Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: One generation screening study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
December 2002 - February 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and OECD Guidelines.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
In a combined, repeated-dose reproductive/developmental toxicity study, Sprague-Dawley rats (10/sex/dose) were administered the test substance orally in the diet at 1000, 2750 or 7500 ppm (ca. 50, 138 or 375 mg/kg bw/day). Treatment began 2 weeks prior to mating and continued throughout maturation, mating, gestation and up today 5 of lactation (ca. 47 days). The dose levels were reduced to 900, 2500 or 6750 ppm (ca. 45, 125 or 338 mg/kg bw/d) on day 29. On study day 33, the high dose group was further reduced to 5500 ppm (ca. 275 mg/kg bw/d) due to observed toxicity. A control group of similar size was given untreated diet only.
Following 2 weeks of dosing, male and female rats were paired within their dose groups to produce litters. At day 5 post partum, all surviving females and offspring together with all adult males were killed and examined macroscopically
At Day 5 post partum, all surviving females and offspring together with all adult males were killed and examined macroscopically. Parental animals were observed daily for clinical signs of toxicity. Bodyweights and food consumption were recorded weekly during the maturation phase, which was continued for males after the mating phase. On the day of initiation of treatment and on Days 8, 15 and 22, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests (motor activity and forelimb/hindlimb grip strength) were also performed on five selected males and five selected females per group on Day 22 for males and Day 4 of lactation for females, together with an assessment of sensory reactivity to auditory, visual and proprioceptive stimuli. Blood sampling for haematology and clinical chemistry was performed on five selected males and five selected females per dose group one day prior to pairing. Post mortem macroscopic examinations were performed on all adults and offspring, including decedents. Selected reproductive organs testes, epididymides, ovaries) were weighed and/or preserved together with any significant abnormalities from all parental animals. In addition an extended list of organs/tissues (adrenals, brain, heart, kidneys, liver, spleen, and thymus) were weighed and/or preserved in fixative for selected males and females. Histopathology was carried out on specific organs from parental animals. Histopathology was also performed on the extended list of tissues preserved from selected males and females.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Date received: 14 August 2006
Description: off-white powder
Storage conditions: ambient temperature and humidity in the dark,

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River UK. On receipt the animals were examined for signs of ill health or injury. The animals were acclimatised for 16 days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of the treatment the males weighed 298 to 375g and the females weighed 200 to 248g.
Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During the mating period animals were transferred to a similar type cage on a one male to one female per cage basis.
Following evidence of successful mating, the males were returned to their original cages and transferred to a separate animal room of comparable conditions. The females were housed, individually, in polypropylene cages with solid floors and stainless steel tops. Mated females were given softwood chips, as bedding, throughout gestation and lactation.
The animals were allowed free access to food and water. A ground diet PMI 5002 was offered ad libitum. Main water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in air conditioned rooms. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system. Both animal rooms used for this study were maintained to operate within a target temperature range of 21 +/-2 degC and a relative humidity range of 55 +/- 15%. On isolated occasions the room temperature and/or humidity fell outside the protocol limits but this was not considered to have affected the purpose or integrity of the study.
The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.
The animals were uniquely identified within the study, by an ear punching system routinely used at the laboratory. Colour coded cage labels were used to assist recognition of dose groups.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
A sufficient number of male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River UK. On receipt the animals were examined for signs of ill health or injury. The animals were acclimatised for 16 days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of the treatment the males weighed 298 to 375g and the females weighed 200 to 248g.
Upon arrival, the animals were housed in groups of four in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays. During the mating period animals were transferred to a similar type cage on a one male to one female per cage basis.
Following evidence of successful mating, the males were returned to their original cages and transferred to a separate animal room of comparable conditions. The females were housed, individually, in polypropylene cages with solid floors and stainless steel tops. Mated females were given softwood chips, as bedding, throughout gestation and lactation.
The animals were allowed free access to food and water. A ground diet PMI 5002 was offered ad libitum. Main water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in air conditioned rooms. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system. Both animal rooms used for this study were maintained to operate within a target temperature range of 21 +/-2 degC and a relative humidity range of 55 +/- 15%. On isolated occasions the room temperature and/or humidity fell outside the protocol limits but this was not considered to have affected the purpose or integrity of the study.
The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.
The animals were uniquely identified within the study, by an ear punching system routinely used at the laboratory. Colour coded cage labels were used to assist recognition of dose groups.
Details on mating procedure:
After the maturation period, the parental generation adults were paired on a one male to one female basis for a period of up to fourteen days. Following pairing, the polypropylene trays beneath each cage were checked for the presence of ejected copulation plugs. Additionally each female was checked for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle 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. Mated females were then separated from the male and housed individually during the period of gestation and lactation. The males were returned to their original holding cages.

