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Toxicological information

Toxicity to reproduction

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 August 2013 - 17 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is GLP compliant and follow the OECD 422 guideline. Acceptable with no restrictions.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
22 August 2013 - 17 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is GLP compliant and follow the OECD 422 guideline. Acceptable with no restrictions.
Justification for type of information:
A discussion and report on the read across strategy is given as an attachment in Section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material: Nonene, branched
- CAS No.:97280-95-0
- Substance type: UVCB
- Physical state: Clear colorless liquid
- Analytical purity: 100%
- Lot/batch No.: TQ-12-2003-23042013
- Data received: 29 April 2013
- Label: Nonene Sampling Date Apr 22.2013
- Expiration date of the lot/batch: 23 April2014

Nonene, branched, CAS# 97280-95-0 used in this study was a typical production sample, according to the details included in the boundary composition in IUCLID section 1.2. Nonene, branched was chosen in the Higher Olefins category testing strategy because it represents a substance with high tri-sub content (category range 1 – 65%), odd carbon number (C9 odd 90%). Please see the testing strategy attached in section 13 for further details.
Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 12 weeks old
- Weight at study initiation: (P) Males: 311 to 375 g; Females: 194 to 222 g.
- Housing: Pre-mating: groups of four animals were housed in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Pairing phase: polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Post 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.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.)
- Water (e.g. ad libitum): free access to food and water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-2°C
- Humidity (%): 55 =/- 15%
- Air changes (per hr): at least 15 air changes per hour. The low intensity fluorescent lighting was controlled to give twelve hours continuous light
- Photoperiod (hrs dark / hrs light): 12 hr dark and 12 hr light

The study was performed between 10 June 2013 and 10 June 2014. The in-life phase of the study was conducted between 22 August 2013 (first day of treatment) and 17 October 2013 (final day of necropsy).



Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Arachis oil BP
- Storage temperature of food: 4 C

VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg
- Concentration in vehicle:
- Control: 0 ml/kg
- Low: 25 mg/ml
- Intermediate: 75 mg/ml
- High: 250 mg/ml
- Lot/batch no. (if required): 2018C-020713MA
- The volume of the test and control item administrated to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation:15 days
- Proof of pregnancy referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually during gestation and lactation in solid floor polypropylene cages.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
The test item was administrated daily by gavage using a stainless cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Arachis oil BP.
Details on study schedule:
i. Groups of twelve male and twelve female animals were treated 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/behavioral 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 the pairing phase (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. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded 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 hematological 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 hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 1000 mg/kg bw/day
No. of animals per sex per dose:
Three groups, each of twelve males and twelve females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selected during the Range-finding experiment - Study Number 41301414

Parental animals: Observations and examinations:
Clinical Observations:
Signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour after dosing throughout the treatment period.

Functional Observations:
All animals were observed for signs of functional/behavioural toxicity prior to the start of treatment and at weekly intervals thereafter.
Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- behavioural assessment
- functional Performance Tests
- Sensory Reactivity

Body Weight:
Individual body weights: Day 1 (prior to dosing), weekly for surviving males until termination and weekly for females until mating was evident.
Female: Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 post partum and terminal kill.

Food Consumption:
Pre-pairing period and after (male): weekly
Females showing evidence of mating: post coitum Days 0-7, 7-14 and 14-20.
Females with live litters: Days 1 and 4 post partum.

Food efficiency:
Males (throughout the study period-with the exception of the mating phase, and females during the pre-pairing phase

Water Consumption:
Daily- visual inspection of the water bottles

Reproductive Performance:
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).

Pregnancy and Parturition:
Pregnant females: observations at approximately 0830, 1230 and 1630 hours and around the period of expected parturition (08.30 and 12.30 hr at weekends and public holidays)

Female data collections:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Laboratory Investigations:
- Haematology
- Blood Chemistry
Litter observations:
Number of live and dead offspring
For each litter the following was recorded:
i. Number of offspring born
ii.Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. 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):
Pathology Females: Uterus (for signs of implantation and the number of uterine implantations in each horn), the corpora lutea. All adult animals and offspring: full external and internal examination. All macroscopic abnormalities were recorded.
- Organ Weights- Histopathology: Tissues from 5 selected male and female animals/group were preserved. Histopathological examination was undertaken on tissues from control and high dost animals.
To clarify possible treatment-related changes, histopathological examination was extended to include similarly prepared sections of the liver, kidneys (males only), stomach, thyroid and pituitary from animals in the low and intermediate groups. In addition, male kidney tissue was subject to immunohistochemical examination to confirm the presence of alph-2-microglobulin.
Postmortem examinations (offspring):
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.
Statistics:
Please see "Any other information on materials and methods incl. tables" for information on statistics.
Reproductive indices:
- Pre-coital Interval
- Fertility Indices
- Gestation length
- Parturition Index

Offspring viability indices:
- Implantation Losses (%)
- Live Birth and Viability Indices
- Sex Ratio (% males)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 and 300 mg/kg bw/day: Increasing salivation in males and females. No effects in animals of either sex treated with 100 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3 of treatment. Overall body weight gain was reduced for these males.

No such effects were detected in treated females or in males treated with 300 or 100 mg/kg bw/day.

Females treated with 300 mg/kg bw/day showed a statistically significant increase in cumulative body weight gain during the final week of gestation. An increase in body weight gain is considered not to represent an adverse effect oftreatment therefore the intergroup difference was considered not to be of toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No adverse effect on food consumption was detected in treated animals when compared to control animals.

