5,7-di-tert-butyl-3-[4-(2-hydroxyethoxy)phenyl]-1-heteropolycycl-2(3H)-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2013 to 14 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

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: Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar Han™:RccHan™:WIST
- Age at study initiation: Approximately twelve weeks old
- Weight at study initiation: males weighed 303 to 351 g; females weighed 185 to 217 g
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Following the pairing phase, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: A pelleted diet was available ad libitum.
- Water: Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 70 % (relative)
- Air changes: The rate of air exchange was at least fifteen air changes per hour
- Photoperiod: Low intensity fluorescent lighting was controlled to give twelve hours of continuous light and twelve hours of darkness.

IN-LIFE DATES: From: 03 April 2013 To: 27 May 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test material was prepared at the appropriate concentrations as a suspension in Corn oil. Formulations were prepared weekly and stored at approximately 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 0, 25, 75 and 250/125 mg/mL
- Amount of vehicle (if gavage): Dose volume 4 mL/kg

Details on mating procedure:

- M/F ratio per cage: Surviving animals were paired on a 1 male: 1 female basis within each dose group
- Length of cohabitation: Up to fourteen days
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
- After successful mating each pregnant female was caged (how): Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHODS
The test material concentration in the test samples was determined by high performance liquid chromatography with UV detection using an external standard technique. The test material gave a chromatographic profile consisting of a single peak; the test material itself was used as the analytical standard.
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test material in the vehicle. The method pf analysis was validated and proven to be suitable for use.

ANALYTICAL PROCEDURE
-Preparation of Standard Solutions
Stock solutions of the test material in methanol were prepared for external standard calibration. 100 mg was exactly weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL.
On each occasion, standard solutions derived from two stock standard solutions were used for calculation.
- Analysis of Samples
The formulations were extracted with methanol. An aliquot of test material formulation was accurately weighed into a volumetric flask and brought to volume with methanol; this was then ultra-sonicated for 15 minutes and centrifuged at 500 rpm for 10 minutes. Where necessary, samples were further diluted with methanol to achieve the working concentration.
- Preparation of Accuracy Samples
Samples of corn oil were accurately fortified with known amounts of the test material equivalent to the highest and lowest concentrations. These samples were then prepared for analysis as the test samples (above).
- Preparation of Linearity Standards
A range of standard solutions were prepared in methanol from a stock solution of 1 mg/mL by serial dilution, covering the concentration range 0 to 0.1608 mg/mL.
- Instrumental Setup
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Sunfire C18 (150 x 4.6 mm i.d.)
Mobile phase: Methanol:water (80:20 v/v)
Flow rate: 1 mL/min
UV detector wavelength: 230 nm
Injection volume: 25 µL
Retention time: ~7.4 minutes

RESULTS - VALIDATION OF ANALYTICAL METHOD
- Specificity
The control dose samples and a solvent blank did not significantly affect the chromatogram at the retention time of the test material (whose area changed accordingly with known concentration).
- Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak area measured and working standard concentrations. The regression coefficients calculated were found to be 0.999.
- Accuracy
The fortified samples of corn oil were found to have a recovery value of ±2 %of the fortification.
- Test material Formulations
The test material was found to be stable in the formulations when kept for 37 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean. The results indicate the accurate use of the test material and corn oil as the vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Up to 8 weeks; 43 days for males animals, up to lactation day 5 for the females.

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

reduced to 500 mg/kg bw/day

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen by the study sponsor based on the results of previous studies on the test material. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour and five hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed individual clinical observations were performed for each animal using a purpose built arena to assess behaviour prior to the start of treatment and at weekly intervals thereafter. The following parameters were observed: gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post-coitum, and on Days 1 and 4 post-partum.
Additional body weight monitoring was performed on a daily basis for health monitoring purposes. Terminal body weights were also recorded for interim sacrifice animals and for full term animals.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post-coitum Days 0 to 7, 7 to 14 and 14 to 20. For females with live litters, food consumption was recorded on Days 1 and 4 post-partum.

FOOD EFFICIENCY: Yes
- Time schedule for examinations: The ratio of body weight change/dietary intake was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was measured daily throughout the study (with the exception of the pairing phase).

OTHER EXAMINATIONS: Yes
Haematology and clinical chemistry parameters were investigated. Blood samples were taken prior to termination (Day 42 for males and Day 4 post-partum for females).
Prior to the start of treatment and at weekly intervals thereafter, all surviving animals were observed for signs of functional toxicity.

Sperm parameters (parental animals):

Parameters examined in male parental generation: testis and epididymis weight; detailed qualitative examination of the testes was undertaken, 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, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Litter observations:

On completion of parturition (Day 0 post-partum), the number of live and dead offspring was recorded.
For each litter the following was recorded:
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1 and 4 post-partum
- Sex of offspring on Days 1 and 4 post-partum
- Clinical condition of offspring from birth to Day 5 post-partum
- Individual offspring weights on Days 1 and 4 post-partum (litter weights were calculated retrospectively from this data)

All live offspring were assessed for surface righting reflex on Day 1 post-partum.

Postmortem examinations (parental animals):

SACRIFICE
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post-partum. Surviving offspring were terminated via intra-cardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post-coitum.

GROSS PATHOLOGY
For all mated females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced, as necessary, by staining the uteri with a 0.5 % ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted. All adult animals and offspring, including those terminated early during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
The following organs were dissected free from fat and weighed before fixation from all surviving males and females from each dose group: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary (post fixation), prostate, seminal vesicles, spleen, testes, thymus, thyroid (weighed post-fixation with parathyroid) and uterus (weighed with cervix).

HISTOPATHOLOGY
Samples of the following tissues were removed from five randomly selected males and females from the 300 mg/kg bw/day dose group and preserved in buffered 10 % formalin, except where stated: adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Bouin’s fluid then transferred to 70 % industrial methylated spirits (IMS) approximately forty-eight hours later), eyes (fixed in Davidson’s fluid), gross lesions, heart, ileum (including Peyer’s patches), jejunum, kidneys, liver, lungs (with bronchi- inflated to approximately normal inspiratory volume with buffered 10 % formalin before immersion in fixative), lymph nodes (mandibular and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach, thyroid/parathyroid, trachea, testes (preserved in Bouin’s fluid then transferred to 70 % industrial methylated spirits (IMS) approximately forty-eight hours later), thymus, urinary bladder, uterus/cervix and vagina.