Each female was observed at 08:30, 12:30 and 16:30 hours at or around the period of expected parturition. At weekends, observations were carried out at 08:30 and 12:30 only. The following was recorded for each female:
i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
1000, 2750, 7500 ppm (ca. 50, 138 or 375 mg/kg bw/day)
900, 2500 or 6750 ppm (ca. 45, 125 or 338 mg/kg bw/d)
5500 ppm (ca. 275 mg/kg bw/d)
Duration of treatment / exposure:
47 days
Frequency of treatment:
daily
Details on study schedule:
-Groups of ten male and ten female rats were dosed according to dose groups for fourteen days prior to pairing.
-All animals were given a detailed clinical examination once every week for four weeks to observe functional/behavioural changes in an open arena.
- One day prior to pairing (Day 13) five males and five females, randomly selected, were sampled for clinical chemistry and haematology.
- On Day 14 all animals were paired on a one male:one female basis within each dose group for a maximum of fourteen days.
- At the observation of mating or at the end of the fourteen day mating period males were returned to their original cages and females were transferred to individual cages.
- At the end of the mating phase five males per dose group were evaluated for functional/sensory responses to various stimuli. The males used were thosed selected for haematology/clinical chemistry.
- Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size and weight were performed during this period.
- At Day 4 post partum, five females per dose group were evaluated for functional/sensory responses to various stimuli. The females used were those selected for haematology/clinical chemistry.
- At Day 5 post partum following completion of functional assessments all females and offspring were killed and examined macroscopically.
- Following completion of the female gestation and lactation phases, the males were killed and examined macroscopically.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Initial: 1000ppm; Adjusted: 900ppm after 29 days
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Initial: 2750ppm; Adjusted: 2500ppm after 29 days
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Initial: 7500ppm; Adjusted: 6750ppm after 29 days; Adjusted: 5500ppm after 33 days
Basis:
nominal in diet
No. of animals per sex per dose:
Control: 10 male, 10 female
1000ppm: 10 male, 10 female
2750ppm: 10 male, 10 female
7500ppm: 10 male, 10 female
Control animals:
yes, plain diet

Examinations

Parental animals: Observations and examinations:
Morbidity/Mortality:
All animals were checked twice daily during the normal working week and once daily at weekends.

Clinical Observations:
All animals were observed daily, immediately before and one hour after dosing, for clinical signs of toxicity.

Functional Observations:
On the day of initiation of treatment and on Days 8, 15 and 22, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected females per group on Day 22 for males and Day 4 of lactation for females, together with an assessment of sensory reactivity to difference stimuli.

Behavioural Assessments:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abonormal/Steriotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lachrymation
Hyper Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elecation

Functional Performance Tests:
- Motor Activity
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was five hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall five hour period and also during the final 20% of the period (considered to be the asymptotic period). The motor activity for females at 2750 ppm was not recorded as females were killed in extremis prior to evaluation.
- Forelimb/Hindlimb Grips Strength
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was drawn along the trough of the meterby the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. THe animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. Fore and hinf limb grip strength was not recorded for femals at 2750 ppm as females were killed in extemis prior to evaluaton.
-
Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed@
Grasp response
Vocalisation
Toe pinch
Tail pinch
Finger approach
Touch escape
Pupil reflex
Startle reflex
Blink reflex
The sensory reactivity for intermediate level females was not recorded as the females were killed in extremis prior to evaluation.

Bodyweight:
During the maturation and mating period the parental generation animals were weighed weekly. Following mating the parental males were weighed weekly until termination. Parental generation females showing evidence of mating were weighed on Days 0, 7, 14 and 20 post coitum. Parental generation females with a live litter were weighted on Days 1 and 4 post partum.