Females treated with 1000 mg/kg bw/day showed a statistically significant increase in food consumption during the second week of gestation. An increase in food consumption is considered not to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effect on food efficiency was detected in treated animals when compared to control animals.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Daily measurements of water bottles revealed a marked increase in water consumption in males treated with 1000 mg/kg bw/day and a slight increase in males treated with 300 or 100 mg/kg bw/day throughout the treatment period. Females from all treatment groups also showed an increase in water consumption throughout the treatment period with statistical significance being achieved throughout gestation and lactation at 1000 mg/kg bw/day.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed a reduction in hemoglobin. Three out of the five individual values were outside of the normal background range for this parameter.

No toxicologically significant effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a reduction in mean corpuscular hemoglobin. Males treated with 1000 and 300 mg/kg bw/day showed a reduction in mean corpuscular hemoglobin concentration. Males from all treatment groups also showed a reduction in
prothrombin time. All of the individual values were within the normal background range for these parameters, therefore the intergroup differences were considered not to be of toxicological importance. Females from all treatment groups showed a statistically significant increase in neutrophil count. The majority of individual values were within the normal background range for these parameters and a true dose related response was not evident, therefore the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically significant effects were detected in the blood chemical parameters examined.

Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in calcium concentration and a statistically significant reduction in alkaline phosphatase. Males treated with 1000 mg/kg bw/day also showed a reduction in cholesterol. All of the individual values were within the normal ranges for rats ofthe strain and age used and in the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological importance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Behavioral Assessments
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.

Functional Performance Tests
There were no toxicologically significant changes in functional performance. Males from all treatment groups showed a statistically significant increase in fore limb grip strength. The statistically significant difference was confined to one out of the three tests and in the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments
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):
Animals of either sex treated with 1000 and 300 mg/kg bw/day and females treated with 100 mg/kg bw/day showed an increase in absolute and relative liver weight. Males treated with 1000 mg/kg bw/day also showed an increase in kidney weight both absolute and relative to terminal body weight. Males from all treatment groups showed an increase in absolute and relative thyroid weight whilst females treated with 1000 mg/kg bw/day showed a reduction in absolute and relative thyroid weight. All of the individual values were within the normal range for rats of the strain and age used and the effect on male thyroid weight also did not show a true dose related response. However with the associated microscopic thyroid changes observed in this study the intergroup differences detected in thyroid weights can not be excluded as an effect of treatment.

Females treated with 1000 mg/kg bw/day showed a reduction in pituitary weight and an increase in kidney weight both absolute and relative to terminal body weight. In the absence of any associated histopathological changes detected in the kidneys or pituitary of females the intergroup differences were considered not to be of toxicological importance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Nine males treated with 1000 mg/kg bw/day had enlarged kidneys at necropsy, eight of which had pale kidneys; four showed a mottled appearance to the kidneys and five of these males also had a dark liver at necropsy. One male treated with 300 mg/kg bw/day had enlarged kidneys at necropsy.

No toxicologically significant effects were detected in females from any treatment group or in males treated with 100 mg/kg bw/day.

One male treated with 100 mg/kg bw/day had small testes and epididymides. One female treated with 300 mg/kg bw/day and two females treated with 100 mg/kg bw/day had reddened lungs at necropsy. Histopathological examination of these tissues did reveal associated microscopic findings to the macroscopic abnormalities however in the absence of a similar effect seen at 1000 mg/kg bw/day, the intergroup differences were considered to be incidental and of no toxicological importance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment related microscopic findings were detected:

Liver:hepatocyte hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Kidneys:proximal tubular basophilia and hyaline droplets were evident in males from all treatment groups. Tubular degeneration/debris was evident in males treated with 1000 and 300 mg/kg bw/day. These findings were demonstrated by immunohistochemical staining to be
due to alpha-2-microglobulin nephropathy. No such effects were detected in any treated female.

Stomach:epithelial hyperplasia was evident in the fore stomach of animals of either sex treated with 1000 mg/kg bw/day and in males treated with 300 mg/kg bw/day. No such effects were detected in females treated with 300 mg/kg bw/day or animals of either sex treated with 100 mg/kg bw/day.

Thyroid:follicular cell hypertrophy and hyperplasia was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Pituitary:increased incidence and severity of hypertrophic/vacuolated cells in pars anterior was evident in males treated with 1000 and 300 mg/kg bw/day. No such effects were detected in any treated female or in males treated with 100 mg/kg bw/day.
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating
There were no treatment-related effects on mating performance.

Fertility
No treatment-related effects on fertility were detected for treated animals, when compared to controls.

One control female and one female treated 100 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female or male reproductive organs which could have been the cause of the infertility. One female treated with 1000 mg/kg bw/day failed to show any positive signs of mating and was non pregnant.

Gestation Length
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. All animals showed gestation lengths of 22 to 24½ days.
Clinical Signs:
1000 and 300 mg/kg bw/day: Increasing salivation in males and females. No effects in animals of either sex treated with 100 mg/kg bw/day.

Body Weight and Weight Changes:
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3 of treatment. Overall body weight gain was reduced for these males.

No such effects were detected in treated females or in males treated with 300 or 100 mg/kg bw/day.

Females treated with 300 mg/kg bw/day showed a statistically significant increase in cumulative body weight gain during the final week of gestation. An increase in body weight gain is considered not to represent an adverse effect oftreatment therefore the intergroup difference was considered not to be of toxicological importance.

Food consumption and Compound Intake:
No adverse effect on food consumption was detected in treated animals when compared to control animals.

Females treated with 1000 mg/kg bw/day showed a statistically significant increase in food consumption during the second week of gestation. An increase in food consumption is considered not to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.

Water Consumption:
Daily measurements of water bottles revealed a marked increase in water consumption in males treated with 1000 mg/kg bw/day and a slight increase in males treated with 300 or 100 mg/kg bw/day throughout the treatment period. Females from all treatment groups also showed an increase in water consumption throughout the treatment period with statistical significance being achieved throughout gestation and lactation at 1000 mg/kg bw/day.