The tissues listed below were preserved from all remaining animals with the following exceptions: at the request of the study sponsor the following modifications were applied to the routine histopathological investigations: histopathological examination of high dose group (500/1000 mg/kg bw/day) animals was limited to examination of the stomach of all twelve males and five females; routine histopathological investigations were extended to include examination of the liver and stomach from five randomly selected males and females from the 100 mg/kg bw/day treatment group together with the prostate and seminal vesicles from these males and ovaries from these females.
Coagulating gland, epididymides (preserved in Bouin’s fluid then transferred to 70 % industrial methylated spirits (IMS) approximately forty-eight hours later), gross lesions, mammary gland, testes (preserved in Bouin’s fluid then transferred to 70 % industrial methylated spirits (IMS) approximately forty-eight hours later) uterus/cervix and vagina.

The tissues from selected control and test group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination.
Since there were indications of treatment-related changes in the adrenal, lungs, mesenteric lymph node, pituitary and thyroid (both sexes) and testes, examination was subsequently extended to include similarly prepared sections of these tissues from five selected males and females from the 100 mg/kg bw/day dose group.

Postmortem examinations (offspring):

All offspring were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

Where appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at p<0.05. Data were analysed using the decision tree from the Provantis™ Tables and Statistics Module:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analysed by Provantis were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test.
Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant this analysis was followed by pair-wise comparisons using Dunnett’s test. Where Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the Kruskal-Wallis test, which if significant, was followed by the Mann-Whitney U test. Dose response relationship was also investigated by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Reproductive indices:

MATING PERFORMANCE AND FERTILITY
The following parameters were calculated from the individual data during the mating period of the parental generation:
- Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

GESTATION AND PARTURITION DATA
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
- Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100

Offspring viability indices:

LITTER RESPONSES
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
- Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) = [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] x 100
Post–implantation loss (%) = [(Number of implantation sites - Total number of offspring born) / Number of implantation sites] x100
- Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100
- Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post-partum, using the following formula:
Sex ratio = (Number of male offspring / Total number of offspring) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Episodes of transient increased salivation (around the time of dosing) were observed in all test group animals during the study.
High dose (1000/500 mg/kg bw/day) animals of either sex displayed occurrences of increased salivation, pilo-erection, staining around the mouth and ano-genital region. These findings were accompanied among the males by staining around the snout, hunched posture, abnormal respiration, ptosis, lethargy dehydration and emaciation and in females by staining around the eyes and generalised fur loss.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Due to a marked weight loss and depreciation in general condition all high dose animals (1000/500 mg/kg bw/day) were terminated for reasons of animal welfare between Study Days 12 and 13.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Low weight gains or weight loss developed in high dose (1000 mg/kg bw/day) males and females during the first week of treatment. Concerns over body weights were slightly alleviated by removing these animals from treatment for two days but once treatment was reinstated, at a reduced dose level (500 mg/kg bw/day), weight loss continued; this led to a decision by the Study Director for an early termination of these animals for reasons of animal welfare.
Males treated at 300 mg/kg bw/day also showed reduced weight gain in comparison with the controls (albeit to a much lesser degree than the high dose group) during the first week of treatment. This situation improved over the following weeks although group mean gains continued to remain lower (ca. 8 %) than that of the control throughout the treatment period.
Body weight development in 100 mg/kg bw/day males remained similar to that of the controls throughout the study.
Female test groups (100 and 300 mg/kg bw/day) did not vary greatly from that of the control females during maturation. However, slightly lower gains were evident in both surviving test groups (100 and 300 mg/kg bw/day) in comparison with controls during the gestation and lactation phases although there was no dose-dependent trend.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Dietary intake in either sex of the high dose group was considerably lower than that of the concurrent controls. Group mean diet intake values for 300 mg/kg bw/day males were below that of the controls during the first two weeks of treatment with recovery thereafter. Dietary intake of males treated at 100 mg/kg bw/day remained unaffected by treatment.
Food consumption in females treated at 100 mg/kg bw/day remained similar to that of the controls during the maturation phase. During the same period, 300 mg/kg bw/day test females consumed approximately 30 % less diet during the first week of dosing but showed recovery by Week 2. Diet intake in both test groups was noticeably lower than that of the control and significantly below that of the control during the lactation phase albeit during the gestation phase there was no dose-dependent trend.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency (the ratio of body weight gain to dietary intake) in both male test groups paralleled the trends seen for dietary intake.
During the period of measurement of food efficiency in female treatment groups (i.e. maturation) group means remained similar to that of the controls.

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

effects observed, treatment-related

Description (incidence and severity):

Gravimetric measurement of water intake recorded throughout the study revealed males at 100 and 300 mg/kg bw/day to be consuming considerably more water than the controls with a similar trend evident during the brief period of treatment of high dose group animals of either sex. In females only the 300 mg/kg bw/day group showed increased water consumption during the maturation phase in comparison with the control females. However, both female test groups showed increased group means during gestation and lactation; attaining statistical significance during the gestation phase (100 mg/kg bw/day p<0.01 or p<0.05, 300 mg/kg bw/day p<0.001).

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were detected in haematological parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An increase in the plasma concentrations of alanine aminotransferase (ALAT) was identified in males treated at 100 mg/kg bw/day (p<0.05) and 300 mg/kg bw/day (p<0.01). Further indication of liver function change was given by a reduction (p<0.01) in cholesterol levels in males treated at 100 and 300 mg/kg bw/day in comparison with the concurrent controls. A similar pattern (albeit of lesser severity) for cholesterol was also evident in 100 and 300 mg/kg bw/day females.
Both sexes exposed to 300 mg/kg bw/day were observed to have increased plasma levels of bile acid (males p<0.01, females p<0.05). Females treated at 300 mg/kg bw/day also showed an increase (p<0.01) in plasma urea levels in comparison with the corresponding controls.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

All inter and intra group differences in behavioural and functional performance scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance. There were no treatment-related changes in sensory reactivity.

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):

The following treatment-related histopathological findings were present:
- Centrilobular hepatocyte hypertrophy of the liver in males receiving 100 mg/kg bw/day or above and in females receiving 300 mg/kg bw/day.
- Follicular cells: hypertrophy/hyperplasia in the thyroid of males receiving 300 mg/kg bw/day and females receiving 100 mg/kg bw/day or above.
- Pituitary: hypertrophic/vacuolated cells (males receiving 100 mg/kg bw/day or above).
- Testicular Sertoli cell vacuolation in males receiving 100 mg/kg bw/day or above and Leydig cell atrophy in males receiving 300 mg/kg bw/day.
- Prostate: atrophy in males receiving 300 mg/kg bw/day.
- Atrophy in the coagulating glands and seminal vesicles of males receiving 100 mg/kg bw/day or above.
- Epithelial hyperplasia in the stomach of both sexes receiving 300 mg/kg bw/day or above accompanied by erosion, inflammation and congestion in the males treated at 1000/500 mg/kg bw/day).
- Haemorrhaging of the mesenteric lymph nodes in both sexes receiving 300 mg/kg bw/day.
- Adrenal cortex vacuolation in males receiving 100 mg/kg bw/day or above.
- Alveolar macrophages in the lungs of males receiving 300 mg/kg bw/day and females receiving 100 mg/kg bw/day or above.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

