Phosphoric acid, mono- and di-C6-10-alkyl esters

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 November 2011 and 21 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: the 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 the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono- and di-C6-10-alkyl esters

EC Number:

269-616-7

EC Name:

Phosphoric acid, mono- and di-C6-10-alkyl esters

Cas Number:

68307-94-8

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Esterification Products of Phosphorus Pentoxide and Alcohols C6-C10 (even numbered)

Test material form:

liquid: viscous

Details on test material:

Sponsor's identification : Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even numbered)
CAS number : 68307-94-8
Identifier : TIS O2891
Description : Amber coloured slightly viscous liquid
Batch number : CI1E0447 solvent free
Label : K92W2 Without Solvent LOT - CI1E0447
Date received : 13 June 2011
Storage conditions : Room temperature in the dark
Expiry date : 29 May 2013

Test animals

Species:

rat

Strain:

other: Wistar Han™:RccHan™:WIST

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: 12 weeks
- Weight at study initiation: males weighed 300 to 345 g, females weighed 189 to 220 g
- Housing: Initially, all animals were housed in groups of 5 in solid floor polypropylene cages with stainless mesh lids and softwood flake bedding. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a 1 male: 1 female basis within each dose group. Following evidence of successful mating, 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.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15
- Air changes (per hr): 15

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results showed the formulations to be stable for 2 hours. Formulations were therefore prepared daily

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 8 ml/kg bw/day of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of Esterification products of Phosphorus Pentoxide and Alcohols C6-C10 (Even Numbered) in the test item formulations was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique.

The test item formulations were sampled and analysed initially and then after storage at ambient conditions for two hours. This was performed as the low level formulations were found to be unstable over eight days and therefore the dose formulations for the study were to be performed prior to each dosing occasion, and dosed within two hours of preparation.


The results indicate that the prepared formulations were within acceptable ranges for the purpose of the study.

Details on mating procedure:

- M/F ratio per cage: 1 male: 1 female
- Length of cohabitation: up to 14 d
- 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 of the presence of sperm was recorded. The presence of sperm within the vagina smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:

8 weeks

Frequency of treatment:

Twice daily

Duration of test:

8 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 160, 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (7-Day Dose Range-Finding Study in the Han Wistar Rat). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item.

Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated twice daily at the appropriate dose level throughout the study (except for females during parturition
where applicable). The first day of dosing was designated as Day 1 of the study.
ii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv) Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
vii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii) Blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43.
ix) Blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes, all animals were examined for overt signs of toxicity, ill-health and behavioural change
- Time schedule: Immediately before the first dose, up to 30 min after each dosing occasion, and 1 and 3 hr after each dosing occasion, during the working week. Animals were observed immediately before the first dose, soon after each dosing occasion, and 1 hr after each dosing occasion at weekends and bank holidays

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.

FOOD CONSUMPTION: during the maturation period, weekly food consumption was recorded for each cage of adults. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY: food efficiency was calculated retrospectively for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily (with the exception of the mating phase)

LABORATORY INVETSIGATIONS: Yes
Haematological and blood chemical investigations were performed 5 females selected from each test and control group prior to termination (Day 4 post partum for females). Blood samples were obtained after the first dosing occasion from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. 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), erythrocyte 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), reticulocyte count ((Retic) methylene blue staining slides were prepared but reticulocytes were not assessed). Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ 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) ration (by calculation), sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol), total bilirubin (Bili), bile acids (Bile).

OTHER:
FUNCTIONAL OBSERVATIONS: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/ behavioural toxicity at least 1 hr after the second dose. Functional performance tests were also performed on the 5 females from each dose level, prior to termination, together with an assessment of sensory reactivity to various 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, hyper/ hypothermia, skin colour, respiration, convulsions, bizarre/ abnormal/ stereotypic behaviour/ salivation/ pilo-erection, exophthalmia, lachrymation, palpebral closure, urination, defecation, transfer arousal, tail elevation.

FUNCTIONAL PERFORMANCE TESTS
Motor activity: purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals 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 30 min for each animal. The percentage of time each animal was active and mobile was recorded for the overall 30 min period and also during the 20 % of the period.

Forelimb/ hindlimb grip strength: an automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of 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 trails were performed for each animal.

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, touch escape, pupil reflex, blink reflex, tail pinch, finger approach, startle reflex.

SACRIFICE
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.


HISTOPATHOLOGY / ORGAN WEIGHTS
Please refer to repeated dose toxicity (7.5.1) and toxicity to reproduction (7.8.1) endpoints for details of histopathology.

Ovaries and uterine content:

The uterine content was examined after termination: Yes
Examinations included:
- Number of implantations: Yes

The number of corpora lutea was recorded.

