14 days recovery. 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 treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, nipple counts (males only), ano-genital distance and macropathology for all offspring were also assessed. 

 

The mean concentrations of the test item in test formulations analyzed for the study were within 11% of nominal concentrations, confirming accurate formulation. Achieved dose, generally maintained the intervals between dietary concentrations.

Dietary administration of the test item at levels of 1500, 4500 and 12000 ppm was generally well tolerated. There were two deaths on Day 20 of gestation at 12000 ppm which were considered of uncertain relationship to treatment.

There was no effect of treatment on mean serum T4 concentrations in F0 males or in F1 male and female offspring on Day 13 of age.

There were no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, ophthalmic examination, hematology, blood chemistry, urinalysis and macroscopic pathology. In addition, the reproductive endpoints assessed which were unaffected by treatment included estrus cycles, pre-coital interval, mating performance, fertility, gestation length, offspring clinical condition, litter size, survival, sex ratio or external development. 

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

Mean bodyweight gain and food consumption of males receiving the test item at 12000 ppm was slightly lower than that of the Control during Days 1-8 of treatment (-32% than the Control for mean body weight gain and -12% for food consumption), and mean bodyweight gain of this group was slightly lower than that of the Control during Days 15-22 of treatment (-33% than the Control). Males receiving 4500 ppm showed slightly low body weight gain during the first week of treatment (-22% than the Control) and low food consumption on Day 1. Toxicity and recovery females receiving the test item at 12000 ppm showed slight bodyweight loss, compared to a bodyweight gain amongst Control females or females at 4500 or 1500 ppm during Days 1-8 of treatment (-1 g at 12000 ppm instead +2 g, +3 g and +7 g at 4500, 1500 and for Control, respectively). Females receiving the test item at 4500 or 1500 ppm showed low weight gain when compared with that of the Control during Days 1-8 of treatment (-71% and -57% than the Control, respectively). 

Overall body weight gain during Days 1-15 of recovery was higher than that of the Control for males and females which had received the test item at 12000 ppm (about 1.5 and 3 times more than Control for males and females, respectively).

Overall body weight gain during Days 1-43 of treatment was lower than that of the Control for males and females receiving the test item at 1500, 4500 or 12000 ppm. 

 

Reproductive phase females at 12000 ppm or 4500 ppm showed initial minor mean bodyweight loss (-2 g at both 4500 and 12000 ppm instead 8 g and 5 g at 1500 ppm and for Control).

Overall body weight gain during gestation for reproductive phase females was similar to that of the Control for females receiving the test item at 1500 or 4500 ppm, and lower than that of the Control for females receiving the test item at 12000 ppm (-10% than the Control).

Overall body weight gain during Days 1-13 of lactation for reproductive phase females was higher than that of the Control for females receiving the test item at 1500, 4500 or 12000 ppm (+24%, +45% and +34% at 1500, 4500 and 12000, respectively). At 12000 ppm, food intake was marginally low on Days 1-9 of lactation.

Macroscopic examination performed after six weeks of treatment, two weeks of recovery or following completion of the reproductive phase, revealed no test-item related lesions. The incidence and distribution of all findings were consistent with the common background seen in Sprague-Dawley rats at these laboratories. 

After 6 weeks of treatment, the mean absolute and adjusted epididymides, prostate and heart weight among males receiving 12000 ppm were marginally low compared with Controls, while the mean absolute and adjusted liver weight for males receiving 1500, 4500 or 12000 ppm was marginally high. Also, the mean absolute and adjusted Cowpers gland, glans penis and LABC weight among males receiving 4500 and 12000 ppm were marginally low compared with Controls. In addition, the mean absolute and adjusted liver weight for females receiving 12000 ppm was marginally high. After 2 weeks recovery, the absolute and mean adjusted liver weight for males and females that previously received 12000 ppm remained marginally higher than in Control, and the absolute and mean Cowpers gland weight for males that previously received 12000 ppm remained marginally lower than in Control. The mean absolute and adjusted Uter+Cerv+Ovid weights among females receiving 1500, 4500 and 12000 ppm were high compared with Controls. There were no differences in the absolute or adjusted organ weights on Day 13 of lactation for females which received test item at 1500, 4500 or 12000 ppm. There were no similar supporting macroscopic or microscopic pathology changes in these organs.

 

The liver centrilobular hypertrophy seen in toxicity phase males and females receivingtest itemat 12000 ppm correlates with the marginally increased absolute and adjusted liver weight in both sexes. Furthermore, changes in liver blood chemistry were seen in all groups: high alanine amino-transferase in all groups of males, high aspartate amino-transferase in all groups of males and females and high bile acid concentration in all groups of males; bile acid values for females were highly variable, with no clear dose response apparent. However, considering the minimal severity and incidence of the histopathological effects and the slight increase in liver weights observed at 4500 ppm in males, toxicological effects observed in the liver can be considered as adaptive at this dose level. Also, after 2 weeks of recovery, no test item-related findings were seen in the liver, indicating recovery.