Food Consumption:
During the maturation period, food consumption was recorded for each cage of adults weekly. For females showing evidence of mating, food consumption was recorded for the periods covering Days 1 to 7, 7 to 14 and 14 to 21 post coitum. For females with live litters, fod consumption was recorded for the period covering Days 1 to 4 post partum.

Laboratory Investigations:
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group on Study Day 13. Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling.

Haematology:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocryte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)
Platelet count (PLT)
Prothrombin time (CT) was assessed by 'Hepato Quick' and Activated partial thromboplastin time (APTT) was assess by 'Preci Clot' using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K+)
Chloride (Cl-)
Calcium (CA++)
Inorganic phosphorus (P)
Aspartate aminotransfease (ASAT)
Alanine aminotransfease (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholestrol (Chol)
Total bilirubin (Bili)
Litter observations:
Litter observations
At the observation of completion of parturition, the number of live and dead offspring was recorded. The subsequent date and time of Day 1 post partum litter observations were standardised.
The following observations were recorded for all individual offspring alive on the particular day of observation:

- Individual offspring weights were recorded on Days 1 and 4 post partum
- The number of offspring was recorded daily up to weaning, and reporting for Days 1 and 4 post partum. Offspring sex was recorded for days 1 and 4 post partum.
The clinical condition of individual offspring was observed daily and any findings recorded.
Postmortem examinations (parental animals):
At Day 5 of lactation all the surviving adults, including non-fertile animals, were killed by carbon dioxide asphyxiation; followed by exsanuination by cardiac puncture for slected animals and cerival dislocation for unselected animals. All animals were examined macroscopically for both internal and external abnormalities. Selected organs and tissues were retained in fixative.
The following list of organs were weighed for all animals at necropsy where applicable:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Thymus
Paired organs were weighed together.

Tissue preservation:
Samples of the following tissues were preserved from all animals 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
Colon
Duodenum
Epididymides (preserved in bouins fluid)
Eyes
Gross lesions
Heart
Ileium
Jejunum
Kidneys
Liver
Lungs (with bronchi) (inflated to normal inspiratory volume with buffered 10% formalin before immersion in fixature)
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal(
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles
Thyroid/parathyroid
Trachea
Urinary bladder
Uterus + Cervix
Vagina
Coagulating gland
Skin (hind limb)
Spinal cord (cerviacl)
Spleen
Stomach
Testes (preservedf in bouins fluid)
Thymus
Urinary bladder
Uterus

Histopathology:
The tissues listed above were examined from five males and five females selected from the control and high dose groups. Histopathology was extended to include examination of the liver, spleen, pituitary, salivary glands and sternum for five males and five females selected from both the low and intermediate dose groups.
The following list of tissues were examined from the remaining unselected males and females from the control and high dose groups:
Epididymides
Ovaries
Pituitary
Prostate
Seminal vescles with coagulating gland
Uterus and cervix
Vagina
Tested.
Postmortem examinations (offspring):
All offspring alive at Day 5 were killed by barbiturate overdose. All these offspring were examined macroscopically for internal and external abnormalities.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

Mortality:
On high dose female (number 71) was killed in extremis during the late gestation phase of the study due to possible dystocia. At the intermediate dose level a total of eight females were killed in extremis during the gestation phase of the study.Six of the eight female mortalities were found to have offspring in utero at post mortum examination indicating possible impairment of parturition.
There were no mortalities at the low dose level or the control level.

Clinical Observations:
The treatment related clinical signs at the high dose were associated with the majority of females and one male. The most prevalent of findings were hunched posture and pilo-erection. Clinical signs of toxicity were seen early in the treatment period with hunched posture observed from week 2 and pilo erection from week 5 along with sporadic instants of tip-toe gait.These findings did not persist thoughout the course of the study as the remaining nine high dose femal animals showed no clinical signs of toxicity at the end of the treatment. One male also showed clinical signs of hunched posture during the treatment period.
Two intermediate dose level females showed hunched posture and three showed pilo erection, but the onset of these findings was later than those seen in high dose females.
No clinical effects were observed for males at this dose level.
At the low dose level there were no clinical signs of reaction to treatment.

Behavioural Evaluations:
The behavioural evaluation showed no evidence to suggest any significant treatment related observations associated with behavioural change. There was no indication of a treatment related effect on sensory reactivity to various stimuli or an effect on motor activity.