Fetal examinations:

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis: quantative functional performance data, body weight and body weight change, food and water consumption during gestation and lactation, pre-coital interval and gestation length, litter size and litter weights, sex ratio, corpora lutea and implantation sites, implantation losses and viability indices, offspring body weight and body weight change, offspring surface righting, haematology, blood chemistry, adult absolute and body weight-relative organ weights.

Probability values (P) were calculated as follows:
P<0.001***
P<0.01**
P<0.05*
P≥0.05 (not significant)

Indices:

Mating performance and fertility
The following parameters were calculated from the individual data during the mating period of the parental generation:
1) Pre-coital interval = the time elapsing between initial pairing and the observation of positive evidence of mating.

2) 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 for individual data during the gestation and parturition period of the parental generation:
1) Gestation length = number of days of gestation including the day for observation of mating and the start of parturition.

2) 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 considered to be the litter, values were first calculated for each litter and the group mean was calculated using the individual litter values. Group mean values included all litters reared to termination (Day 5 of age).
1) Implantation losses (%)
Group mean percentile pre-implantation and post-implantation losses 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) x 100

2) Live birth and viability indices
The following indices were calculated for each litter as follows:

Live birth index (%)= (Number of offspring alive on Day 4/ Number of offspring born) x 100

Viability index (%)= (Number of offspring alive on Day 4/ Number of offspring alive on Day 1) x 100

3) Sex ratio (% males) = (Number of male offspring/ Total number of offspring)x100

Historical control data:

Normal range data for the different parameters examined were used in comparison against the test data.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Refer to details on maternal toxic effects section for details.

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
Animals treated with 500 mg/kg bw/day showed increased salivation and noisy respiration throughout the treatment period.

One female treated with 500 mg/kg bw/day showed laboured respiration on Day 1 and a further female from this treatment group showed laboured and gasping respiration on Day 3.

Isolated incidences of staining around the mouth or snout were evident in animals treated with 500 mg/kg bw/day later in the treatment period (Days 10-11 females).

No such effects were detected in animals treated with 160 or 50 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant effects were detected in females treated with 500 or 160 mg/kg bw/day or animals treated with 50 mg/kg bw/day.

FOOD CONSUMPTION AND FOOD EFFICIENCY
No adverse effect on food consumption was detected for females during the pre-mating, gestation or lactation phases of the study. No adverse effect on food efficiency was detected for females during pre-mating. Statistical analysis of gestation and lactation data revealed no significant differences.

WATER CONSUMPTION
Females treated with 500 mg/kg bw/day showed an increase in water consumption during the maturation phase of the study.

No adverse effect on water consumption was detected in animals treated with 160 or 50 mg/kg bw/day.

HAEMATOLOGY
There were no toxicologically significant effects detected in the haematological parameters examined.

Females from all treatment groups showed a statistically significant reduction in activated partial thromboplastin time. In the absence of a true dose related response the intergroup differences were considered not to be of toxicological importance.

CLINICAL CHEMISTRY
Animals treated with 500 mg/kg bw/day and 160 mg/kg bw/day showed a statistically significant increase in albumin/globulin ratio with all the individual values being outside the normal ranges for rats of the strain and age used.

No such effects were detected in animals treated with 50 mg/kg bw/day.

Females treated with 500 and 160 mg/kg bw/day showed a statistically significant increase in glucose levels. The majority of individual values within the normal ranges for rats of the strain and age used, and in the absence of any true dose related responses the intergroup differences were considered not to be of toxicological importance.

ORGAN WEIGHTS
Animals treated with 500 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Females from the 500 mg/kg bw/day treatment group showed an increase in absolute and relative thyroid weight, however a true dose related response was not evident.

No such effects were detected in animals treated with 160 or 50 mg/kg bw/day.

Females treated with 160 or 50 mg/kg bw/day showed a statistically significant increase in thyroid weight both absolute and relative to terminal body weight. In the absence of any associated histology correlates at these levels the intergroup differences were considered of no toxicological importance.

NECROPSY
Adults
Two females treated with 500 mg/kg bw/day had sloughing on the non glandular region of the stomach. One female treated with 160 mg/kg bw/day had sloughing on the glandular region of the stomach.

No toxicologically significant effects were detected in animals treated with 50 mg/kg bw/day.

One female treated with 500 mg/kg bw/day had increased pelvic space in both or left kidney(s) (respectively). In the absence of any histology correlates the intergroup differences were considered of no toxicological importance.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment related microscopic abnormalities were detected:
Liver: Minimal to slight centrilobular hepatocellular hypertrophy was evident in animals treated with 500 mg/kg bw/day.

Thyroid: Minimal diffuse follicular cell hypertrophy was evident in 2 females treated with 500 mg/kg bw/day.