 

In the kidneys of all toxicity phase males receiving the test item at all dose levels, a constellation of findings was indicative of alpha 2μ-globulin nephropathy. A statistically significant increase in urea and creatinine was present in males receiving test item at 12000 ppm and correlates with the microscopic evaluation, as the severity was slightly higher in this group. After 2 weeks of recovery, the same constellation of findings was seen in males previously given test item at 12000 ppm, but the overall severity was slightly lower, indicating partial recovery. After 2 weeks of recovery, although there was still a statistically significant increase in blood levels of urea and creatinine, there was a reduction from what was seen in the treatment phase.

There was no effect of treatment on the pre coital interval: all animals mated within 4 days of pairing at the first estrus. Mating performance and fertility was 100% in all groups. There was no effect of treatment on gestation length. The gestation index at 12000 ppm was lower due to the 2 decedent pregnant females on Day 20 of gestation.

There was considered to be no effect of treatment on litter size, offspring survival or the sex ratio. At 12000 ppm, mean offspring bodyweights on Day 1 of age were slightly lower than in Controls as was subsequent offspring body weight gain. Ano-genital distance in male and female offspring was unaffected by treatment.

 

Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) of the test item for systemic toxicity was 4500 ppm (mean achieved doses of 267 mg/kg bw/day for males, 271 mg/kg bw/day for toxicity phase females, 283 mg/kg bw/day for females during gestation and 699 mg/kg bw/day for females during lactation). The NOAEL of the test item for reproductive/developmental effects was concluded to be 4500 ppm.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

271 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study conducted acording to OECD guideline 422

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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, the test item wasadministered to groups of Crl:CD(SD) rats at dietary concentrations of 1500, 4500 and 12000 ppm.

 

Reproductive phase females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 13 of lactation. The F1 generation was killed on Day 4 or Day 13 of age, but received no direct administration ofthe test item; any exposure wasin uteroor via the milk. Toxicity phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks. Toxicity phase females were treated daily for a minimum of six consecutive weeks.Recovery phase males were treated daily for three weeks before pairing up to necropsy after a minimum of six consecutive weeks followed by a minimum of 14 days recovery. Recovery phase females were treated daily for a minimum of six consecutive weeks followed by a minimum of 14 days recovery. 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 treatment period. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, urinalysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, nipple counts (males only), ano-genital distance and macropathology for all offspring were also assessed. 

 

The mean concentrations ofthe test itemin test formulations analyzed for the study were within 11% of nominal concentrations, confirming accurate formulation.Achieved dose, generally maintained the intervals between dietary concentrations.

Dietary administration of the test item at levels of 1500, 4500 and 12000 ppm was generally well tolerated. There were two deaths on Day 20 of gestation at 12000 ppm which were considered of uncertain relationship to treatment.

There was no effect of treatment on mean serum T4 concentrations in F0 males or in F1 male and female offspring on Day 13 of age.

There were no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, ophthalmic examination, hematology, blood chemistry, urinalysis and macroscopic pathology. In addition, the reproductive endpoints assessed which were unaffected by treatment included estrus cycles, pre-coital interval, mating performance, fertility, gestation length, offspring clinical condition, litter size, survival, sex ratio or external development. 

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

Mean bodyweight gain and food consumption of males receiving the test item at 12000 ppm was slightly lower than that of the Control during Days 1-8 of treatment (-32% than the Control for mean body weight gain and -12% for food consumption), and mean bodyweight gain of this group was slightly lower than that of the Control during Days 15-22 of treatment (-33% than the Control). Males receiving 4500 ppm showed slightly low body weight gain during the first week of treatment (-22% than the Control) and low food consumption on Day 1. Toxicity and recovery females receiving the test item at 12000 ppm showed slight bodyweight loss, compared to a bodyweight gain amongst Control females or females at 4500 or 1500 ppm during Days 1-8 of treatment (-1 g at 12000 ppm instead +2 g, +3 g and +7 g at 4500, 1500 and for Control, respectively). Females receiving the test item at 4500 or 1500 ppm showed low weight gain when compared with that of the Control during Days 1-8 of treatment (-71% and -57% than the Control, respectively). 

Overall body weight gain during Days 1-15 of recovery was higher than that of the Control for males and females which had received the test item at 12000 ppm (about 1.5 and 3 times more than Control for males and females, respectively).

Overall body weight gain during Days 1-43 of treatment was lower than that of the Control for males and females receiving the test item at 1500, 4500 or 12000 ppm. 