Bodyweight:
- Maturation/post mating
At the high dose level there was a reduction in group mean bodyweight for males during the first week of treatment which resulted in a statistically significant difference (p<0.001) compared to control values at Week 2 and 3 of the study. Subsequent bodyweight gain for males and females prior to mating was lower than control value and the difference in group mean bodyweight remained statistically significant (p<0.001). Post mating bodyweight gain was inconsistent and differences in group mean values remained statistically significant (p<0.001) compared to control values.
At the intermediate dose level there was a reduction in malebodyweight gain, which resulted in lower group mean bodyweight compared to control values. The difference was statistically significant (p<0.001) from Week 4 of the study until termination. There was no significant effect on femal bodyweight gain prior to mating.
At the low dose level there was a reduction in malke bodyweight gain which resulted in lower group mean bodyweight compared to control values. The difference was statistically significant (p<0.o5) from Week 4 until termination. There were no significant effects upon female bodyweight gain priot to mating.

- Gestation
The two high dose females that were pregnant showed a significantly lower bodyweight gain during pregnancy than controls.
The females of the intermediate level showed a statistically lower bodyweight gain during gestation when compared to the controls. This resulted in statistically significant differences (p<0.001) in group mean bodyweight for Days 7, 14 and 20 of gestation.
At the low dose level there was a slight reduction in female bodyweight gain during gestation compared to controls. This resulted in a statistically significant difference in group mean bodyweight (p<0.01) for Day 20 of gestation.

- Lactation
At the high (one animal) and intermediate (two animals) dose level there were insufficient females with live litters to allow for meaningful evaluation of bodyweight change during lactation.
At the low dose level, while female bodyweights during lactation were lower than control values, there was no dignificant difference in bodyweight gain between Days 1 and 4 of lactation.

Food Consumption:
- Maturation/Post Mating
At the high dose level there was a marked reduction in group mean food consumption for males and females prior to mating. This reduction in food consumption was also evident for males during the post mating phase of the study.
At the intermediate dose level there was a reduction in group mean food consumption for males and females prior to mating. The reduction in food consumption was also evident for males during the post mating phase of the study.
At the low dose levele there was a reduction in make and female food consumption prior to mating. Post mating male food consumption was reduced compared to control values.

- Gestation
At the high dose level from the small number of pregnant females, there was a notably lower food consumption throughout gestation when compared to control values.
At the intermediate dose level there was a lower group mean food consumption throughout gestation when compared with control values. The difference was statistically significant (p<0.001) of each measurement point.
At the low dose level there was a lower group mean food consumption throughout gestation when compared with control values. The differences were statistically significant (p<0.01 - Days 1 to 7 of gestation, p<0.001 Days 7 to 14 and 14 to 21 of gestation).

- Lactation
At the high and intermediate dose levels there were too few females (two females at the intermediate dose level and one female at the higyh dose level) with live litters to allow for a meaningful evaluation of food consumption. Of the females evaluated, the food consumption values were notably lower than controls.
At the low dose level there was no significant difference in female food consumption between Days 1 and 4 of lactation when compared ith control values.

- Food conversion ratios
Due to the low number of females with live litters, food conversion ratio evaluation is restricted to pre-mating only.
At the high dose level the foo conversion ration for females was notably lower than control values during the first week of treatment whereas the male value was higher than control. The post mating food conversion ratios for males were inconsistent compared with controls.
At the intermediate dose level both male and, more obviously, female values were lower than controls prior to mating. Post mating male values were more comparable with controls.
At the low dose level male or female food conversion ratios prior to mating or for males post mating were comparable with control values.

- Chemical intake
As the high and intermediate dose level, there was an increase in test material intake for males and females in the second week of treatment prior to mating which reflects the increased food consumption seen. This was also seen for females at the low dose level, Post mating male test material intake was lower during the sixth week of the study which reflects the reduction in dose levels for all the treated groups.

Haematology:
At the high dose level there was a slight decrease in platlet count for males compared to control values. The differences was statistically significant (p<0.01). There was a slight increase in clotting time for females compared to ctrol values. The difference was statistically signicant (p<0.05). There was a reduction in mean cell volume and an increase in activated partial thromboplastin time for males only, which was statistically significant (p<0.01) conpared to control values.
At the intermediate dose level there was a slight reduction in mean cell volume for males only. The difference was statistically significant (p<0.05) compared to control values.
At the low dose level there were no significant treatment related effects seen in the haematological parameters examined.