Stomach: Diffuse minimal to moderate squamous cell hyperplasia was evident in the forestomach (non-glandular region) in 3 females treated with 50 mg/kg bw/day, 4 females treated with 160 mg/kg bw/day and 6 females treated with 500 mg/kg bw/day. This finding was associated in all animals with minimal to marked diffuse hyperkeratosis and in animals treated with 500 mg/kg bw/day with minimal to moderate multifocal parakeratosis and minimal to moderate multifocal epithelial vesicles. Minimal to moderate submucosal inflammation was also evident in animals from all treated groups. This was associated in one 500 mg/kg bw/day female with minimal focal lymphoid cell infiltration.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence of severity between control and treatment groups that were considered to be of toxicological significance.

OTHER FINDINGS: FUNCTIONAL OBSERVATIONS
BEHAVIOURAL ASSESSMENTS
The open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls. All inter and intr group differences in urination, defecation and transfer arousal 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.

FUNCTIONAL PERFORMANCE TESTS
There were no treatment related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

SENSORY REACTIVITY ASSESSMENTS
There were no treatment-related changes in sensory reactivity.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Remarks:

reproductive/developmental toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Remarks:

(systemic toxicity)

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Reproductive Performance

Mating

There were no treatment-related effects on mating performance.

Fertility

No treatment effects on fertility were detected for treated animals when compared to controls.

One control female and two females treated with 500 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female reproductive organs. The control male partner revealed bilateral marked diffuse tubular atrophy with slight diffuse edema and multifocal multinucleated spermatid giant cells in the testes. This was accompanied by a slight increase in intratubular cellular debris in the epididymides with marked oligospermia. One male treated with 500 mg/kg bw/day revealed bilateral marked diffuse tubular atrophy with slight diffuse edema in the testes which was associated with aspermia in the epididymides. These findings were considered to be the cause of the non pregnancies however all findings in the testes and epididymides were considered to be incidental and within the normal background range and therefore the non pregnancies were considered unrelated to the test item. No histopathological correlates were evident in the reproductive organs of the remaining 500 mg/kg bw/day male. A possible cause for the non pregnancy however may have been the forestomach findings in both animals.

Gestation Length

No treatement-related effects were detected in the length of gestation for treated females when compared to controls. All animals showed gestation lengths of 22 to 23½ days.

Litter Response

In total nine females from the control group, ten females from the 50 and 160 mg/kg bw/day dose groups and seven females from the 500 mg/kg bw/day gave birth to a live litter and successfully reared young to Day 5 age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size and Viability

No toxicologically significant effects were detected.

No significant differences were detected for corpora lutea, implantation counts, litter size or litter viability for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

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

No toxicologically significant effects were detected.

There were no significant differences in litter weights, offspring weights or surface righting reflex.

Statistical analysis of the data did not reveal any significant intergroup differences.

No obvious clinical signs of toxicity were detected for offspring from treated females when compared to controls. The incidental clinical signs detected throughout the control and treated groups, consisting of small size, offspring found dead or missing, cold,

scattered and physical injuries were considered to be low incidence findings observed in offspring in studies of this type, and were unrelated to test item toxicity.

Applicant's summary and conclusion

Conclusions:

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive/developmental toxicity was considered to be 500 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is compatible with the requirements of the OECD Guidelines for Testing Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

This study was also designed to be compatible with the 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 the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered twice daily by gavage to 3 groups, each of 10 male and 10 female Wistar Han™:RccHam™:WIST strain rats, for up to 8 weeks (including a 2 week maturation phase, pairing, gestation and early lactation for females), at dose levels of 50, 160 and 500 mg/kg bw/day. A control group of 10 males and 10 females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a 1 male: 1 female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on 5 selected males from each dose group after the completion of the mating phase, and for 5 selected parental females from each dose group on Day 4post partum.Haematology and blood chemistry were evaluated prior to termination on 5 selected males and females from each dose group.

Adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses

Mortality:There were no unscheduled deaths.

Clinical Observations:Animals of either sex treated with 500 mg/kg bw/day showed increased salivation and noisy respiration throughout the treatment period. Incidences of either laboured respiration, decreased respiratory rate, gasping respiration, lethargy or pilo-erection were also evident in these animals during the first 3 days of treatment and isolated incidences of staining around the mouth or snout were evident later in the study. No such effects were detected in animals of either sex treated with 160 or 50 mg/kg bw/day.

Behavioural Assessment:Three males treated with 500 mg/kg bw/day showed noisy respiration during week 5.

There were no toxicologically significant changes in the behavioural parameters measured for females treated with 500 mg/kg bw/day or for animals of either sex treated with 160 or 50 mg/kg bw/day.

Functional Performance Tests:There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments:There were no treatment-related changes in sensory reactivity.