Hematology:
Males treated with 1000 mg/kg bw/day showed a reduction in hemoglobin. Three out of the five individual values were outside of the normal background range for this parameter.

No toxicologically significant effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a reduction in mean corpuscular hemoglobin. Males treated with 1000 and 300 mg/kg bw/day showed a reduction in mean corpuscular hemoglobin concentration. Males from all treatment groups also showed a reduction in
prothrombin time. All of the individual values were within the normal background range for these parameters, therefore the intergroup differences were considered not to be of toxicological importance. Females from all treatment groups showed a statistically significant increase in neutrophil count. The majority of individual values were within the normal background range for these parameters and a true dose related response was not evident, therefore the intergroup differences were considered not to be of toxicological significance.

Clinical Biochemistry:
No toxicologically significant effects were detected in the blood chemical parameters examined.

Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in calcium concentration and a statistically significant reduction in alkaline phosphatase. Males treated with 1000 mg/kg bw/day also showed a reduction in cholesterol. All of the individual values were within the normal ranges for rats ofthe strain and age used and in the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological importance.

Behaviour:
Functional Performance Tests - There were no toxicologically significant changes in functional performance. Males from all treatment groups showed a statistically significant increase in fore limb grip strength. The statistically significant difference was confined to one out of the three tests and in the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Organ Weight:
Animals of either sex treated with 1000 and 300 mg/kg bw/day and females treated with 100 mg/kg bw/day showed an increase in absolute and relative liver weight. Males treated with 1000 mg/kg bw/day also showed an increase in kidney weight both absolute and relative to terminal body weight. Males from all treatment groups showed an increase in absolute and relative thyroid weight whilst females treated with 1000 mg/kg bw/day showed a reduction in absolute and relative thyroid weight. All of the individual values were within the normal range for rats of the strain and age used and the effect on male thyroid weight also did not show a true dose related response. However with the associated microscopic thyroid changes observed in this study the intergroup differences detected in thyroid weights can not be excluded as an effect of treatment.

Females treated with 1000 mg/kg bw/day showed a reduction in pituitary weight and an increase in kidney weight both absolute and relative to terminal body weight. In the absence of any associated histopathological changes detected in the kidneys or pituitary of females the intergroup differences were considered not to be of toxicological importance.

Necropsy:
Nine males treated with 1000 mg/kg bw/day had enlarged kidneys at necropsy, eight of which had pale kidneys; four showed a mottled appearance to the kidneys and five of these males also had a dark liver at necropsy. One male treated with 300 mg/kg bw/day had enlarged kidneys at necropsy.

No toxicologically significant effects were detected in females from any treatment group or in males treated with 100 mg/kg bw/day.

One male treated with 100 mg/kg bw/day had small testes and epididymides. One female treated with 300 mg/kg bw/day and two females treated with 100 mg/kg bw/day had reddened lungs at necropsy. Histopathological examination of these tissues did reveal associated microscopic findings to the macroscopic abnormalities however in the absence of a similar effect seen at 1000 mg/kg bw/day, the intergroup differences were considered to be incidental and of no toxicological importance.

Histopathology:
Liver:hepatocyte hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Kidneys:proximal tubular basophilia and hyaline droplets were evident in males from all treatment groups. Tubular degeneration/debris was evident in males treated with 1000 and 300 mg/kg bw/day. These findings were demonstrated by immunohistochemical staining to be
due to alpha-2-microglobulin nephropathy. No such effects were detected in any treated female.

Stomach:epithelial hyperplasia was evident in the fore stomach of animals of either sex treated with 1000 mg/kg bw/day and in males treated with 300 mg/kg bw/day. No such effects were detected in females treated with 300 mg/kg bw/day or animals of either sex treated with 100 mg/kg bw/day.

Thyroid:follicular cell hypertrophy and hyperplasia was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Pituitary:increased incidence and severity of hypertrophic/vacuolated cells in pars anterior was evident in males treated with 1000 and 300 mg/kg bw/day. No such effects were detected in any treated female or in males treated with 100 mg/kg bw/day.

Reproductive Performance:
Fertility - No treatment-related effects on fertility were detected for treated animals, when compared to controls.

One control female and one female treated 100 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female or male reproductive organs which could have been the cause of the infertility. One female treated with 1000 mg/kg bw/day failed to show any positive signs of mating and was non pregnant.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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, cold, weak, pale, no milk in stomach, physical injury, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Litter weights on Day 4 post partum from females treated with 1000 mg/kg bw/day were reduced when compared to control litters however statistical significance was not achieved. Offspring body weight gain at 1000 mg/kg bw/day was also reduced between Days 1 and 4 post partum. Statistical significance was achieved for male off spring only. No such effects were detected in litters from females treated with 300 or 100 mg/kg bw/day.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.
Histopathological findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Statistical analysis of surface righting reflex data did not reveal any significant intergroup differences.
Offspring Litter Size, Sex Ratio and Viability
No significant differences were detected for corpora lutea, implantation counts or implantation losses for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Of the litters born, litter size at birth and subsequently on Day 1 post partumwere comparable to controls. Litter size on Day 4 post partumhowever was reduced in litters from females treated with 1000 mg/kg bw/day although statistical significance was not achieved. Offspring viability on Day 4 post partumwas statistically significantly reduced in these litters. A total litter loss was also observed for one female treated with 1000 mg/kg bw/day. No such effects were detected in litters from females treated with 300 or 100 mg/kg bw/day.

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
Litter weights on Day 4 post partumfrom females treated with 1000 mg/kg bw/day were reduced when compared to control litters however statistical significance was not achieved. Offspring body weight gain at 1000 mg/kg bw/day was also reduced between Days 1 and 4 post partum. Statistical significance was achieved for male offspring only.