ORGAN WEIGHTS
Males and females treated at 300 mg/kg bw/day showed increased (p<0.01) liver weight (both absolute and relative to terminal body weight) in comparison with controls. Increased liver weight (p<0.01) was also identified in the males treated at 100 mg/kg bw/day.
Further findings in the surviving male test groups involved a reduction in prostate weights (100 mg/kg bw/day p<0.05; and 300 mg/kg bw/day p<0.01) accompanied in both test groups by low (p<0.01) seminal vesicle weights. Additional findings in females tested at 300 mg/kg bw/day included an increase (p<0.01) in relative brain weight (reduction in absolute weight) and both female test groups displayed a reduction for ovary and pituitary weights (100 mg/kg bw/day) p<0.05 and 300 mg/kg bw/day (p<0.01) in comparison with controls. However, individual values in the greater number of animals were within the normal historical ranges and as such considered not to be toxicologically significant.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

- Mating
A summary of adult performance is presented in Table 1. A summary incidence for mating performance is presented in Table 2.
No treatment-related effects were detected in mating performance. All paired animals from the 100 mg/kg bw/day and 300 mg/kg bw/day treatment groups mated within the first five days of pairing.
Statistical analysis of the pre-coital interval data did not reveal any significant intergroup differences.
- Fertility
A summary of adult performance is presented in Table 1. Group values for fertility, litter data and implantation losses are given in Tables 2, 3 and 4.
No treatment-related effects on fertility were detected for treated animals, when compared to controls.
Two control females (Nos. 21 and 24) and two females (Nos. 63 and 66) treated with 300 mg/kg bw/day did not achieve pregnancy following positive evidence of mating. Macroscopic examination of the male partners revealed small epididymides and testes in the partner to control female No.24. Histopathological correlates identified in the male partners of the non-pregnant females was confined to minimal vacuolation in the sertoli cells of one control male and in one 300 mg/kg bw/day male and in the remaining test male (300 mg/kg bw/day) by minimal atrophy of the seminal vesicles and coagulation gland.
- Gestation Length
A summary of gestation lengths is presented in Table 2.
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22½ and 24½ days.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: There were no treatment-related effects detected in the reproductive parameters in females treated at 100 or 300 mg/kg bw/day.

Dose descriptor:

NOAEL

Sex:

male

Basis for effect level:

other: Histological changes in the male reproductive organs of both surviving test groups were considered to be associated with treatment and therefore a NOAEL for reproductive toxicity could not be determined in males

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

LITTER RESPONSES
In total ten females from the control, eleven females from the 100 mg/kg bw/day dose group and ten females from the 300 mg/kg bw/day dose group gave birth to a live litter. From these, nine females from the control, eight females from the 100 mg/kg bw/day dose group and all ten females from the 300 mg/kg bw/day successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

OFFSPRING LITTER SIZE, SEX RATIO AND VIABILITY
Group mean corpora lutea and implantation counts, litter size, implantation losses and survival indices are given in Tables 3 and 4.
No statistically significant differences were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for pregnant females when compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.
However, when group means were adjusted to exclude females that showed total litter loss there was a minimal difference in the group mean for corpora lutea which resulted in the 100 mg/kg bw/day value attaining statistical significance (reduction p<0.01) in comparison with control means. This was considered to have no toxicological significance because of the absence of a dose-response relationship.
There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences

OFFSPRING GROWTH AND DEVELOPMENT
There were no toxicologically significant effects. Statistical analysis of the offspring weights data did not reveal any significant intergroup differences.
No obvious clinical signs of toxicity were detected in offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, no milk in stomach, found dead or missing, cold, weak, cannibalised, scattered litter and damaged digits were considered to be low-incidence findings observed in offspring in studies of this type and were considered unrelated to treatment with the test material.

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Table 1: Summary of Reproductive Performance - Group Values

Parameter	Dose Group (mg/kg)
	0	11	300	1000/500
Males
Initial group size	12	12	12	12
Paired	12	11	12	-
Failed to mate	0	0	0	-
Failed to induce pregnancy in female partner	2	0	2	-
Induced pregnancy in female partner	10	10	10	-
Surviving to terminal necropsy	12	12	12	0
Females
Initial group size	12	12	12	12
Paired	12	11	12	-
Failed to mate	0	0	0	-
Non-pregnant	2	0	0	-
Total litter loss (Pre-day 1 post-partum)	0	1	0	-
Total litter loss (Day 2 post-partum)	1	1	0	-
Total litter loss (Day 3 post-partum)	0	1	0	-
Rearing young to Day 5 of age	9	8	10	-

 

Table 2: Summary Incidence of Mating Performance, Fertility and Gestation Lengths

Dose Group (mg/kg)	No. of Males Paired	No. of Females			Pre-coital Interval (Days)
		Paired	Mated	Pregnant	1	2	3	4	5	13	Day not confirmed
0	12	12	12	10	2	1	5	3	0	1	0
100	11	11	11	11	0	3	5	2	0	0	1
300	12	12	10	10	3	2	1	5	1	0	0
1000/500	-	-	-	-	-	-	-	-	-	-	-
Dose Group (mg/kg)	Mating Index (%)		Pregnancy index (%)		Gestation length (Days)				Females with Live Offspring		Parturition Index (%)
					22.5	23	23.5	24.5
0	83		100		6	3	0	1	9		90
100	100		100		4	1	4	1	10		90
300	83		100		1	3	6	0	10		100
1000/500	-		-		-	-	-	-	-		-

 

Table 3: Group Mean Litter Size and Litter Weights

Dose Group (mg/kg)	No. Corpora Lutea	No. Implantation Sites	Total No. Offspring Born	No. Live Offspring		Litter Weight (g)		Offspring weight (g)				Offspring Body weight Change (g) Days 1 to 4
								Day 1		Day 4
				Day 1	Day 4	Day 1	Day 4	Male	Female	Male	Female	Male	Female
0	13.0 (14.1)	12.0 (13.0)	10.6 (11.6)	10.5 (11.4)	11.3 (11.3)	62.02 (67.76)	92.92	5.99 (6.08)	5.75 (5.81)	8.43	8.11	1.59 (2.35)	1.55 (2.30)
100	11.7 (11.4**)	11.5 (11.1)	10.5 (10.5)	10.2 (10.4)	10.1 (10.1)	56.05 (59.06)	78.38	5.56 (5.82)	5.45 (5.67)	7.85	7.80	0.49 (1.78)	0.79 (2.13)
300	11.1	10.5	9.4	9.3	8.5	54.60	63.81	6.09	5.82	7.86	7.40	1.77	1.58
1000/500	-	-	-	-	-	-	-	-	-	-	-	-	-

( ) - Values in parentheses are group mean values excluding total litter loss females

 

Table 4: Group Mean Implantation Losses and Survival Indices Values

Dose Group (mg/kg)	Pre-implantation Loss (%)	Post-implantation Loss (%)	Live Birth Index (%)	Viability Index (%)
0	7.1 (7.9)	12.3 (10.0)	98.0 (97.8)	89.2 (99.1)
100	2.6 (2.5)	10.7 (6.3)	87.2 (98.4)	77.1 (96.4)
300	5.5	8.3	99.1	90.3
1000/500	-	-	-	-

( ) - Values in parentheses are group mean values excluding total litter loss females

Conclusions:

In relation to the test groups that went on to rear offspring there were no treatment-related effects detected in the reproductive parameters in females treated at 100 or 300 mg/kg bw/day, the NOEL for reproductive toxicity in females was therefore considered to be 300 mg/kg bw/day. However, the histological changes in the male reproductive organs of both surviving test groups were considered to be associated with treatment and therefore a NOAEL for reproductive toxicity could not be determined in males.