Body weight:Males treated with 500 mg/kg bw/day showed a reduction in body weight gain between Weeks 1 and 5 compared to controls. Males treated with 160 mg/kg bw/day also showed a reduction in body weight gain during Weeks 3, 4 and 5. Subsequent overall body weight gain for these males was reduced. No toxicologically significant effects were detected in females treated with 500 160 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

Food Consumption:Males treated with 500 mg/kg bw/day showed a reduction in overall food consumption. Food efficiency in these males was also affected.

No such effects were detected in females treated with 500 mg/kg bw/day or animals of either sex treated with 160 or 50 mg/kg bw/day.

Water Consumption:Females treated with 500 mg/kg bw/day showed a slight increase in water consumption during the maturation phase of the study.

No adverse effect on water consumption was detected in males treated with 500 mg/kg bw/day or animals of either sex treated with 160 or 50 mg/kg bw/day.

Reproductive Performance

Mating and Fertility:There were no treatment-related effects on mating or conception rates for treated animals.

Gestation Lengths:There were no treatment-related differences in gestation lengths.

The distribution for treated females was comparable to controls.

Litter Responses

Offspring Litter Size and Viability:Of the litters born, litter size at birth and subsequently on Day 1 and 4post partum were comparable to controls. There were no significant intergroup differences in sex ratio.

Offspring Growth and Development:Offspring body weight gain and litter weights at birth and subsequently on Day 1 and 4post partum were comparable to controls.

Offspring Observations:No clinically observable signs of toxicity were detected for offspring from all treatment groups.

Laboratory Investigations

Haematology:There were no toxicologically significant effects detected in the haematological parameters examined.

Blood Chemistry:Animals of either sex treated with 500 mg/kg bw/day and females treated with 160 mg/kg bw/day showed a statistically significant increase in albumin/ globulin ratio. No such effects were detected in males treated with 160 mg/kg bw/day or in animals or either sex treated with 50 mg/kg bw/day.

Pathology

Necropsy:Nine males and 2 females treated with 500 mg/kg bw/day had sloughing on the non glandular region of the stomach. One female treated with 160 mg/kg bw/day had sloughing on the glandular region of the stomach. No such effects were detected in males treated with 160 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

Organ weights:Animals of either sex treated with 500 mg/kg bw/day showed an increase in liver weight both absolute and relative to terminal body weight. Males treated with 500 mg/kg bw/day also showed an increase in absolute and relative adrenal weight whilst females also showed an increase in absolute and relative thyroid weight.

No such effects were detected in animals of either sex treated with 160 or 50 mg/kg bw/day.

Histopathology:The following treatment related microscopic abnormalities were detected:

Liver:Minimal to slight centrilobular hepatocellular hypertrophy was evident in animals of either sex treated with 500 mg/kg bw/day. Minimal to slight glycogen depletion was also evident in 3 males from this treatment group. At 160 mg/kg bw/day, 3 males showed minimal centrilobular hepatocellular hypertrophy.

Thyroid:Minimal diffuse follicular cell hypertrophy was evident in 2 females treated with 500 mg/kg bw/day.

Stomach:Diffuse minimal to moderate squamous cell hyperplasia was evident in the forestomach (non-glandular region) in 4 males and 3 females treated with 50 mg/kg bw/day, in 4 males and 4 females treated with 160 mg/kg bw/day and in 9 males and 6 females treated with 500 mg/kg bw/day. This finding was associated in all animals with minimal to marked diffuse hyperkeratosis and in animals or of either sex treated with 500 mg/kg bw/day and males treated with 160 and 50 mg/kg bw/day with minimal to moderate multifocal parakeratosis and minimal to moderate multifocal epithelial vesicles. Minimal to moderate submucosal inflammation was also evident in animals of either sex from all treated groups. This was associated in one 500 mg/kg bw/day female with minimal focal lymphoid cell infiltration.

Adrenals:Diffuse fatty change, mainly affecting the zona fasciculata, was slightly increased in males treated with 500 mg/kg bw/day.

Conclusion

The oral administration of the test item to rats by gavage, at dose levels 50, 160 and 500 mg/kg bw/day, resulted in treatment related effects detected in animals of either sex from all treatment groups.

The microscopic changes identified in the liver and thyroid together with the associated blood chemical and organ weight changes seen at 500 or 160 mg/kg bw/day were considered to be an adaptive response rather than adverse effect of treatment.

The stomach changes identified together with the associated reductions in body weight and food consumption at 500 mg/ka bw/day may be considered to be an adverse effect of treatment, however they are also considered to be a result of local irritation of the test item rather than a true effect of systemic toxicity. The effects were sufficient to elicit an adaptive response in the adrenal glands.

Based on these findings the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 500 mg/kg bw/day.

No treatment related effects were detected in the reproductive parameters examined therefore the ‘No Observed Effect Level’ (NOEL) for reproductive/ developmental toxicity was considered to be 500 mg/kg bw/day.