1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422, GLP, K, rel.1): NOAEL Fertility and development (foetal growth until day 14) = 3750 ppm in rats (i.e. equivalent to daily intakes between 223 and 598 mg/kg bw/day depending on the sex, age and weight of the animals). No adverse effect reported at the highest dose level tested in this study.

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 April 2016 to 20 March 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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, 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)

Remarks:

UK GLP compliance programme (inspected on December 05, 2016 / signed on March 15, 2017)

Limit test:

no

Justification for study design:

- Basis for dose level selection: Dose levels were selected on the basis of 14-day range-finding study (Envigo Study Number QC66HW).
- Route of administration: The oral (dietary), route of administration was chosen as it is a possible route of human exposure during manufacture, handling or use of the test item.

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: 323 to 383 g; Females: 229 to 299 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing (treatment), gestation, littering lactation and recovery 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:
Main study (pre-pairing), toxicity phase and recovery phase animals: up to five animals of one sex; Pairing: one male and one female; Main study males after mating: up to five animals; Gestation: one female; Lactation: one female with litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology, blood chemistry, urine collection and adult thyroid hormone investigations)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Males: Six days before commencement of treatment; Females: 20 days before 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 hours light : 12 hours 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: 28 April 2016 to 20 March 2017

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prepared weekly and frozen in aliquots to allow daily feeding
- Mixing appropriate amounts with SDS VRF1 certified diet: On each occasion of the preparation of the premix the required amount of test substance was weighed into a suitable container. An amount of sieved diet that approximately equaled 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.
- Storage temperature of food: Frozen until immediately prior to feeding daily

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups
- Pairing commenced: After a minimum of two 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 were analysed to assess the stability and homogeneity of the test item in the diet matrix. Stability and homogeneity of the formulations was confirmed at dietary concentrations of 100 ppm for 40 days when stored frozen (-10 to -30 °C) and at dietary concentrations of 100 and 20000 ppm for 30 hours at ambient temperature (15 to 25 °C).
Achieved concentration: Samples of each formulation prepared for administration in Week 1 and the final week of treatment were analysed for achieved concentration of the test item.

Duration of treatment / exposure:

Main study males: Two weeks before pairing up to necropsy after minimum of six weeks.
Main study females: Two weeks before pairing, then throughout pairing and gestation until Day 14 of lactation (approximately seven weeks).
Toxicity phase females: A minimum of six weeks.
Recovery phase animals: A minimum of six weeks (followed by at least 14 days recovery).
Animals of the F1 generation were not dosed. There was no significant direct treatment (there was a possibility of minimal ingestion mid lactation).

Frequency of treatment:

Continuously

Dose / conc.:

0 ppm

Remarks:

Group 1 (control)

Dose / conc.:

417 ppm

Remarks:

Group 2

Dose / conc.:

1 250 ppm

Remarks:

Group 3

Dose / conc.:

3 750 ppm

Remarks:

Group 4

No. of animals per sex per dose:

Main study animals: 10 animals/sex/dose
Toxicity phase females: 5 females/dose in control and high 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: The doses used in this study (0, 417, 1250 and 3750 ppm) were selected in conjunction with the Sponsor. Dose levels were selected following the completion of a 14-day range-finding study (Envigo Study Number QC66HW). In this study, rats (3/sex/dose) were administered test substance at the dose levels of 0, 2500, 5000 and 7500 ppm in the diet for 14 days. The test item was shown to be less palatable than control diet in females, resulting in a non-dose related reduction in food consumption, and a dose-related reduction in overall body weight gain when compared with Controls. No clear dose-related effect on food consumption or body weight gain was observed in males. The dose level of 7500 ppm was considered to be too high as females initially lost weight and took 14 days to regain parity with their pre-treatment body weight. At 5000 ppm the females showed only 70% of the weight gain of the Control group over the 14 day exposure period, whilst at 2500 ppm the animals gained 86% of the Control weight gain. Several factors were considered in the selection of dose levels for this main study, including: the small group size and the use of a staggered start for each dose level in the range-finding study, the absence of an adverse effect on palatability in the males, and the need to demonstrate some toxicity at the high dose level whilst ensuring normal nutrition was not compromised by persistent palatability issues. On this basis a high dose level of 3750 ppm was selected, with intermediate and low dose levels of 1250 and 417 ppm, respectively.