 

Reproductive phase females at 12000 ppm or 4500 ppm showed initial minor mean bodyweight loss (-2 g at both 4500 and 12000 ppm instead 8 g and 5 g at 1500 ppm and for Control).

Overall body weight gain during gestation for reproductive phase females was similar to that of the Control for females receiving the test item at 1500 or 4500 ppm, and lower than that of the Control for females receiving the test item at 12000 ppm (-10% than the Control).

Overall body weight gain during Days 1-13 of lactation for reproductive phase females was higher than that of the Control for females receiving the test item at 1500, 4500 or 12000 ppm (+24%, +45% and +34% at 1500, 4500 and 12000, respectively). At 12000 ppm, food intake was marginally low on Days 1-9 of lactation.

Macroscopic examination performed after six weeks of treatment, two weeks of recovery or following completion of the reproductive phase, revealed no test-item related lesions. The incidence and distribution of all findings were consistent with the common background seen in Sprague-Dawley rats at these laboratories. 

After 6 weeks of treatment, the mean absolute and adjusted epididymides, prostate and heart weight among males receiving 12000 ppm were marginally low compared with Controls, while the mean absolute and adjusted liver weight for males receiving 1500, 4500 or 12000 ppm was marginally high. Also, the mean absolute and adjusted Cowpers gland, glans penis and LABC weight among males receiving 4500 and 12000 ppm were marginally low compared with Controls. In addition, the mean absolute and adjusted liver weight for females receiving 12000 ppm was marginally high. After 2 weeks recovery, the absolute and mean adjusted liver weight for males and females that previously received 12000 ppm remained marginally higher than in Control, and the absolute and mean Cowpers gland weight for males that previously received 12000 ppm remained marginally lower than in Control. The mean absolute and adjusted Uter+Cerv+Ovid weights among females receiving 1500, 4500 and 12000 ppm were high compared with Controls. There were no differences in the absolute or adjusted organ weights on Day 13 of lactation for females which received test item at 1500, 4500 or 12000 ppm. There were no similar supporting macroscopic or microscopic pathology changes in these organs.

 

The liver centrilobular hypertrophy seen in toxicity phase males and females receivingtest itemat 12000 ppm correlates with the marginally increased absolute and adjusted liver weight in both sexes. Furthermore, changes in liver blood chemistry were seen in all groups: high alanine amino-transferase in all groups of males, high aspartate amino-transferase in all groups of males and females and high bile acid concentration in all groups of males; bile acid values for females were highly variable, with no clear dose response apparent.However, considering the minimal severity and incidence of the histopathological effects and the slight increase in liver weights observed at 4500 ppm in males, toxicological effects observed in the liver can be considered as adaptive at this dose level. Also, after 2 weeks of recovery, no test item-related findings were seen in the liver, indicating recovery.

 

In the kidneys of all toxicity phase males receiving the test item at all dose levels, a constellation of findings was indicative of alpha 2μ-globulin nephropathy. A statistically significant increase in urea and creatinine was present in males receiving test item at 12000 ppm and correlates with the microscopic evaluation, as the severity was slightly higher in this group. After 2 weeks of recovery, the same constellation of findings was seen in males previously given test item at 12000 ppm, but the overall severity was slightly lower, indicating partial recovery. After 2 weeks of recovery, although there was still a statistically significant increase in blood levels of urea and creatinine, there was a reduction from what was seen in the treatment phase.

There was no effect of treatment on the pre coital interval: all animals mated within 4 days of pairing at the first estrus. Mating performance and fertility was 100% in all groups. There was no effect of treatment on gestation length. The gestation index at 12000 ppm was lower due to the 2 decedent pregnant females on Day 20 of gestation.

There was considered to be no effect of treatment on litter size, offspring survival or the sex ratio.At 12000 ppm, mean offspring bodyweights on Day 1 of age were slightly lower than in Controls as was subsequent offspring body weight gain.Ano-genital distance in male and female offspring was unaffected by treatment.

 

Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) of the test item for systemic toxicity was 4500 ppm (mean achieved doses of 267 mg/kg bw/day for males, 271 mg/kg bw/day for toxicity phase females, 283 mg/kg bw/day for females during gestation and 699 mg/kg bw/day for females during lactation).The NOAEL of the test item for reproductive/developmental effects was concluded to be 4500 ppm.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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 studies (OECD Guideline 422), systemic toxicity changes were observed in males and females rats exposed to the registered substance: mainly liver centrilobular hypertrophy seen in toxicity phase males and females receiving the test item at 12000 ppm associated with marginally increased absolute and adjusted liver weight in both sexes.

No effects on fertility and reproductive parameters were observed up to the highest dose tested. Furthermore, only decrease in bodyweight and bodyweight gain of offspring were observed at 12000 ppm, probably due to the decrease in food consumption of dams during the lactation period.

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