Clinical Chemistry:
At the high dose level notable statistically differences, compared to control values were associated with decreased plasma phosphorus values for both sexes (p<0.01), decreased plasma chloride for males and females (p<0.01 and p<0.001 respectively), increased plasma chlesterol for both sexes (p<0.001) and an increased plasma creatinine (p<0.001) for males only. The statistically significant difference for polasma bilirubin for females (p<0.05) was sisidered to be incidental.
At the intermediate dose level notable statistically significant differences comparted to control values were associated with a decreased plasma phosphorus for males and females (p<0.01 and p<0.001 respectively), an increased plasma cholesterol for males and females (p<0.01 and p<0.05 respectively) a decreased plasma chloride for females only (p<0.01) and an increased plasma creatinine for males only (p<0.05). A reduction in plasma asparate aminotransferase for females (p<0.01) was also observed which is considered to be incidental.
At the low dose level statistically significant differences when compared to control values were associated with a decreased plasma phosphorus for both sexes (p<0.05) and an increased plasma cholesterol for both sexes (p<0.01) and an increased plasma creatinine for males only (p<0.05).

Reproductive Perfomances:
- Fertility
At the high dose lecel there was a marked reduction in the number of mating pairs with positive evidence of mating (four females mated). In adidtion, of those females with positive evidence of mating, only 50% acheived pregnancy (two females acheived pregnancy). The females with no evidence of mating generally showed a lack of oestrous cyclicity. Two of the mating pairs with positive evidence of mating also showed an increased pre coital interval. These findings were condsidered to be of toxicological importance and treatment-related.
At the intermediate and low dose levels there were no treatment related effects upon fertility. All mating pairs showed positive evidence mating and pregnancy.

- Gestation and Parturition
At the high dose level one of the two pregnant females was killed due to possible dystocia.
At the intermediate dose level eight females were killed in extremis during late gestation. The appearance of offspring, in utero in six of these females at post mortem examination was indicative of difficulties at parturation. Of the females that started delivery, there was an apparent increase in the gestation length.
At the low dose level all females produced a live litter but there was a slight increase in the gestation lengths compared to controls.

Organ Weights:
At the high dose group the low number of pregnant females resulted in too few animals of the same physiological status as the control group females to allow direct comparison of organ weights. Statistically significant reductions in male abolute organ weigth values compared to control values were observed for kidneys, epididymides, heart, thymus, spleen (p<0.001) and adrenals (p<0.01). Only the spleen and thymus weight relative to bodyweight was significantly lower than controls (p<0.01). The weight of testis and brain relative to bodyweight were significantly higher (p<0.001) than controls but this is likely to be fortuitous and a result of reduced bodyweight among males. The increase in liver weight, relative to bodyweight (p<0.001), may well represent a true effect of treatment.
At the intermediate dose group the low number of females at termination resulted in too few animals to allow for a meaningful evaluation. Statistically significant reductions in male absolute organ weight values were observed for kidneys, epididymides, spleen and thymus (p<0.01) plus adrenals and heart (p<0.01). Only the difference for the thymus weight, relative to bodyweight, remained statistically significant (p<0.001). Liver weight, relative to bodyweight was significantly higher (p<0.05) than controls. An increased brain and testis weight, relative to bodyweight, was considered to be fortuitous and directly related to lower bodyweights among males.
At the low dose group statistically significant reductions in organ weights were observed for thymus and spleen (p<0.001) kidneys (p<0.01) and heart (p<0.05) for males and adrenals (p<0.001) and heart (p<0.05) for females. Concomitant statistically significant reductions in organ weight, relative to bodyweight, were seen for spleen (p<0.01) and thymum (p<0.001) in males and females (p<0.01).

Histopathology:
LIVER:
Centrilobular hepatocyte enlargement was observed in relation to treatment for rats of with sex of all treatment levels. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.

SPLEEN:
Generally lower grades of severity of extramedullary haemopoiesis and higher grades of severity of pigment accumulation were observed in relation to treatment in high dose females. An effect at the intermediate dose level was difficult to determine but at the low dose level the effect on haemopoiesis was in the opposite direction with generally higher grades of severity of the condition prevailing compared with the control group. The toxicological significance, if any, of this finding is uncertain. There was probaly no effect on pigment deposition at the intermediate dose level and no effect at the low dose level. Male rats were not similarly affected.