- 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.
- Rationale for selecting satellite groups: Subgroups of males and females were used to assess recovery, persistence or delayed occurrence of systemic effects (recovery group; non-paired animals). Another group of non-paired females were treated for at least 6 weeks.
- Post-exposure recovery period in satellite groups: 14 days for the recovery groups
- 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 treatment: Abnormal estrus cycle - six females; Body weight range extremes - one female; Unexpected deaths - one 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.

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

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Main study males, Toxicity phase females 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 the day of necropsy.
F0 Main study females: Weekly during acclimatization; Before feeding of the treated diets on the day that treatment commenced (Day 1) and weekly until mating detected; Days 0, 6, 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):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for examinations:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Recorded daily, including the recovery phase and reported weekly. Food consumption was not recorded for Main study males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. Food consumption was recorded continuously for Toxicity phase females and Recovery phase animals.
For Main study females after mating food consumption was recorded daily and reported to match the body weight recording: Days 0-6, 7-13 and 14-19 after mating; Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

OTHER:

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule:
Sensory reactivity and grip strength assessments were performed on the five lowest numbered surviving main study males in Groups 2 and 3 and all recovery phase animals in Groups 1 and 4 during Week 5 of treatment and on the first five lactating main study females in each group at Days 7-9 of lactation.
Motor activity: The motor activity of the five lowest numbered surviving main study males in Groups 2 and 3 and all recovery phase animals in Groups 1 and 4 during Week 5 of treatment and the first five lactating main study females in each group at Days 7-9 of lactation.

HAEMATOLOGY AND CLINICAL CHEMISTRY:
- Time schedule for collection of blood:
At termination: the five lowest numbered surviving males per group; all toxicity phase females; all recovery phase males only.
Day 14 of lactation: the first five lactating females with a litter per group
With the exception of lactating females, sampling performed on the morning after overnight collection of urine.
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food (no more than 20 hours); 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.
- 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, Blood urea nitrogen (BUN), 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: At termination: the five lowest numbered surviving males per group; all toxicity phase females; all recovery phase males only.
- 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 - by visual assessment; Volume - using a measuring cylinder*; pH - using a pH meter; Specific gravity (SG) - by direct refractometry using a SG meter*
Only parameters marked * were examined for recovery males.
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 - All adults
Day 4 of age - F1 offspring, two females per litter (where possible); one for T4 (serum)#; one for TSH (plasma)
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
Analysis for thyroxine (T4) was limited to samples obtained from offspring on Day 13 of age and main study male animals; in the absence of an effect further analysis for T4 or TSH was considered unnecessary.

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
Dry smears: Main study - For 15 days before pairing using cotton swabs.
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 study.
- Main study females after pairing until mating.
- For four days before scheduled termination (all females).

Sperm parameters (parental animals):

Parameters examined in male parental generation:
- testis weight
- epididymis weight
- glans penis weight
- For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Litter observations:

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 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
- Main study males and toxicity phase females: After Week 6 investigations completed (Week 7).
- Main study females: Day 14 of lactation
- Recovery phase animals: After at least 14 days following completion of treatment.
- Method of sacrifice: All adult animals were killed by Carbon dioxide asphyxiation (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 if none were visible at visual inspection.
For apparently empty uterine horns: The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski, E, 1964)].

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

HISTOPATHOLOGY: : yes (See Table 7.8.1/1 and Table 7.8.1 /2)
- 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: The five lowest surviving Main study males and females and all toxicity phase females in Groups 1 and 4 at scheduled termination.
Abnormalities: All remaining Main study and Recovery phase adult animals.
Liver: All Recovery phase animals; Groups 2 and 3: Five lowest numbered surviving males and first five females with a live litter at scheduled termination.
Thyroid: All main and Recovery study 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.
Light Microscopy: Tissues preserved for examination were examined as per table 7.8.1/4

Postmortem examinations (offspring):