No such effects were detected in litters from females treated with 300 or 100 mg/kg bw/day. Statistical analysis of surface righting reflex 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, cold, weak, pale, no milk in stomach, physical injury, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced post natal offspring viability, offspring body weight gain and litter size at 1000 mg/kg bw/day.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Table 2. Summary of Reproductive Performance - Groups Values

 

Dose Group (mg/Kg bw/day

0 (Control)

100

300

1000

Males

 

Initial group size

12

12

12

12

Paired

12

12

12

12

Failed to mate

0

0

0

1

Failed to induce pregnancy in female partner

1

1

0

0

Induced pregnancy in female partner

11

11

12

11

Surviving to terminal necropsy

12

12

12

12

 

Females

 

Initial group size

12

12

12

12

Paired

12

12

12

12

Failed to mate

0

0

0

1

Non-pregnant

1

1

0

0

Rearing young to Day 5 of age

11

11

12

10

Total Litter Loss

0

0

0

1

 

Table 3. Group Mean Functional Test Values and Standard Deviations - Males

Group

(sex)

 

Test 1 Forelimb

(g)

Test 1 Hindlimb

(g)

Test 2 Forelimb

(g)

Test 2 Hindlimb

(g)

Test 3 Forelimb

(g)

Test 3 Hindlimb

(g)

Overall

Activity

Overall Mobile

Last 20%

Activity

Last 20% Mobile

1 (M)

Mean

1143.2

422.6

1004.2

524.4

985.2

445.0

377.2

0.6

3.4

0.0n

S.D.

234.7

131.5

113.9

66.2

91.6

76.7

122.9

0.9

4.0

0.0

N

5

5

5

5

5

5

5

5

5

5

 

2 (M)

Mean

1176.8

510.2

1303.0*

581.4

1084.8

628.2

384.0

0.6

8.2

0.0n

S.D.

237.0

140.4

313.6

105.2

146.7

157.4

214.5

0.5

9.5

0.0

N

5

5

5

5

5

5

5

5

5

5

 

3 (M)

Mean

1353.0

593.8

1450.4*

472.0

1378.2

528.2

334.6

0.2

4.2

0.0n

S.D.

297.1

121.5

200.0

147.6

442.9

177.5

141.7

0.4

5.2

0.0

N

5

5

5

5

5

5

5

5

5

5

 

4 (M)

Mean

1071.2

620.8

1271.0*

473.4

1118.6

555.0

250.8

0.2

36.2

0.0n

S.D.

257.2

184.4

250.8

230.9

185.2

151.8

118.6

0.4

58.3

0.0

N

5

5

5

5

5

5

5

5

5

5

General Footnote: Unit = Time (seconds) for Motor Activity Assessments

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

* Significantly different from control group p<0.05

n Data not appropriate for statistical analysis

 

Table 4. Group Mean Body Weight Values - Males

 

Group

(sex)

 

Increase in Body Weight (g)

 

Day Numbers Relative to Start Date

Abs Gain

% Gain

From:

1

8

15

22

29

36

1

1

To:

8

15

22

29

36

43

43

43

1 (M)

Mean

14.4

13.0

9.7

14.6

10.2

6.8

68.6

20.9

S.D.

5,8

6.5

6.6

6.0

4.5

3.7

21.7

5.9

N

12

12

12

12

12

12

12

12

 

 

 

2 (M)

Mean

13.9

12.5

6.9

13.3

12.0

4.3

63.0

19.0

S.D.

3.8

3.9

6.3

4.4

3.4

5.1

16.9

4.8

N

12

12

12

12

12

12

12

12

 

 

 

3 (M)

Mean

13.4

15.3

7.3

14.1

11.9

8.1

70.2

21.3

S.D.

5.5

5.0

3.9

4.6

5.6

3.4

18.8

5.3

N

12

12

12

12

12

12

12

12

 

 

 

4 (M)

Mean

5.5**

10.0

1.7**

12.4

8.1

5.1

42.8

13.0

S.D.

9.8

7.5

6.8

3.8

4.8

4.8

18.0

5.3

N

12

12

12

12

12

12

12

12

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

** Significantly different from control group p<0.01

 

Table 5. Group Mean Body Weight Gains - Females

 

Group

(sex)

 

Increase in Body Weight (g)

Cumulative Body Weight Change (g)

 

Days

Days

 

Gestation

Lactation

Gestation

From:

0

7

14

1

0

0

To:

7

14

20

4

14

20

1 (F)

Mean

29.8

28.9

60.4

7.9

58.7

119.1

S.D.

4.4

3.9

13.0

5.4

7.3

16.3

N

11

11

11

11

11

11

 

2 (F)

Mean

27.4

28.3

60.8

11.8

55.6

116.5

S.D.

4.8

5.0

12.5

10.3

6.1

14.9

N

11

11

11

11

11

11

 

3 (F)

Mean

33.3

33.1

69.1

12.8

66.4

135.5*

S.D.

5.2

5.2

10.9

7.1

7.8

15.2

N

12

12

12

12

12

12

 

4 (F)

Mean

29.8

27.9

57.3

6.4

57.7

115.0

S.D.

6.8

6.5

11.7

12.2

11.4

17.1

N

11

11

11

10

11

11

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

* Significantly different from control group p<0.05

 

Table 6. Group Mean Food Consumptions - Females

Group

(sex)

 

Day Numbers

Gestation

Lactation

From

0

7

14

1

To

7

14

20

4

1 (F)

Mean

19.7

22.3

25.1

26.5

S.D.