Executive summary:

A study was conducted to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) in accordance with the standardised guideline OECD 422 under GLP conditions. The study was also designed to be compatible with Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on REACH.

The test material was administered by gavage to three groups of twelve male and twelve female Wistar rats for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 (reduced to 500) mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil).

Due to a marked adverse response that developed in high dose group (1000 mg/kg bw/day) animals during the first week of treatment, these animals were at first removed from treatment for two days, then recommenced at 500 mg/kg bw/day. However, the condition of the animals deteriorated further soon after resumption of treatment and a decision was then taken on welfare grounds to sacrifice these animals.

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations, haematology and clinical chemistry investigations were performed on a random selection of animals.

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post-partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post-coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Treatment with the test material led to low weight gains or weight loss in all male animals. Weight gains of the female test groups (100 and 300 mg/kg bw/day) did not vary greatly from that of the control females during maturation. Food consumption was reduced in the high and 300 mg/kg dose groups; food efficiency in both male and female test groups paralleled the trends seen for dietary intake in the controls. All treatment groups showed an increase in water consumption, though for the 100 mg/kg females this was only during gestation and lactation.

No treatment-related effects were detected in mating performance. All paired animals from the 100 mg/kg bw/day and 300 mg/kg bw/day treatment groups mated within the first five days of pairing. No treatment-related effects on fertility were detected for treated animals, when compared to controls. There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22½ and 24½ days.

In total ten females from the control, eleven females from the 100 mg/kg bw/day dose group and ten females from the 300 mg/kg bw/day dose group gave birth to a live litter. From these, nine females from the control, eight females from the 100 mg/kg bw/day dose group and all ten females from the 300 mg/kg bw/day successfully reared young to Day 5 of age.

No statistically significant differences were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for pregnant females when compared to controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences. There were no toxicologically significant effects on offspring growth and development. No obvious clinical signs of toxicity were detected in offspring from treated females when compared to controls.

No treatment-related changes were detected in haematological parameters. Statistically significant findings were observed in alanine aminotransferase (increase) and cholesterol (decrease) in either sex at 100 and 300 mg/kg bw/day. These changes were accompanied in males from both test groups by elevated values for albumin/globulin ratio and in 300 mg/kg bw/day females by an increase in plasma urea levels. In addition, both sexes that received 300 mg/kg bw/day showed heightened levels of bile acids.

There were no treatment-related changes in the behavioural parameters measured or functional performance assessments or sensory reactivity assessments.

The predominant finding at necropsy in early termination high dose (1000/500 mg/kg bw/day) animals of either sex was local irritation in the stomach.

Males and females treated at 300 mg/kg bw/day showed increased liver weight (both absolute and relative to terminal body weight) in comparison with controls. This was accompanied in the males by a slight but statistically significant increase in adrenal weights. Females from this test group showed a slight reduction in pituitary weight and reduced ovary weights in comparison with controls together with minor changes in brain weight. In addition, males treated at 100 and 300 mg/kg bw/day showed reduced absolute and relative prostate and seminal vesicle values in comparison with control males.

Treatment-related histopathological findings were present: centrilobular hepatocyte hypertrophy of the liver in males (100 mg/kg or above) and females (300 mg/kg); follicular cells: hypertrophy/hyperplasia in the thyroid of males (300 mg/kg) and females (100 mg/kg or above); pituitary: hypertrophic/vacuolated cells in males (100 mg/kg or above); testicular Sertoli cell vacuolation in males (100 mg/kg or above) and Leydig cell atrophy (300 mg/kg); prostate: atrophy in males (300 mg/kg); atrophy in the coagulating glands and seminal vesicles of males (100 mg/kg or above); epithelial hyperplasia in the stomach of both sexes (300 mg/kg or above) accompanied by erosion, inflammation and congestion in the males treated at 1000/500 mg/kg; haemorrhaging of the mesenteric lymph nodes in both sexes (300 mg/kg); adrenal cortex vacuolation in males (100 mg/kg or above); and alveolar macrophages in the lungs of males (300 mg/kg) and females (100 mg/kg or above).

In relation to the test groups that went on to rear offspring there were no treatment-related effects detected in the reproductive parameters in females treated at 100 or 300 mg/kg bw/day, the NOEL for reproductive toxicity in females was therefore considered to be 300 mg/kg bw/day. However, the histological changes in the male reproductive organs of both surviving test groups were considered to be associated with treatment and therefore a NOAEL for reproductive toxicity could not be determined in males.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted in accordance with standardised guidelines and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). The quality of the database is therefore considered to be high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study was conducted to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) in accordance with the standardised guideline OECD 422 under GLP conditions. The study was also designed to be compatible with Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on REACH.

The test material was administered by gavage to three groups of twelve male and twelve female Wistar rats for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 (reduced to 500) mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil).

Due to a marked adverse response that developed in high dose group (1000 mg/kg bw/day) animals during the first week of treatment, these animals were at first removed from treatment for two days, then recommenced at 500 mg/kg bw/day. However, the condition of the animals deteriorated further soon after resumption of treatment and a decision was then taken on welfare grounds to sacrifice these animals.

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations, haematology and clinical chemistry investigations were performed on a random selection of animals.

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post-partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post-coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Treatment with the test material led to low weight gains or weight loss in all male animals. Weight gains of the female test groups (100 and 300 mg/kg bw/day) did not vary greatly from that of the control females during maturation. Food consumption was reduced in the high and 300 mg/kg dose groups; food efficiency in both male and female test groups paralleled the trends seen for dietary intake in the controls. All treatment groups showed an increase in water consumption, though for the 100 mg/kg females this was only during gestation and lactation.

No treatment-related effects were detected in mating performance. All paired animals from the 100 mg/kg bw/day and 300 mg/kg bw/day treatment groups mated within the first five days of pairing. No treatment-related effects on fertility were detected for treated animals, when compared to controls. There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22½ and 24½ days.