SACRIFICE
- F1 offspring: Day 13 of age
- Offspring - selected for thyroid hormone sampling on Day 4 or 13 of age: Decapitation.
- Offspring - not selected for thyroid hormone sampling: Intraperitoneal injection of sodium pentobarbitone.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:
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 identified on despatch to necropsy, examined externally, and retained pending possible future examination.
Offspring at scheduled termination: 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. Abnormal tissues were retained in an appropriate fixative. Thyroid glands were preserved from one male and one female in each litter.
Table 7.8.1/3 for "Pathology procedures for Offspring on Day 13 of age"

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 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 of lactation / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 of lactation (before blood sampling) / Number live offspring on Day 1) 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 observations at routine physical examination or during arena observations that could be attributed to treatment with the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Group 1 male number 36 was found dead during Week 7 of treatment. Prior to its demise there had been no signs at routine physical examination and the animal was considered to be in good general condition. No macroscopic abnormalities were seen at necropsy. Following microscopic examination, no findings were seen that would have contributed to the animal’s death and so the cause of death could not be determined.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- During treatment bodyweight and bodyweight gain for main study males, for main study females before pairing and for toxicity/recovery phase females showed no adverse effects of treatment.
- Following completion of treatment recovery phase males that had previously received 3750 ppm showed body weight gains that were similar to Controls, however for recovery phase females that had previously received 3750 ppm body weight gain was high when compared with Controls.
- Bodyweight change for main study females during both gestation and lactation was unaffected by treatment. Absolute body weights for females at 417 ppm on Days 4, 7 and 13 of lactation were significantly high when compared with Controls but a similar difference was not apparent at the higher dose levels.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Throughout treatment no significant scatter of the diet was observed.
- During Week 1 of treatment mean food consumption at 3750 ppm for main study and recovery phase males and for main study and toxicity/recovery phase females were approximately 92% and 89% of Controls, respectively; thereafter for the remainder of the treatment period values were generally similar to Controls.
- Following completion of treatment food consumption for males that previously received 3750 ppm was similar to concurrent Controls, however for females that previously received 3750 ppm food consumption was high when compared with Controls; approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery.

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- After 6 weeks of treatment hematological findings were restricted to slightly extended mean prothrombin clotting time for males at 3750 ppm. As this finding was not evident in toxicity phase females after 6 weeks of treatment or in main study females on Day 14 of lactation it was considered to be related to natural variation rather than effect of treatment.
- There were no other consistent inter-group differences which were considered to relate to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- After 6 weeks of treatment males at 3750 ppm showed slightly elevated total protein and males at 1250 and 3750 ppm showed low plasma bile acid concentration (no dose response was apparent); these findings were not evident in toxicity phase females after 6 weeks of treatment or in main study females on Day 14 of lactation.
- After two weeks off dose the total protein, albumin concentration and albumin/globulin ratio for males that previously received 3750 ppm was essentially similar to Controls.
- There were no other consistent inter-group differences which were considered to relate to treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

- After 6 weeks of treatment males at all dose levels showed low urinary specific gravity with males at 3750 ppm showing increased urinary output and females at 3750 ppm showing high urinary pH when compared with Control values.
- After two weeks of recovery males that previously received 3750 ppm showed urinary output and specific gravity that was essentially similar to Control values.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Sensory Reactivity and Grip Strength
- Three males at 1250 ppm and three females at 3750 ppm during Week 5 of treatment showed a weak response to the tail pinch, however, all five males at 3750 ppm showed a normal response. Group mean forelimb grip strength values for all treated males and females and hindlimb values for treated females during Week 5 of treatment were similar to Controls.
- There was some inter-group variation in group mean hindlimb grip strength values for some groups of treated males, with the values at 417 and 3750 ppm achieving statistical significance. However, as the value at 1250 ppm was lower than the value at 417 ppm and the fact that an increase in grip strength due to treatment is unlikely; both of these values are within the historical control data range therefore these differences are attributed to natural variation.
- Sensory reactivity and grip strength values for all groups of treated females at Day 7-9 of lactation are similar to Controls.

Motor Activity
- Many of the group mean high (rearing) and low (ambulatory) beam activity scores for males at 3750 ppm during Week 5 of treatment were marginally low compared with Controls, however, these differences did not achieve statistical significance and there was no dose-relationship. High and low beam activity scores for treated females during Week 5 of treatment were similar to Controls. The high and low beam activity scores for treated females at Day 7-9 of lactation were variable (as expected during this physiological stage), but these differences did not achieve statistical significance and there was no dose-relationship.