1.8

1.9

2.4

4.6

N

11

11

11

11

 

2 (F)

Mean

20.2

22.5

24.6

29.5

S.D.

2.0

2.3

1.2

8.3

N

11

11

11

11

 

3 (F)

Mean

20.7

23.6

25.5

28.2

S.D.

1.2

1.2

1.1

3.3

N

12

12

12

12

 

4 (F)

Mean

20.9

24.2*

26.8

25.4

S.D.

1.6

2.2

2.3

6.2

N

11

11

11

10

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

* Significantly different from control group p<0.05

 

Table 7. Group Mean Water Consumption - Females

Group

(sex)

 

Day Numbers

Gestation

Lactation

From

0

7

14

1

To

7

14

20

4

1 (F)

Mean

27.6

31.0

36.0

39.9

S.D.

3.8

5.1

6.8

4.3

N

11

11

11

11

 

2 (F)

Mean

32.1

36.9

39.9

44.2

S.D.

5.3

5.9

4.8

10.6

N

11

11

11

11

 

3 (F)

Mean

36.5

42.9

44.8

45.8

S.D.

2.2

13.9

10.7

7.1

N

12

12

12

12

 

4 (F)

Mean

52.6***

67.8***

76.4***

54.1**

S.D.

20.4

22.5

34.1

12.5

N

11

11

11

11

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

 

Table 8. Group Mean Hematological Values - Males

Group

 

Hb (g/dL)

MCH (pg)

MCHC (g/dL)

CT (seconds)

Group 1 (0 – Control)

Mean

16.92

18.86

35.66

9.92

S.D.

0.44

0.44

1.49

0.47

N

5

5

5

5

 

Group 2 (100 mg/Kg bw/day)

Mean

16.86

18.64

35.18

9.40*

S.D.

0.74

0.11

0.67

0.23

N

5

5

5

5

 

Group 3 (300 mg/Kg bw/day)

Mean

16.08

18.36

34.12**

9.34*

S.D.

0.31

0.48

0.53

0.26

N

5

5

5

5

 

Group 4 (1000 mg/Kg bw/day)

Mean

14.94**

17.58**

33.72**

9.34*

S.D.

1.56

0.63

0.28

0.36

N

5

5

5

5

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 9. Group Mean Hematological Values - Females

Group

 

Neut (109/L)

Group 1 (0 – Control)

Mean

1.360

S.D.

0.417

N

5

 

Group 2 (100 mg/Kg bw/day)

Mean

2.500*

S.D.

0.568

N

5

 

Group 3 (300 mg/Kg bw/day)

Mean

2.146*

S.D.

0.463

N

5

 

Group 4 (1000 mg/Kg bw/day)

Mean

1.872*

S.D.

0.509

N

5

* Significantly different from control group p<0.05

 

Table 10. Group Mean Blood Chemical Values - Males

Group

 

Ca++(mmol/L)

AP (IU/L)

Chol (mg/dL)

Group 1 (0 – Control)

Mean

2.448

234.0

95.2

S.D.

0.258

52.1

5.3

N

5

5

5

 

Group 2 (100 mg/Kg bw/day)

Mean

2.522

201.6

102.6

S.D.

0.146

26.1

14.4

N

5

5

5

 

Group 3 (300 mg/Kg bw/day)

Mean

2.730*

150.6**

99.4

S.D.

0.077

10.8

10.7

N

5

5

5

 

Group 4 (1000 mg/Kg bw/day)

Mean

2.644*

157.6**

78.0*

S.D.

0.129

55.2

13.3

N

5

5

5

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 11. Summary Incidence of Necropsy Findings - Males

 

Males

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Number of animals examined

12

12

12

12

Epididymides

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

11

12

12

Small; Left

0

1

0

0

 

Kidneys

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

12

11

3

Enlarged

0

0

1

9

Mottled Appearance

0

0

0

4

Pallor

0

0

0

3

Pale

0

0

0

6

 

Liver

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

12

12

7

Dark

0

0

0

5

 

Testes

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

11

12

12

Small; Left

0

1

0

0

 

Table 12. Summary Incidence of Necropsy Findings - Females

 

Females

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Number of animals examined

12

12

12

12

Liver

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

12

12

11

Dark

0

0

0

1

 

Lungs (With Bronchi)

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

10

11

12

Reddened

0

2

1

0

  

Table 13. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

 

 

Males

Females

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Kidneys

Mean (g)

2.29348

2.31240

2.38234

3.16702**

1.54460

1.65510

1.66136

1.97002**

S.D.

0.32697

0.14310

0.16218

0.44018

0.14343

0.11476

0.20712

0.11782

N

5

5

5

5

5

5

5

5

 

Mean (%)

0.574

0.585

0.616

0.830**

0.565

0.610

0.602

0.701**

S.D.

0.046

0.013

0.051

0.102

0.047

0.035

0.055

0.041

N

5

5

5

5

5

5

5

5

 

Liver

Mean (g)

12.7830

13.2647

14.3898**

19.3412**

10.5429

11.7936*

12.7876**

16.1219**

S.D.

1.51792

0.98038

1.33119

1.23327

0.51191

0.52983

1.29755

1.61466

N

5

5

5

5

5

5

5

5

 

Mean (%)

3.203

3.355

3.710**

5.070**

3.857

4.353*

4.638**

5.742**

S.D.

0.125

0.111

0.164

0.230

0.149

0.194

0.424

0.639

N

5

5

5

5

5

5

5

5

 

Pituitary

Mean (g)

 

 

 

 

0.01793

0.01529

0.01783

0.01467**

S.D.

 

 

 

 

0.00277

0.00241

0.00264

0.00180

N

 

 

 

 

11

11

12

10

 

 

Mean (%)

 

 

 

 

0.006

0.006

0.006

0.005**

S.D.