In total ten females from the control, eleven females from the 100 mg/kg bw/day dose group and ten females from the 300 mg/kg bw/day dose group gave birth to a live litter. From these, nine females from the control, eight females from the 100 mg/kg bw/day dose group and all ten females from the 300 mg/kg bw/day successfully reared young to Day 5 of age.

No statistically significant differences were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for pregnant females when compared to controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences. There were no toxicologically significant effects on offspring growth and development. No obvious clinical signs of toxicity were detected in offspring from treated females when compared to controls.

No treatment-related changes were detected in haematological parameters. Statistically significant findings were observed in alanine aminotransferase (increase) and cholesterol (decrease) in either sex at 100 and 300 mg/kg bw/day. These changes were accompanied in males from both test groups by elevated values for albumin/globulin ratio and in 300 mg/kg bw/day females by an increase in plasma urea levels. In addition, both sexes that received 300 mg/kg bw/day showed heightened levels of bile acids.

There were no treatment-related changes in the behavioural parameters measured or functional performance assessments or sensory reactivity assessments.

The predominant finding at necropsy in early termination high dose (1000/500 mg/kg bw/day) animals of either sex was local irritation in the stomach.

Males and females treated at 300 mg/kg bw/day showed increased liver weight (both absolute and relative to terminal body weight) in comparison with controls. This was accompanied in the males by a slight but statistically significant increase in adrenal weights. Females from this test group showed a slight reduction in pituitary weight and reduced ovary weights in comparison with controls together with minor changes in brain weight. In addition, males treated at 100 and 300 mg/kg bw/day showed reduced absolute and relative prostate and seminal vesicle values in comparison with control males.

Treatment-related histopathological findings were present: centrilobular hepatocyte hypertrophy of the liver in males (100 mg/kg or above) and females (300 mg/kg); follicular cells: hypertrophy/hyperplasia in the thyroid of males (300 mg/kg) and females (100 mg/kg or above); pituitary: hypertrophic/vacuolated cells in males (100 mg/kg or above); testicular Sertoli cell vacuolation in males (100 mg/kg or above) and Leydig cell atrophy (300 mg/kg); prostate: atrophy in males (300 mg/kg); atrophy in the coagulating glands and seminal vesicles of males (100 mg/kg or above); epithelial hyperplasia in the stomach of both sexes (300 mg/kg or above) accompanied by erosion, inflammation and congestion in the males treated at 1000/500 mg/kg; haemorrhaging of the mesenteric lymph nodes in both sexes (300 mg/kg); adrenal cortex vacuolation in males (100 mg/kg or above); and alveolar macrophages in the lungs of males (300 mg/kg) and females (100 mg/kg or above).

In relation to the test groups that went on to rear offspring there were no treatment-related effects detected in the reproductive parameters in females treated at 100 or 300 mg/kg bw/day, the NOEL for reproductive toxicity in females was therefore considered to be 300 mg/kg bw/day. However, the histological changes in the male reproductive organs of both surviving test groups were considered to be associated with treatment and therefore a NOAEL for reproductive toxicity could not be determined in males.

Short description of key information:

ORAL

NOEL 300 mg/kg bw/day in females, no NOAEL identified in males; Wistar rat; dose levels of 100, 300 and 1000/500 mg/kg; OECD 422

Justification for selection of Effect on fertility via oral route:

Only one study available.

Effects on developmental toxicity

Description of key information

ORAL

NOEL 300 mg/kg bw/day in females; Wistar rat; dose levels of 100, 300 and 1000/500 mg/kg; OECD 422

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2013 to 14 July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar Han™:RccHan™:WIST
- Age at study initiation: Approximately twelve weeks old
- Weight at study initiation: males weighed 303 to 351 g; females weighed 185 to 217 g
- Fasting period before study: No
- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Following the pairing phase, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.
- Diet: A pelleted diet was available ad libitum.
- Water: Mains drinking water was supplied ad libitum from polycarbonate bottles attached to the cage.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 70 % (relative)
- Air changes: The rate of air exchange was at least fifteen air changes per hour
- Photoperiod: Low intensity fluorescent lighting was controlled to give twelve hours of continuous light and twelve hours of darkness.

IN-LIFE DATES: From: 03 April 2013 To: 27 May 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test material was prepared at the appropriate concentrations as a suspension in Corn oil. Formulations were prepared weekly and stored at approximately 4 °C in the dark.

VEHICLE
- Concentration in vehicle: 0, 25, 75 and 250/125 mg/mL
- Amount of vehicle (if gavage): Dose volume 4 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHODS
The test material concentration in the test samples was determined by high performance liquid chromatography with UV detection using an external standard technique. The test material gave a chromatographic profile consisting of a single peak; the test material itself was used as the analytical standard.
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test material in the vehicle. The method pf analysis was validated and proven to be suitable for use.

ANALYTICAL PROCEDURE
-Preparation of Standard Solutions
Stock solutions of the test material in methanol were prepared for external standard calibration. 100 mg was exactly weighed into a 100 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL.
On each occasion, standard solutions derived from two stock standard solutions were used for calculation.
- Analysis of Samples
The formulations were extracted with methanol. An aliquot of test material formulation was accurately weighed into a volumetric flask and brought to volume with methanol; this was then ultra-sonicated for 15 minutes and centrifuged at 500 rpm for 10 minutes. Where necessary, samples were further diluted with methanol to achieve the working concentration.
- Preparation of Accuracy Samples
Samples of corn oil were accurately fortified with known amounts of the test material equivalent to the highest and lowest concentrations. These samples were then prepared for analysis as the test samples (above).
- Preparation of Linearity Standards
A range of standard solutions were prepared in methanol from a stock solution of 1 mg/mL by serial dilution, covering the concentration range 0 to 0.1608 mg/mL.
- Instrumental Setup
HPLC: Agilent Technologies 1200, incorporating autosampler and workstation
Column: Sunfire C18 (150 x 4.6 mm i.d.)
Mobile phase: Methanol:water (80:20 v/v)
Flow rate: 1 mL/min
UV detector wavelength: 230 nm
Injection volume: 25 µL
Retention time: ~7.4 minutes

RESULTS - VALIDATION OF ANALYTICAL METHOD
- Specificity
The control dose samples and a solvent blank did not significantly affect the chromatogram at the retention time of the test material (whose area changed accordingly with known concentration).
- Linearity
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak area measured and working standard concentrations. The regression coefficients calculated were found to be 0.999.
- Accuracy
The fortified samples of corn oil were found to have a recovery value of ±2 %of the fortification.
- Test material Formulations
The test material was found to be stable in the formulations when kept for 37 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean. The results indicate the accurate use of the test material and corn oil as the vehicle during this study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Details on mating procedure:

- M/F ratio per cage: Surviving animals were paired on a 1 male: 1 female basis within each dose group
- Length of cohabitation: Up to fourteen days
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
- After successful mating each pregnant female was caged (how): Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

Up to 8 weeks; up to lactation day 5 for the females

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

reduced to 500 mg/kg bw/day

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen by the study sponsor based on the results of previous studies on the test material. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour and five hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed individual clinical observations were performed for each animal using a purpose built arena to assess behaviour prior to the start of treatment and at weekly intervals thereafter. The following parameters were observed: gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post-coitum, and on Days 1 and 4 post-partum.
Additional body weight monitoring was performed on a daily basis for health monitoring purposes. Terminal body weights were also recorded for interim sacrifice animals and for full term animals.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post-coitum Days 0 to 7, 7 to 14 and 14 to 20. For females with live litters, food consumption was recorded on Days 1 and 4 post-partum.