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):

After 6 Weeks of Treatment (Main study males and Toxicity phase females):
Changes related to treatment were seen in the liver and thyroids.
- Liver: Minimal centrilobular hepatocyte hypertrophy was seen in animals treated with 3750 ppm.
- Thyroids: Minimal follicular cell hypertrophy was seen in animals treated with 3750 ppm and in males treated with 417 or 1250 ppm.
- Spermatogenesis: Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.
All other histological changes were considered to be unrelated to 6 weeks of treatment.

After 14 Days of Recovery
Changes related to previous treatment were seen in the liver and thyroids.
- Liver: Minimal centrilobular hepatocyte hypertrophy was seen in two of five males (Nos. 21 and 25) and in one of five females (No. 104) previously treated with 3750 ppm.
- Thyroids: Minimal follicular cell hypertrophy was seen in two of five males (Nos. 22 and 23) previously treated with 3750 ppm.
All histological changes were considered to be unrelated to treatment.

Females on Day 14 of Lactation (Main study females)
Treatment Related Findings - Changes related to treatment were seen in the liver and thyroids.
- Liver: Minimal or slight centrilobular hepatocyte hypertrophy was seen in the majority of females treated with 1250 or 3750 ppm and in one female treated with 417 ppm and demonstrated a dose-relationship.
- Thyroids: Minimal follicular cell hypertrophy was seen in two animals treated with 3750 ppm.

Incidental Findings
- Thymus: Minimal or slight involution/atrophy was seen in a few control females and females treated with 3750 ppm. As this change was not seen in any unmated control females or females treated with 3750 ppm for 6 weeks, it was considered that the involution/atrophy was related to the ‘stress’ of pregnancy and lactation in rodents (Everds et al., 2013; Kendall et al., 2000; Pearse, 2006) and unrelated to treatment. The involution/atrophy was considered to have contributed to the recorded decreased body weight adjusted group mean thymus weights in females treated with 3750 ppm, compared with female controls.
- All other histological changes were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones: Analyses of samples for thyroxine (T4) obtained from Main study male animals and F1 offspring on Day 13 of age did not reveal any differences that could be attributed to treatment; therefore further assessment of T4 and thyroid stimulating hormone (TSH) was not considered necessary.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- All females allocated to study showed normal 4/5 day estrous cycles during the acclimatisation period.
- The majority of main study females showed regular 4/5 day estrous cycles during the treatment period prior to mating; one Control female showed an irregular cycle and one female at 3750 ppm was acyclic.
- Examination of vaginal smears prior to termination of main study, toxicity and recovery phase females did not reveal any adverse effects of treatment.
- Pre-coital interval for main study females was unaffected by treatment with all females, including the acyclic female at 3750 ppm, mating at the first estrus after pairing.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, fertility, gestation length and gestation index were unaffected by treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic and reproductive toxicity

Effect level:

3 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 223 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effect observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 225 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effect observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs that could be attributed to parental treatment.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- On Day 1 of age the mean body weights for both male and female offspring derived from groups receiving the test item were essentially similar to Control and considered unaffected by parental treatment.
- Subsequent weight gain up to Day 7 of age was similar to Controls. However between Days 7 and 13 of age body weight gain for male and female offspring at 1250 and 3750 ppm were low when compared with Controls, with the differences attaining statistical significance for males at 3750 ppm (p<0.01) and for females at both 1250 ppm (p<0.05) and 3750ppm (p<0.01); a dose response was apparent. This resulted in significantly low absolute mean bodyweight on Day 13 of age for male offspring at 3750 ppm (p<0.05) and for female offspring at 1250ppm (p<0.05) and 3750ppm (p<0.05). This was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Macroscopic examination of offspring that died prior to scheduled termination did not reveal any findings related to treatment.
- On Day 13 of age offspring from two out of the ten litters at 3750 ppm were observed with patchy coat, which may be related to a slight delay in development because of the slightly larger mean litter size at this dose level; there were no other findings that could be related to parental treatment.