PITUITARY:
Higher grades of severity of cellular vacuolation in the pars anterior were seen for rats of either sec treated with the high and intermediate dose levels and for male rats only of the low dose group.

THYROIDS:
Generally higher grades of severity of follicular cell hypertrophy were seen in relation to treatment for rats of either sex of all treatment groups.

SALIVARY GLANDS:
Lower severity grades of serous secretion in the submandibular glands were more prevalent among rats of either sex of all treatment groups compared with the controls.

BONE MARROW:
Higher grades of severity of adipose infiltration, representing myeloid hypoplasia, were seen in relation to treatment for rats of either sex treated at the high and intermediate dose levels, and for male rats only of the low dose level.

HEART:
Focal myocarditis was observed in several control and treated rats and is a common background entity in laboratory maintained rats. The severity of the condition was never greater than minimal, or one or two foci, and should not be interpreted as being indicative of any ongoing myocardial disease.

LIVER:
Scattered mononuclear cell foci were observed in the majority of animals examined in the study. Such are commonly observed in the rodent liver and are not indicative of any adverse condition at the severities encounted.

KIDNEYS:
Isolated groups of basophilic tubules are frequently encountered in the renal cortex of laboratory maintained rats and have no pathological significance at the severities or frequencies reported in this study. Similarly focal mineralisation is a commonly observed background condition amongst female rats.

LUNGS:
A minimal severity of bronchus associated lymphoid tissue was reported for most animals examined in the study and is not indicative of respiriratory disease. Minor severities and low incidences of focal pneumonitis and accumulations of alveolar macrophages are commonly observed pulmonary changes in laboratory maintained rats of this age and are not suggestive of significant respiratory disease.

MAMMARY GLAND:
Glandular hyperplasia was observed in the mammary tissue of all femaled control rats examined and in two female high dose rats. The appearance of the mammary tissue is consistent with pregnancy and lactation and the group distribution of the condition in this study is not a primary effect of treatment but related to the incidence of pregnancy.

UTERUS:
Areas of haemorrhage and fibrosis were seen in the myometrium of the uterus in all control terminal death animals and in one high dose terminal death animal. These conditions are consistent with normal post partum uterine changes in the rat and the group distribution is not a primary effect of treatment. Areas of haemorrhage and fibrosis were also seen for most female premature death animals from Group 3 and in single premature death female rat from Group 4, together with uterine dilatition in all cases. Pyometra was reported for two Group 3 female rats.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Effect levels (P0)

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Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
>= 900 - <= 1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
gross pathology
Dose descriptor:
LOAEL
Remarks:
fertility
Effect level:
>= 900 - <= 1 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
not specified
Description (incidence and severity):
(Too few numbers of live litters to allow meaningful evaluation.)
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

Live Litter Size and Viability:
At high and intermediate dose levels there were a small number of litters for evaluation; of those available there was a notable decrease in litter size at birth and the live litter birth index was reduced for both groups. At the intermediate dose level there was one total litter loss during lactation.
At the low dose level there was a slight reduction in group mean live litter size at birth. This did not achieve statistical significance due to the high intragroup variability but is considered to be of toxicological importance.

Offspring Clinical Condition:
There were no significant clinical findings associated with live offspring during the study.

Offspring Bodyweight Gain:
At the high and intermediate dose level, there were limited numbers of live litters to allow for meaningful evaluation of offspring weight gain.
At the low dose level there were slightly lower live litter weights compared to control values but this did not attain statistical significance and was due to lower group mean litter sizes. Group mean individual offspring bodyweights were comparable with control values.

Offspring Sex Ratio:
At the hight and intermediate dose group the limited number of live litters did not allow meaningful evaluation.
At the low dose group there were no significant treatment related differences in offspring sex ratios.

Macroscopic post mortum findings:
At high and intermediate dose level there were limited numbers of offspring available for evaluation, but it is of note that the offspring of the intermediate dose level showed an increase in the number that had no milk in the stomach at examination.
At the low dose group there were no significant findings.