FOOD EFFICIENCY: Yes
- Time schedule for examinations: The ratio of body weight change/dietary intake was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was measured daily throughout the study (with the exception of the pairing phase).

OTHER EXAMINATIONS: Yes
Haematology and clinical chemistry parameters were investigated. Blood samples were taken prior to termination (Day 4 post-partum for females).
Prior to the start of treatment and at weekly intervals thereafter, all surviving animals were observed for signs of functional toxicity.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
For all mated females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced, as necessary, by staining the uteri with a 0.5 % ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.

Fetal examinations:

On completion of parturition (Day 0 post-partum), the number of live and dead offspring was recorded.
For each litter the following was recorded:
- Number of offspring born
- Number of offspring alive recorded daily and reported on Days 1 and 4 post-partum
- Sex of offspring on Days 1 and 4 post-partum
- Clinical condition of offspring from birth to Day 5 post-partum
- Individual offspring weights on Days 1 and 4 post-partum (litter weights were calculated retrospectively from this data)

All live offspring were assessed for surface righting reflex on Day 1 post-partum.

Statistics:

Where appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at p<0.05. Data were analysed using the decision tree from the Provantis™ Tables and Statistics Module:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analysed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Data not analysed by Provantis were assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test.
Where Levene’s test was shown to be non-significant (p≥0.05), parametric analysis of the data was applied, incorporating analysis of variance (ANOVA). If this data was shown to be significant this analysis was followed by pair-wise comparisons using Dunnett’s test. Where Levene’s test was significant, non-parametric analysis of the data was analysed incorporating the Kruskal-Wallis test, which if significant, was followed by the Mann-Whitney U test. Dose response relationship was also investigated by linear regression. Where the data was unsuitable for these analyses, then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Indices:

MATING PERFORMANCE AND FERTILITY
The following were calculated from the individual data:
- Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
- Fertility Indices
For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

GESTATION AND PARTURITION DATA
The following parameters were calculated from individual data:
- Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
- Parturition Index
The following was calculated for each group:
Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100

LITTER RESPONSES
The standard unit of assessment was the litter; values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
- Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) = [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] x 100
Post–implantation loss (%) = [(Number of implantation sites - Total number of offspring born) / Number of implantation sites] x100
- Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100
- Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post-partum, using the following formula:
Sex ratio = (Number of male offspring / Total number of offspring) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Episodes of transient increased salivation (around the time of dosing) were observed in all test group animals during the study.
High dose (1000/500 mg/kg bw/day) animals displayed occurrences of increased salivation, pilo-erection, staining around the mouth and ano-genital region. These findings were accompanied by staining around the eyes and generalised fur loss.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Due to a marked weight loss and depreciation in general condition all high dose animals (1000/500 mg/kg bw/day) were terminated for reasons of animal welfare between Study Days 12 and 13.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Low weight gains or weight loss developed in high dose (1000 mg/kg bw/day) females during the first week of treatment. Concerns over body weights were slightly alleviated by removing these animals from treatment for two days but once treatment was reinstated, at a reduced dose level (500 mg/kg bw/day), weight loss continued; this led to a decision by the Study Director for an early termination of these animals for reasons of animal welfare.
Test groups 100 and 300 mg/kg bw/day did not vary greatly from that of the control females during maturation. However, slightly lower gains were evident in both surviving test groups (100 and 300 mg/kg bw/day) in comparison with controls during the gestation and lactation phases although there was no dose-dependent trend.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Dietary intake of the high dose group was considerably lower than that of the concurrent controls. Food consumption in females treated at 100 mg/kg bw/day remained similar to that of the controls during the maturation phase. During the same period, 300 mg/kg bw/day test females consumed approximately 30 % less diet during the first week of dosing but showed recovery by Week 2. Diet intake in both test groups was noticeably lower than that of the control and significantly below that of the control during the lactation phase albeit during the gestation phase there was no dose-dependent trend.

Food efficiency:

no effects observed

Description (incidence and severity):

During the period of measurement of food efficiency in female treatment groups (i.e. maturation) group means remained similar to that of the controls.

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

effects observed, treatment-related

Description (incidence and severity):

The 300 mg/kg bw/day group showed increased water consumption during the maturation phase in comparison with the control females. However, both female test groups showed increased group means during gestation and lactation; attaining statistical significance during the gestation phase (100 mg/kg bw/day p<0.01 or p<0.05, 300 mg/kg bw/day p<0.001).

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were detected in haematological parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A reduction (p<0.01) in cholesterol levels was evident in 100 and 300 mg/kg bw/day females. Animlas exposed to 300 mg/kg bw/day were observed to have increased plasma levels of bile acid (p<0.05). Females treated at 300 mg/kg bw/day also showed an increase (p<0.01) in plasma urea levels in comparison with the corresponding controls.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

All inter and intra group differences in behavioural and functional performance scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance. There were no treatment-related changes in sensory reactivity.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females treated at 300 mg/kg bw/day showed increased (p<0.01) liver weight (both absolute and relative to terminal body weight) in comparison with controls.
Additional findings in females tested at 300 mg/kg bw/day included an increase (p<0.01) in relative brain weight (reduction in absolute weight) and both female test groups displayed a reduction for ovary and pituitary weights (100 mg/kg bw/day) p<0.05 and 300 mg/kg bw/day (p<0.01) in comparison with controls. However, individual values in the greater number of animals were within the normal historical ranges and as such considered not to be toxicologically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of animals sacrificed at study termination revealed the following findings: examination of early termination animals (1000/500 mg/kg bw/day) revealed local irritation in the stomach of 5 of 12 females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related histopathological findings were present:
- Centrilobular hepatocyte hypertrophy of the liver in females receiving 300 mg/kg bw/day.
- Follicular cells: hypertrophy/hyperplasia in the thyroid of females receiving 100 mg/kg bw/day or above.
- Epithelial hyperplasia in the stomach of animals receiving 300 mg/kg bw/day or above.
- Haemorrhaging of the mesenteric lymph nodes in animals receiving 300 mg/kg bw/day.
- Alveolar macrophages in the lungs of females receiving 100 mg/kg bw/day or above.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects. Statistical analysis of the offspring weights data did not reveal any significant intergroup differences.
No obvious clinical signs of toxicity were detected in offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, no milk in stomach, found dead or missing, cold, weak, canibalised, scattered litter and damaged digits were considered to be low-incidence findings observed in offspring in studies of this type and were considered unrelated to treatment with the test material.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