Other effects:

no effects observed

Description (incidence and severity):

Litter Size, Sex Ratio and Survival Indices: Litter size, offspring survival and sex ratio were unaffected by parental treatment. However, the average litter size at 1250 and 3750 ppm was slightly high when compared with Controls, particularly after Day 4 of age.
Ano-Genital Distance: Ano-genital distance of both male and female offspring on Day 1 of age showed no adverse effects of parental treatment.
Offspring Nipple Count: A few male offspring were observed with nipples on Day 13 of age, however the distribution across the groups showed no relationship to treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

3 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Formulation Analysis

The homogeneity was confirmed for the test item in SDS VRF1 diet formulations at nominal concentrations of 100 ppm and 20000 ppm. Stability was confirmed at ambient storage for up to 30 hours for 100 ppm and up to 2 days at 20000 ppm. Stability was confirmed at frozen storage for up to 40 days for 100 ppm. Frozen stability at 20000 ppm was not assessed in error, however degradation and formulation instability is more pronounced at lower levels. As frozen stability was confirmed at 100 ppm for 40 days and all subsequent testing of achieved concentration samples were well within acceptable limits after being stored frozen, it is considered that there is sufficient evidence to suggest that the test diets with a concentration of 20000 ppm are stable for 40 days under frozen conditions.

The mean concentrations of the test item in formulations analyzed for the study were within ±5% of nominal concentrations, confirming accurate formulation.

Achieved Dose

The mean achieved dose for males receiving 417, 1250 and 3750 ppm during the 6 week treatment period was 25.1, 75.0 and 223 mg/kg bw/day, respectively.

Overall the mean achieved dose for females receiving 3750 ppm during 6 weeks was 232 mg/kg bw/day (Toxicity and Recovery phase animals and main phase females for two weeks pre-pairing).

The mean achieved doses for females receiving 417, 1250 and 3750 ppm during main study phases were:

- 26.9, 75.6 and 225 mg/kg bw/day, respectively, during the two week pre-pairing treatment period (Main, Toxicity and Recovery phase animals)

- 28.7, 87.0 and 259 mg/kg bw/day, respectively, during the gestation phase (Main phase females only)

- 67.3, 192 and 598 mg/kg bw/day respectively, during the lactation phase (Main phase females only)

See the attached document for information on Tables of results 422-Toxicity to reproduction

Conclusions:

Under the test conditions, the NOAEL for reproductive and developmental toxicity was 3750 ppm (equivalent to daily intakes between 223 and 598 mg/kg bw/day depending on the sex, age and weight of the animals) based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below.

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, test substance was administered to groups of Crl:CD(SD) rats at dietary concentrations of 417, 1250 or 3750 ppm. Main study males (10 animal/dose) were treated daily for two weeks before pairing and for a minimum total of six weeks prior to necropsy. Main study females (10 animal/dose) were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 14 of lactation. The F1 generation was killed on Day 4 or Day 13 of age, but received no direct administration to the test item; any exposure was in utero or via the milk. High dose and control toxicity phase females (5 animal/dose) were treated daily for a minimum of six consecutive weeks. and were not paired. High dose and control recovery phase animals (5 animal/sex/dose) were treated daily for a minimum of six consecutive weeks followed by a minimum of two weeks recovery to assess the potential of any treatment related change to recover. Recovery phase animals were not paired. A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet, 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, water consumption (visual assessment), hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, 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, ano-genital distance and nipple counts (males only), and macropathology for all offspring were also recorded.

 

F0 responses

The mean achieved dose for males receiving 417, 1250 and 3750 ppm during the 6 week treatment period was 25.1, 75.0 and 223 mg/kg bw bw/day, respectively. Overall the mean achieved dose for females receiving 3750 ppm during 6 weeks was 232 mg/kg bw/day (Toxicity and Recovery phase animals and main phase females for two weeks pre-pairing). The mean achieved doses during each main study phase for females receiving 417, 1250 and 3750 ppm were: 26.9, 75.6 and 225 mg/kg bw/day, respectively, during the two week pre-pairing treatment period (Main, Toxicity and Recovery phase animals); 28.7, 87.0 and 259 mg/kg bw/day, respectively, during the gestation phase (Main phase females only); 67.3, 192 and 598 mg/kg bw/day respectively, during the lactation phase; mean achieved doses were higher due to the increased physiological demand of lactation(Main phase females only). This was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment.