 

 

 

 

0.001

0.001

0.001

0.001

N

 

 

 

 

11

11

12

10

 

Thyroid/Parathyroid

Mean (g)

0.01552

0.02172**

0.02162**

0.02112**

0.002238

0.01972

0.02046

0.01776*

S.D.

0.00377

0.00176

0.00140

0.00207

0.00395

0.00339

0.00202

0.00259

N

5

5

5

5

5

5

5

5

 

Mean (%)

0.004

0.005**

0.006**

0.006**

0.008

0.007

0.007

0.006*

S.D.

0.001

0.000

0.000

0.000

0.002

0.001

0.001

0.001

N

5

5

5

5

5

5

5

5

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 14. Histopathology – Treatment-related Findings

 

Male

Female

Dosage

(mg/Kg bw/day)

0

100

300

1000

0

100

300

1000

 

Liver

Number Examined

5

5

5

9

5

5

5

6

- Hypertrophy; hepatoc.

0

5 (1.0)

5 (1.0)

9 (1.7)

0

0

3 (1.0)

6 (1.0)

- Congestion

0

0

0

5 (1.0)

0

0

0

1(3.0)

 

Thyroid

Number Examined

5

5

5

5

5

5

5

5

- Hypertrophy / Hyperplasia;

Follicular Cell

2 (1.0)

5 (1.4)

5 (1.4)

5 (1.8)

2 (1.0)

2 (1.0)

3 (1.3)

5 (1.2)

 

Pituitary Gland

Number Examined

12

12

12

12

12

12

12

10

-Hypertrophic / Vacuolated cells in

Pars Anterior

10 (1.0)

9 (1.2)

12 (1.4)

9 (1.6)

0

0

3 (1.0)

0

 

Kidney

Number Examined

5

5

5

9

5

5

5

5

- Tubular Basophilia

0

3 (1.0)

4 (1.3)

9 (2.0)

0

0

0

1 (1.0)

- Tubular Degeneration

/ Debris

0

0

4 (1.0)

7 (2.0)

0

0

0

0

- Hyaline Droplets

0

5 (1.0)

5 (1.8)

9 (2.1)

0

0

0

0

 

Kidney Immunohistochemistry

Number Examined

3

3

3

3

0

0

0

0

- alpha-2-microglobulin

3 (1.0)

3 (2.0)

3 (2.3)

3 (2.7)

0

0

0

0

 

Stomach - Forestomach (microscopic changes recorded in the stomach)

Number Examined

5

5

5

5

5

5

5

5

- Hyperplasia, epithelial

0

0

2 (1.0)

2 (1.0)

0

0

0

3 (1.0)

(): Mean Severity

Findings in Bold were considered treatment-related

Table 15. Group Mean offspring Weights

Group

 

Offspring Body Weight Change (g) – Days 1-4

Males

Females

Control (0 mg/Kg bw/day)

Mean (g)

3.18

3.07

S.D.

0.90

0.82

N

10

11

 

100 mg/Kg bw/day

Mean (g)

3.07

3.02

S.D.

0.67

0.61

N

11

11

 

300 mg/Kg bw/day

Mean (g)

2.60

2.54

S.D.

0.62

0.48

N

12

12

 

1000 mg/Kg bw/day

Mean (g)

2.30*

2.42

S.D.

0.85

0.78

N

10

9

* Significantly different from control group p<0.05

 

Table 16. Group Mean Implantation Losses and Survival Indices Values

Group

 

Pre-Implantation

Loss (%)

Post-Implantation

Loss (%)

Live Birth

Index (%)

Viability

Index (%)

Control

(0 mg/Kg bw/day)

Mean

13.9

8.5

100.0

98.9

S.D.

13.7

9.2

0.0

3.8

N

11

11

11

11

 

100 mg/Kg bw/day

Mean

7.9

12.6

100.0

99.2

S.D.

16.2

15.3

0.0

2.5

N

11

11

11

11

 

300 mg/Kg bw/day

Mean

3.4

5.4

100.0

99.4

S.D.

4.9

6.0

0.0

1.9

N

12

12

12

12

 

1000 mg/Kg bw/day

Mean

12.2

12.1

97.1

91.9*

S.D.

18.6

8.2

7.2

9.5

N

11

11

11

10

* Significantly different from control group p<0.05

Conclusions:
Oral administration of Nonene, branched to rats for 8 weeks, at levels of 100, 300 and 1000 mgk/kg/day, resulted in treatment related effects in both sexes and at all treatment levels. The effects seen were however either adaptive in nature or were not considered toxicologically relevant for man. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day based on reduced offspring viability, offspring body weight gain, litter size and litter weigh at 1000 mg/kg bw/day.
Executive summary:

In a key Guideline (OECD 422) combined repeated dose reproductive/developmental toxicity study, the test material (Nonene, branched; CAS# 97280-95-0) was administered by gavage to three groups of Wistar Han™:RccHan™:WIST strain rats (12/sex/dose) 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 mg/Kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

 

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one 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 five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post-partum. Hematology and blood chemistry were evaluated prior to termination on five 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 5 post-partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. One female which did not show positive evidence of mating and did not produce a pregnancy was terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

No test material related mortality was observed through the study period. Clinical signs were detected in animals of either sex treated with 300 and 1000 mg/Kg bw/day during the study. Episodes of increased salivation were reported from Day 8 onwards. The physical condition of males treated with 1000 mg/Kg bw/day was also affected with reductions in body weight development during Weeks 1 and 3 of treatment. Subsequently, a reduction in overall body weight gain in these males and a slight reduction in food efficiency during week 1, was evident. A reduction in haemoglobin was also observed in these males. Water consumption was also significantly increased for animals of either sex from all treatment groups throughout the treatment period. Observations of this nature are often reported when a test material formulation is unpalatable or irritant and can be associated with gastric irritancy rather than be attributable to systemic toxicity. This was supported microscopically with stomach changes identified as epithelial hyperplasia in the forestomach in animals of either sex treated with 1000 mg/Kg bw/day and in males treated with 300 mg/Kg bw/day. This finding was considered to be the result of local irritancy of the test material and therefore cannot be considered indicative of true systemic toxicity.