Effect levels (F1)

Dose descriptor:
LOAEL
Generation:
F1
Effect level:
>= 900 - <= 1 000 ppm
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: increased post-implantation loss, decreased litter size; no external or internal abnormalities.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

During the course of the study there were a total of nine females that were killed in extremis. One female from the high dose and eight from the intermediate dose level. All mortalities were associated with pregnant females particularliy during late gestation at and around the time of parturition.

At the high dose level, clinical signs of toxicity were observed during treatment for both sexes; these signs were considered not to be indicative of behavioural or neurotoxic effect.

There was also evidence of significant reductions in both bodyweight gain and food consumption prior to mating and persisted for males post mating. The findings from the blood chemistry evaluations showed alterations in plasma concentrations of phosphorus and chloride for both sexes plus increased plasma creatinines for males. These findings were not associated with any significant renal pathology. The observed blood chemistry changes together with an increase plasma cholesterol, reduced adrenal weights for both sexes plus histopathological changes to the liver and thyroid glands are symptomatic of altered metabolic state. This may well be a direct consequence of test material metabolism or as a consequence of the reductions in weight gain and food consumption. The splenic changes for males and females seen at histopathology and with the organ weight decrements does not appear to be associated with any significant haematological changes. The slight variations in clotting time and platlet count for males or females may be a consequence of potential alterations in hepatic or general metabolism that may have a secondary effect upon clotting factors.

At histopathology, the hypertrophy observed for the liver of both sexes may be indicative of enhanced metabolism from xenobiotic administration. The thyroid gland hypertophy may also be a consequence of a negative feedback via enhanced metabolism of circulating thyroid hormones. The histopathological changes tend to support the proposal of an altered metabolism being the principal sytemic effect seen following administration of test material. The splenic changes seen may be associated with the altered physiologic status of the animals from reductions in bodyweight and food consumption. Other treatment related histopathological changes to the salivary glands and pituitary are of note because of the treatment and dosage relationship but are not indicative of a specific toxic effect. Organ weight analysis showed decrements for a number of organs for males at termination, which were primarily a consequence of reduced bodyweight. Elevated relative liver weight was connected to they hypertrophy observed at histopathology, which may be a result of alterations in metabolic status.

The results of the mating performance of the males and females show significant treatment-related effects. There is evidence to suggest that normal reproductive behaviour has been affected at this dose level. The mating pairs with no positive evidence of mating showed the females to generally not exhibit normal oestrous cycling. Of the mating pairs where positive evidence of mating was observed, pregnancy was established in 50% of these. Histopathology of the reproductive organs showed no specific effects. The live litter size at birth was low for the small number of females that gave birth. Offspring bodyweight appeared unaffected by treatment but the number of offspring evaluated did not allow a meaningful assessment to be conducted. The overall conclusion was that reproductive performance was affected from the conception phase to parturition. These effects were seen at a dose level that was toxic to the adult and therefore reproductive failure was a consequence of the toxicity seen.

At the intermediate dose level similar clinical signs of toxicity were observed but at a lower incidence. Effects upon bodyweight gain and food consumptions were also observed. A similar profile of effects were seen for blood chemistry, organ weight and histopathological changes indicating a treatment and dosage relationship. There were no effects upon mating performance of fertility when compared to the highest dose level. The most important finding was sufficient to inhibit delivery of the offspring. An evaluation of offspring bodyweight at birth shows that the foetus/neonate has not been directly affected by the test material.

At the low dose level there were indications of test material effects upon the adult. There were slight reductions in bodyweight change at various time points during the study for either or both sexes. In addition, there were slight reductions in food consumption at various time points including females during gestation. Similar changes to that seen at higher dose levels were observed for the blood chemistry and histopathology evaluations. There appears to be a treatment and dosage relationship.

Mating performance and fertility were not affected by treatment. There was a slight reduction in group mean live litter size at birth together with an increase in post implantation offspring loss. These results were not statistically significant because of a large intragroup variability. These results are of significance because of reproductive effects seen at higher dose levels and cannot therefore be ignored.

Applicant's summary and conclusion

Conclusions:
The administration of the substance to male and female rats at dose levels up to 7500 ppm (adjusted to 6750 ppm and then to 5500 ppm), for a period of up to 47 days, resulted in treatment-related toxic effects upon adults. The No Observed Adverse Effect Level (NOAEL) was not established.