 

The test item was generally well tolerated at doses up to 3750 ppm, there were no unscheduled deaths. Clinical condition, behaviour in the arena, sensory reactivity, grip strength and motor activity were unaffected by treatment.

 

During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment. Following completion of treatment recovery phase females that had previously received 3750 ppm showed body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males that previously received 3750 ppm was similar to Controls. During Week 1 of treatment mean food consumption at 3750 ppm was approximately 92% of Controls for males and approximately 89% of Controls for females; thereafter for the remainder of the treatment period (including gestation and lactation) values were generally similar to Controls. Following completion of treatment, food consumption for females that had previously received 3750 ppm was high when compared with Controls (approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery); males that previously received 3750 ppm showed food consumption that was similar to concurrent Controls.

 

Hematological investigations did not reveal any differences that could be attributed to treatment.

After 6 weeks of treatment males at 3750 ppm showed slightly elevated total protein and males at 1250 and 3750 ppm showed low plasma bile acid concentration (no dose response was apparent); these findings were not evident in toxicity phase females after 6 weeks of treatment or in main study females on Day 14 of lactation. After two weeks off dose the total protein, albumin concentration and albumin/globulin ratio for recovery phase males that previously received 3750 ppm was comparable with Controls. After 6 weeks of treatment males at all dose levels showed low urinary specific gravity with males at 3750 ppm showing increased urinary output and females at 3750 ppm showing high urinary pH when compared with Control values. After two weeks of recovery males that previously received 3750 ppm showed urinary output and specific gravity that were essentially comparable with Control values.

 

Estrous cycles, pre-coital interval, mating performance, fertility and gestation length were unaffected by treatment.

 

After 6 weeks of treatment males that received 3750 ppm showed low adjusted mean heart and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). After two weeks of recovery adjusted heart, adrenal and thyroid weights for males that received 3750 ppm were not significantly different from Controls. After 6 weeks of treatment toxicity phase females at 3750 ppm showed significantly high adjusted mean liver weight when compared with Controls (p<0.01; approximately 108% of Controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05; approximately 115% of Controls). On Day 14 of lactation females at 3750 ppm had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls.

 

At scheduled termination of main study males, toxicity phase females or recovery phase animals there were no macroscopic findings that could be related to treatment.

 

Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Follicular cell hypertrophy in the thyroids had not completely recovered in males but complete recovery was seen in females.

Analyses of samples for thyroxine (T4) obtained from Main study male animals did not reveal any differences that could be attributed to treatment; therefore further assessment of T4 and thyroid stimulating hormone (TSH) was not considered necessary.

 

Mating performance, fertility, gestation length and gestation index were unaffected by treatment. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

 

F1 responses

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment. Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment.

 

On Day 1 of age the mean body weights for both male and female offspring derived from groups receiving the test item were essentially similar to Control and considered unaffected by parental treatment. Subsequent weight gain up to Day 7 of age was similar to Controls. However between Days 7 and 13 of age body weight gain for male and female offspring at 1250 and 3750 ppm were low when compared with Controls, with the differences attaining statistical significance for males at 3750 ppm and for females at 1250 ppm and 3750 ppm. This resulted in significantly low absolute mean bodyweight on Day 13 of age for male offspring at 3750 ppm and for female offspring at 1250ppm and 3750ppm. This was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment.

 

Macroscopic examination of offspring that died prior to scheduled termination did not reveal any findings related to treatment. On Day 13 of age offspring from two out of the ten litters at 3750 ppm were observed with patchy coat; which may be related to a slight delay in development because of the slightly larger mean litter size of this group. There were no other findings that could be related to parental treatment.

 

In conclusion dietary administration of test substance to rats at dose levels of 417, 1250 and 3750 ppm doses for 5 weeks to males and non-pregnant females and to females for 2 weeks before pairing, throughout gestation up to Day 14 of lactation was well-tolerated and did not cause any adverse change in the adult animals. Reproductive performance, fertility and offspring survival were unaffected by parental treatment. Therefore under the test conditions, the NOAEL for reproductive and developmental toxicity was 3750 ppm (equivalent to daily intakes between 223 and 598 mg/kg bw/day depending on the sex, age and weight of the animals) based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

223 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1).