 

Although there were some statistically significant differences in treated animals from controls for the blood chemical parameters measured, these differences were considered not to be of toxicological significance. Macroscopic findings detected at necropsy were confined to enlarged, pale and mottled kidneys and a dark liver in a number of males treated with 1000 mg/Kg bw/day. One male treated with 300 mg/Kg bw/day also had enlarged kidneys.

 

Histopathological examination of the liver revealed hepatocellular hypertrophy in animals of either sex treated with 1000 and 300 mg/Kg bw/day and in males treated with 100 mg/Kg bw/day. Organ weight data supported this finding with increased absolute and relative liver weights observed in these animals. In the absence of any degenerative or inflammatory changes, this condition is considered to be adaptive in nature.

 

Microscopic examination of the thyroid revealed increased incidence and severity of follicular cell hypertrophy/hyperplasia in animals of either sex treated with 1000 and 300 mg/Kg bw/day and in males treated with 100 mg/Kg bw/day. Males treated with 1000 and 300 mg/Kg bw/day also showed hypertrophic/vacuolated cells in the pituitary. The thyroid, liver and pituitary changes are characteristic of a consequence of hepatocellular induction as a result of enhanced hepatic metabolism. As a side effect of hepatic induction an increased liver metabolism of thyroid hormones T3 and T4 can occur. This subsequently leads to an enhanced thyroid gland production of these hormones as a consequence of a negative feedback stimulation of TSH production. The appearance of thyroid follicular cell hypertrophy and hypertrophic/vacuolated cells in the pituitary are themselves considered to be a result of this process. The thyroid and pituitary changes were considered to be adaptive in nature.

 

Microscopic examination also revealed effects in the kidneys of males from all treatment groups. Tubular basophilia and hyaline droplets was present in males from all treated groups. These tubular findings were also accompanied by tubular degeneration/debris in males treated with 1000 and 300 mg/Kg bw/day. The hyaline droplets can be directly linked to accumulation of alpha 2u-globulin, which is unique to the male rat. This finding is commonly observed in male rats following treatment with some hydrocarbons and is not predictive of any adverse effect in humans. The remaining kidney findings consisting of tubular degeneration/debris may be considered to represent an adverse effect of the test item, however immunohistochemical staining demonstrated that these findings were correlated to the same condition as hyaline droplets.

 

Mating performance and fertility was unaffected by treatment. Offspring viability was however reduced in litters from females treated with 1000 mg/Kg bw/day on Day 4 post-partum. Subsequently reduced litter size and litter weights were evident in these litters on Day 4 post-partum when compared to controls. One female treated with 1000 mg/Kg bw/day also had a total litter loss between Days 2 and 4 post-partum. The mean offspring body weight gains for litters which survived to Day 5 post-partum were also reduced between Days 1 and 4 post-partum at this treatment level.

 

Based on the results observed, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 1000 mg/Kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/Kg bw/day, based on reduced offspring viability, offspring body weight gain, litter size and litter weights on Day 4 post-partum at 1000 mg/Kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear colorless liquid
Details on test material:
- Name of test material: Nonene, branched
- CAS No.:97280-95-0
- Substance type: UVCB
- Physical state: Clear colorless liquid
- Analytical purity: 100%
- Lot/batch No.: TQ-12-2003-23042013
- Data received: 29 April 2013
- Label: Nonene Sampling Date Apr 22.2013
- Expiration date of the lot/batch: 23 April2014
Specific details on test material used for the study:
- Name of test material: Nonene, branched
- CAS No.:97280-95-0
- Substance type: UVCB
- Physical state: Clear colorless liquid
- Analytical purity: 100%
- Lot/batch No.: TQ-12-2003-23042013
- Data received: 29 April 2013
- Label: Nonene Sampling Date Apr 22.2013
- Expiration date of the lot/batch: 23 April2014

Nonene, branched, CAS# 97280-95-0 used in this study was a typical production sample, according to the details included in the boundary composition in IUCLID section 1.2. Nonene, branched was chosen in the Higher Olefins category testing strategy because it represents a substance with high tri-sub content (category range 1 – 65%), odd carbon number (C9 odd 90%). Please see the testing strategy attached in section 13 for further details.

Test animals

Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 12 weeks old
- Weight at study initiation: (P) Males: 311 to 375 g; Females: 194 to 222 g.
- Housing: Pre-mating: groups of four animals were housed in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Pairing phase: polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Post 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.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.)
- Water (e.g. ad libitum): free access to food and water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-2°C
- Humidity (%): 55 =/- 15%
- Air changes (per hr): at least 15 air changes per hour. The low intensity fluorescent lighting was controlled to give twelve hours continuous light
- Photoperiod (hrs dark / hrs light): 12 hr dark and 12 hr light

The study was performed between 10 June 2013 and 10 June 2014. The in-life phase of the study was conducted between 22 August 2013 (first day of treatment) and 17 October 2013 (final day of necropsy).