In total ten females from the control, eleven females from the 100 mg/kg bw/day dose group and ten females from the 300 mg/kg bw/day dose group gave birth to a live litter. From these, nine females from the control, eight females from the 100 mg/kg bw/day dose group and all ten females from the 300 mg/kg bw/day successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

No statistically significant differences were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for pregnant females when compared to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.
However, when group means were adjusted to exclude females that showed total litter loss there was a minimal difference in the group mean for corpora lutea which resulted in the 100 mg/kg bw/day value attaining statistical significance (reduction p<0.01) in comparison with control means. This was considered to have no toxicological significance because of the absence of a dose-response relationship.

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Summary of Reproductive Performance - Group Values

Parameter	Dose Group (mg/kg)
	0	11	300	1000/500
Males
Initial group size	12	12	12	12
Paired	12	11	12	-
Failed to mate	0	0	0	-
Failed to induce pregnancy in female partner	2	0	2	-
Induced pregnancy in female partner	10	10	10	-
Surviving to terminal necropsy	12	12	12	0
Females
Initial group size	12	12	12	12
Paired	12	11	12	-
Failed to mate	0	0	0	-
Non-pregnant	2	0	0	-
Total litter loss (Pre-day 1 post-partum)	0	1	0	-
Total litter loss (Day 2 post-partum)	1	1	0	-
Total litter loss (Day 3 post-partum)	0	1	0	-
Rearing young to Day 5 of age	9	8	10	-

 

Table 2: Summary Incidence of Mating Performance, Fertility and Gestation Lengths

Dose Group (mg/kg)	No. of Males Paired	No. of Females			Pre-coital Interval (Days)
		Paired	Mated	Pregnant	1	2	3	4	5	13	Day not confirmed
0	12	12	12	10	2	1	5	3	0	1	0
100	11	11	11	11	0	3	5	2	0	0	1
300	12	12	10	10	3	2	1	5	1	0	0
1000/500	-	-	-	-	-	-	-	-	-	-	-
Dose Group (mg/kg)	Mating Index (%)		Pregnancy index (%)		Gestation length (Days)				Females with Live Offspring		Parturition Index (%)
					22.5	23	23.5	24.5
0	83		100		6	3	0	1	9		90
100	100		100		4	1	4	1	10		90
300	83		100		1	3	6	0	10		100
1000/500	-		-		-	-	-	-	-		-

 

Table 3: Group Mean Litter Size and Litter Weights

Dose Group (mg/kg)	No. Corpora Lutea	No. Implantation Sites	Total No. Offspring Born	No. Live Offspring		Litter Weight (g)		Offspring weight (g)				Offspring Body weight Change (g) Days 1 to 4
								Day 1		Day 4
				Day 1	Day 4	Day 1	Day 4	Male	Female	Male	Female	Male	Female
0	13.0 (14.1)	12.0 (13.0)	10.6 (11.6)	10.5 (11.4)	11.3 (11.3)	62.02 (67.76)	92.92	5.99 (6.08)	5.75 (5.81)	8.43	8.11	1.59 (2.35)	1.55 (2.30)
100	11.7 (11.4**)	11.5 (11.1)	10.5 (10.5)	10.2 (10.4)	10.1 (10.1)	56.05 (59.06)	78.38	5.56 (5.82)	5.45 (5.67)	7.85	7.80	0.49 (1.78)	0.79 (2.13)
300	11.1	10.5	9.4	9.3	8.5	54.60	63.81	6.09	5.82	7.86	7.40	1.77	1.58
1000/500	-	-	-	-	-	-	-	-	-	-	-	-	-

( ) - Values in parentheses are group mean values excluding total litter loss females

 

Table 4: Group Mean Implantation Losses and Survival Indices Values

Dose Group (mg/kg)	Pre-implantation Loss (%)	Post-implantation Loss (%)	Live Birth Index (%)	Viability Index (%)
0	7.1 (7.9)	12.3 (10.0)	98.0 (97.8)	89.2 (99.1)
100	2.6 (2.5)	10.7 (6.3)	87.2 (98.4)	77.1 (96.4)
300	5.5	8.3	99.1	90.3
1000/500	-	-	-	-

( ) - Values in parentheses are group mean values excluding total litter loss females

Table 5: Summary Incidence of Offspring Necropsy Findings

Dose Group (mg/kg)	Post-partum day of Termination	Number of Litters Affected	Number of offspring	Macroscopic Findings
Interim Deaths
0	Pre-day 1 Pre-day 1	1 1	1F 1M	No milk in stomach Autolysis
100	Pre-day 1 Pre-day 1 Pre-day 1 2	2 1 1 1	5M, 2F 1* 2M 2M, 1F	Autolysis Cannibalised No milk in stomach Autolysis
300	5 Pre-day 1	1 1	1F 1F	No abnormalities detected Autolysis
1000/5000	-	-	-	-
Terminal Kill (Day 5 Post-partum)
0	-	10		No abnormalities detected
100	-	7 1	11M, 3F	No abnormalities detected Small
300	-	8 1	1M 6M, 5F	No abnormalities detected Damaged digit one hind paw Small
1000/5000	-	-	-	-

*Sex undetermined

Conclusions:

In relation to the test groups that went on to rear offspring there were no treatment-related effects detected in the reproductive parameters in females treated at 100 or 300 mg/kg bw/day, the NOEL for reproductive toxicity in females was therefore considered to be 300 mg/kg bw/day.

Executive summary:

A study was conducted to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) in accordance with the standardised guideline OECD 422 under GLP conditions. The study was also designed to be compatible with Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on REACH.

The test material was administered by gavage to three groups of twelve male and twelve female Wistar rats for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 (reduced to 500) mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil).

Due to a marked adverse response that developed in high dose group (1000 mg/kg bw/day) animals during the first week of treatment, these animals were at first removed from treatment for two days, then recommenced at 500 mg/kg bw/day. However, the condition of the animals deteriorated further soon after resumption of treatment and a decision was then taken on welfare grounds to sacrifice these animals.

Clinical signs, behavioural assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations, haematology and clinical chemistry investigations were performed on a random selection of animals.