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, test substance was administered to groups of Crl:CD(SD) rats at dietary concentrations of 417, 1250 or 3750 ppm.Main study males (10 animal/dose) were treated daily for two weeks before pairing and for a minimum total of six weeks prior to necropsy. Main study females(10 animal/dose) were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 14 of lactation. Females were allowed to litter and rear their offspring to weaning and were killed on Day 14 of lactation. The F1 generation was killed on Day 4 or Day 13 of age, but received no direct administration to the test item; any exposure wasin utero or via the milk. High dose and control toxicity phase females (5 animal/dose) were treated daily for a minimum of six consecutive weeks.and were not paired. High dose and control recovery phase animals (5 animal/sex/dose) were treated daily for a minimum of six consecutive weeks followed by a minimum of two weeks recovery to assess the potential of any treatment related change to recover. Recovery phase animals were not paired. A similarly constituted Control group was assigned to each phase, and received the vehicle, powdered SDS VRF1 Certified diet, 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, water consumption (visual assessment), hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone analysis, 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, ano-genital distance and nipple counts (males only), and macropathology for all offspring were also recorded.

 

F0 responses

The mean achieved dose for males receiving 417, 1250 and 3750 ppm during the 6 week treatment period was 25.1, 75.0 and 223 mg/kg bw bw/day, respectively. Overall the mean achieved dose for females receiving 3750 ppm during 6 weeks was 232 mg/kg bw/day (Toxicity and Recovery phase animals and main phase females for two weeks pre-pairing). The mean achieved doses during each main study phase for females receiving 417, 1250 and 3750 ppm were: 26.9, 75.6 and 225 mg/kg bw/day, respectively, during the two week pre-pairing treatment period (Main, Toxicity and Recovery phase animals); 28.7, 87.0 and 259 mg/kg bw/day, respectively, during the gestation phase (Main phase females only); 67.3, 192 and 598 mg/kg bw/day respectively, during the lactation phase; mean achieved doses were higher due to the increased physiological demand of lactation (Main phase females only).

 

The test item was generally well tolerated at doses up to 3750 ppm, there were no unscheduled deaths. Clinical condition, behaviour in the arena, sensory reactivity, grip strength and motor activity were unaffected by treatment.

 

During treatment (including females during gestation and lactation) bodyweight gain was unaffected by treatment. Following completion of treatment recovery phase females that had previously received 3750 ppm showed body weight gain that was high when compared with Controls, however mean weight gain for recovery phase males that previously received 3750 ppm was similar to Controls. During Week 1 of treatment mean food consumption at 3750 ppm was approximately 92% of Controls for males and approximately 89% of Controls for females; thereafter for the remainder of the treatment period (including gestation and lactation) values were generally similar to Controls. Following completion of treatment, food consumption for females that had previously received 3750 ppm was high when compared with Controls (approximately 118% of Controls during Week 1 of recovery and approximately 109% of Controls during Week 2 of recovery); males that previously received 3750 ppm showed food consumption that was similar to concurrent Controls.

 

Hematological investigations did not reveal any differences that could be attributed to treatment.

After 6 weeks of treatment males at 3750 ppm showed slightly elevated total protein and males at 1250 and 3750 ppm showed low plasma bile acid concentration (no dose response was apparent); these findings were not evident in toxicity phase females after 6 weeks of treatment or in main study females on Day 14 of lactation. After two weeks off dose the total protein, albumin concentration and albumin/globulin ratio for recovery phase males that previously received 3750 ppm was comparable with Controls. After 6 weeks of treatment males at all dose levels showed low urinary specific gravity with males at 3750 ppm showing increased urinary output and females at 3750 ppm showing high urinary pH when compared with Control values. After two weeks of recovery males that previously received 3750 ppm showed urinary output and specific gravity that were essentially comparable with Control values.

 

Estrous cycles, pre-coital interval, mating performance, fertility and gestation length were unaffected by treatment.

 

After 6 weeks of treatment males that received 3750 ppm showed low adjusted mean heart and adrenal weight (p<0.05) and high adjusted mean thyroid weight (p<0.05). After two weeks of recovery adjusted heart, adrenal and thyroid weights for males that received 3750 ppm were not significantly different from Controls. After 6 weeks of treatment toxicity phase females at 3750 ppm showed significantly high adjusted mean liver weight when compared with Controls (p<0.01; approximately 108% of Controls) and after two weeks of recovery adjusted mean liver weight for females that received 3750 ppm remained high when compared with Controls (p<0.05; approximately 115% of Controls). On Day 14 of lactation females at 3750 ppm had high adjusted mean liver weight (p<0.01) and low adjusted mean thymus weight (p<0.05) when compared with Controls.

 

At scheduled termination of main study males, toxicity phase females or recovery phase animals there were no macroscopic findings that could be related to treatment.

 

Microscopic findings related to treatment for 6 weeks (males and females) or until Day 14 of lactation (females) were seen in the liver (centrilobular hepatocyte hypertrophy) and thyroids (follicular cell hypertrophy) at all dietary concentrations. Following the 6 week treatment period and 2 week recovery period, centrilobular hepatocyte hypertrophy demonstrated partial and near full recovery in males and females respectively. Follicular cell hypertrophy in the thyroids had not completely recovered in males but complete recovery was seen in females.

Analyses of samples for thyroxine (T4) obtained from Main study male animals did not reveal any differences that could be attributed to treatment; therefore further assessment of T4 and thyroid stimulating hormone (TSH) was not considered necessary.

 

Mating performance, fertility, gestation length and gestation index were unaffected by treatment.Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

 

F1 responses

The clinical condition of the offspring, litter size, offspring survival and sex ratio were unaffected by parental treatment. Ano-genital distance of both male and female offspring on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment.

 

On Day 1 of age the mean body weights for both male and female offspring derived from groups receiving the test item were essentially similar to Control and considered unaffected by parental treatment. Subsequent weight gain up to Day 7 of age was similar to Controls. However between Days 7 and 13 of age body weight gain for male and female offspring at 1250 and 3750 ppm were low when compared with Controls, with the differences attaining statistical significance for males at 3750 ppm and for females at 1250 ppm and 3750 ppm. This resulted in significantly low absolute mean bodyweight on Day 13 of age for male offspring at 3750 ppm and for female offspring at 1250ppm and 3750ppm. This was associated with the slightly larger litter sizes in these treatment groups and was considered not to be an effect of treatment.

 

Macroscopic examination of offspring that died prior to scheduled termination did not reveal any findings related to treatment. On Day 13 of age offspring from two out of the ten litters at 3750 ppm were observed with patchy coat; which may be related to a slight delay in development because of the slightly larger mean litter size of this group. There were no other findings that could be related to parental treatment.

 

In conclusion dietary administration of test substance to rats at dose levels of 417, 1250 and 3750 ppm doses for 5 weeks to males and non-pregnant females and to females for 2 weeks before pairing, throughout gestation up to Day 14 of lactation was well-tolerated and did not cause any adverse change in the adult animals. Reproductive performance, fertility and offspring survival were unaffected by parental treatment.

Therefore under the test conditions, the NOAEL for reproductive and developmental toxicity was 3750 ppm (equivalent to daily intakes between 223 and 598 mg/kg bw/day depending on the sex, age and weight of the animals) based on the absence of significant effects that could be considered to be adverse at the highest dose tested and below.

Effects on developmental toxicity

Description of key information

Since no alerts for reproductive toxicity were observed in the screening study, no additional study is proposed at this tonnage level.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

223 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1).

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

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data on the source substance, no additional classification is proposed for the target substance regarding specific target organ toxicity after oral dose-repeated exposure. Indeed, adverse effects were not observed at the dose level of 3500 ppm (mean daily test item intake levels of 223 mg/kg bw/day in males, and 225 mg/kg bw/day in females), i.e. above the treshold for Category 1 or 2 classification for a study duration of 6 weeks (ie. 42 days) (corresponding to LOAEL of 21.4 and 214 mg/kg bw/day, respectively, as calculated according to the Haber's rule).