Surviving males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5 post-partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post-coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Weight gains of the 100 and 300 mg/kg bw/day test groups did not vary greatly from that of the control females during maturation. Food consumption was reduced in the high and 300 mg/kg dose groups; food efficiency paralleled the trends seen for dietary intake in the controls. All treatment groups showed an increase in water consumption, though for the 100 mg/kg females this was only during gestation and lactation.

No treatment-related effects were detected in mating performance. All paired animals from the 100 mg/kg bw/day and 300 mg/kg bw/day treatment groups mated within the first five days of pairing. No treatment-related effects on fertility were detected for treated animals, when compared to controls. There were no differences in gestation lengths. The distribution for treated females was comparable to controls. The gestation lengths were between 22½ and 24½ days.

In total ten females from the control, eleven females from the 100 mg/kg bw/day dose group and ten females from the 300 mg/kg bw/day dose group gave birth to a live litter. From these, nine females from the control, eight females from the 100 mg/kg bw/day dose group and all ten females from the 300 mg/kg bw/day successfully reared young to Day 5 of age.

No statistically significant differences were detected for corpora lutea, implantation counts, implantation losses, litter size or litter viability for pregnant females when compared to controls. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences. There were no toxicologically significant effects on offspring growth and development. No obvious clinical signs of toxicity were detected in offspring from treated females when compared to controls.

No treatment-related changes were detected in haematological parameters. Statistically significant findings were observed in alanine aminotransferase (increase) and cholesterol (decrease) at 100 and 300 mg/kg bw/day. These changes were accompanied in 300 mg/kg bw/day females by an increase in plasma urea levels. In addition, animals that received 300 mg/kg bw/day showed heightened levels of bile acids.

There were no treatment-related changes in the behavioural parameters measured or functional performance assessments or sensory reactivity assessments.

The predominant finding at necropsy in early termination high dose (1000/500 mg/kg bw/day) animals was local irritation in the stomach.

Animals treated at 300 mg/kg bw/day showed increased liver weight (both absolute and relative to terminal body weight) in comparison with controls. Additionally a slight reduction in pituitary weight and reduced ovary weights in comparison with controls together with minor changes in brain weight were seen.

The following treatment-related histopathological findings were present: centrilobular hepatocyte hypertrophy of the liver in females receiving 300 mg/kg bw/day; follicular cells: hypertrophy/hyperplasia in the thyroid of females receiving 100 mg/kg bw/day or above; epithelial hyperplasia in the stomach of animals receiving 300 mg/kg bw/day or above; haemorrhaging of the mesenteric lymph nodes in animals receiving 300 mg/kg bw/day; and alveolar macrophages in the lungs of females receiving 100 mg/kg bw/day or above.

In relation to the test groups that went on to rear offspring there were no treatment-related effects detected in the reproductive parameters in females treated at 100 or 300 mg/kg bw/day, the NOEL for reproductive toxicity in females was therefore considered to be 300 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted in accordance with standardised guidelines and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). The quality of the database is therefore considered to be high.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 (reduced to 500) mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Corn oil). Due to a marked toxic response that developed in the high dose group (1000 mg/kg bw/day) animals during the first week of treatment; these animals were at first removed from treatment for two days dosing, then dosing recommenced at 500 mg/kg bw/day. However, the condition of the animals deteriorated further soon after resumption of treatment and a decision was then taken on welfare grounds to terminate these animals. The histopathology results showed that these effects were caused by severe local irritation to the stomach resulting in erosion, inflammation and congestion in the high dose group and epithelial hyperplasia at the mid-dose group. In addition, all test animals induced persistent episodes of excessive salivation around the time of dosing indicating the test item to have irritative properties.

 

 

Reproductive Effects and Classification

 

The study concluded that there were no treatment-related effects on fertility, no effect on gestation length, no effect on litter size, no effect on sex-ratio, no effect on viability or development of the offspring.Two control females (Nos. 21 and 24) and two females (Nos. 63 and 66) treated with 300 mg/kg bw/day did not achieve pregnancy following positive evidence of mating. Macroscopic

examination of the male partners revealed small epididymides and testes in the partner to control female No.24. Histopathological correlates identified in the male partners of the non-pregnant females were confined to minimal vacuolation in the Sertoli cells of one control male and in one 300 mg/kg bw/day male and in the remaining test male (300 mg/kg bw/day) by minimal atrophy of the seminal vesicles and coagulation gland. Small seminal vesicles were identified in one 100 mg/kg bw/day male (No. 35) and in three males (Nos. 49, 50 and 57) at 300 mg/kg bw/day. A small prostate was also identified in one male (No. 49). Small epididymides and testes were noted in one control male (No. 12) and in one 300 mg/kg bw/day male (No. 49). Further findings in the surviving male test groups involved a reduction in mean prostate weight (100 mg/kg bw/day p<0.05; and 300 mg/kg bw/day p<0.01) accompanied in both test groups by low (p<0.01) mean seminal vesicle weights. On the contrary, the qualitative examination of the stages of spermatogenesis in the testes revealedno treatment-relatedabnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.

 

In this study type, mating take place after 2 weeks of exposure to the males, whereas the animals were terminated after 4 weeks of exposure. Consequently, it is feasible that effects on male fertility may not be detected if such effects are delayed until after ~3 weeks of exposure. However, the minor male accessory organ effects observed in these studies may be consequential to the reduced food consumption and/or reduced food conversion efficiency, and consequent reduced weight gain. Dietary restriction has been shown to cause changes in animal bodyweight, rate and nature of weight change, and the timing of these effects, can cause a secondary effect on the age of sexual development in both males and females (Ashby and Lefevre, 2000). Also, mild reductions in food intake not only lowered body weight gain, reduced reproductive organ weights (seminal vesicles, ventral prostate, epididymis), and decreased serum testosterone, but also caused specific stage-dependent, microscopic testicular alterations (Rehm et al., 2008). Furthermore, common stress-related effects were observed in this study (decreased adrenal glands and thymus weights, etc.) and both mild and severe stressors are associated with decreased prostate and seminal vesicle weights but not with histological changes to the testes (Everds, 2013), which is exactly the profile seen in this study.

 

 

 

 

References

 

Ashby J. and Lefevre PA (2000) The prepubertal male rat assay as an alternative to the Hershberger castrated male rat assay for the detection of anti-androgens, oestrogens and metabolic modulators. J. Appl. Toxicol. 20: 35-47.

 

Everds, N E, et al.(2013) Interpreting Stress Responses during Routine Toxicity Studies: A Review of the Biology, Impact, and Assessment. Toxicologic Pathology. Mar;41(4):560-614

 

Rehm, S, White, TE, Zahalka EA, Stanislaus DJ, Boyce RW and Wier PJ (2008) Effects of food restriction on testis and accessory sex glands in maturing rats. Toxicologic Pathology. 36: 687-694.

 

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to reprotoxicity.