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Arachis oil BP
- Storage temperature of food: 4 C

VEHICLE
- Amount of vehicle (if gavage): 4 ml/kg
- Concentration in vehicle:
- Control: 0 ml/kg
- Low: 25 mg/ml
- Intermediate: 75 mg/ml
- High: 250 mg/ml
- Lot/batch no. (if required): 2018C-020713MA
- The volume of the test and control item administrated to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation:15 days
- Proof of pregnancy referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually during gestation and lactation in solid floor polypropylene cages.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
The test item was administrated daily by gavage using a stainless cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Arachis oil BP.
Details on study schedule:
i. Groups of twelve male and twelve female animals were treated 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/behavioral 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 the pairing phase (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. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded 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 hematological 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 hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Doses / Concentrations:

Basis: actual ingested 1000 mg/kg bw/day
No. of animals per sex per dose:
Three groups, each of twelve males and twelve females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selected during the Range-finding experiment - Study Number 41301414

Examinations

Parental animals: Observations and examinations:
Clinical Observations:
Signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour after dosing throughout the treatment period.

Functional Observations:
All animals were observed for signs of functional/behavioural toxicity prior to the start of treatment and at weekly intervals thereafter.
Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- behavioural assessment
- functional Performance Tests
- Sensory Reactivity

Body Weight:
Individual body weights: Day 1 (prior to dosing), weekly for surviving males until termination and weekly for females until mating was evident.
Female: Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 post partum and terminal kill.

Food Consumption:
Pre-pairing period and after (male): weekly
Females showing evidence of mating: post coitum Days 0-7, 7-14 and 14-20.
Females with live litters: Days 1 and 4 post partum.

Food efficiency:
Males (throughout the study period-with the exception of the mating phase, and females during the pre-pairing phase

Water Consumption:
Daily- visual inspection of the water bottles

Reproductive Performance:
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).

Pregnancy and Parturition:
Pregnant females: observations at approximately 0830, 1230 and 1630 hours and around the period of expected parturition (08.30 and 12.30 hr at weekends and public holidays)

Female data collections:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Laboratory Investigations:
- Haematology
- Blood Chemistry
Litter observations:
Number of live and dead offspring
For each litter the following was recorded:
i. Number of offspring born
ii.Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. 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):
Pathology Females: Uterus (for signs of implantation and the number of uterine implantations in each horn), the corpora lutea. All adult animals and offspring: full external and internal examination. All macroscopic abnormalities were recorded.
- Organ Weights- Histopathology: Tissues from 5 selected male and female animals/group were preserved. Histopathological examination was undertaken on tissues from control and high dost animals.
To clarify possible treatment-related changes, histopathological examination was extended to include similarly prepared sections of the liver, kidneys (males only), stomach, thyroid and pituitary from animals in the low and intermediate groups. In addition, male kidney tissue was subject to immunohistochemical examination to confirm the presence of alph-2-microglobulin.
Postmortem examinations (offspring):
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.
Statistics:
Please see "Any other information on materials and methods incl. tables" for information on statistics.
Reproductive indices:
- Pre-coital Interval
- Fertility Indices
- Gestation length
- Parturition Index

Offspring viability indices:
- Implantation Losses (%)
- Live Birth and Viability Indices
- Sex Ratio (% males)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 and 300 mg/kg bw/day: Increasing salivation in males and females. No effects in animals of either sex treated with 100 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3 of treatment. Overall body weight gain was reduced for these males.

No such effects were detected in treated females or in males treated with 300 or 100 mg/kg bw/day.

Females treated with 300 mg/kg bw/day showed a statistically significant increase in cumulative body weight gain during the final week of gestation. An increase in body weight gain is considered not to represent an adverse effect oftreatment therefore the intergroup difference was considered not to be of toxicological importance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No adverse effect on food consumption was detected in treated animals when compared to control animals.

Females treated with 1000 mg/kg bw/day showed a statistically significant increase in food consumption during the second week of gestation. An increase in food consumption is considered not to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.
Food efficiency:
no effects observed
Description (incidence and severity):
No adverse effect on food efficiency was detected in treated animals when compared to control animals.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Daily measurements of water bottles revealed a marked increase in water consumption in males treated with 1000 mg/kg bw/day and a slight increase in males treated with 300 or 100 mg/kg bw/day throughout the treatment period. Females from all treatment groups also showed an increase in water consumption throughout the treatment period with statistical significance being achieved throughout gestation and lactation at 1000 mg/kg bw/day.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed a reduction in hemoglobin. Three out of the five individual values were outside of the normal background range for this parameter.

No toxicologically significant effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a reduction in mean corpuscular hemoglobin. Males treated with 1000 and 300 mg/kg bw/day showed a reduction in mean corpuscular hemoglobin concentration. Males from all treatment groups also showed a reduction in
prothrombin time. All of the individual values were within the normal background range for these parameters, therefore the intergroup differences were considered not to be of toxicological importance. Females from all treatment groups showed a statistically significant increase in neutrophil count. The majority of individual values were within the normal background range for these parameters and a true dose related response was not evident, therefore the intergroup differences were considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically significant effects were detected in the blood chemical parameters examined.

Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in calcium concentration and a statistically significant reduction in alkaline phosphatase. Males treated with 1000 mg/kg bw/day also showed a reduction in cholesterol. All of the individual values were within the normal ranges for rats ofthe strain and age used and in the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological importance.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Behavioral Assessments
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.

Functional Performance Tests
There were no toxicologically significant changes in functional performance. Males from all treatment groups showed a statistically significant increase in fore limb grip strength. The statistically significant difference was confined to one out of the three tests and in the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments
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):
Animals of either sex treated with 1000 and 300 mg/kg bw/day and females treated with 100 mg/kg bw/day showed an increase in absolute and relative liver weight. Males treated with 1000 mg/kg bw/day also showed an increase in kidney weight both absolute and relative to terminal body weight. Males from all treatment groups showed an increase in absolute and relative thyroid weight whilst females treated with 1000 mg/kg bw/day showed a reduction in absolute and relative thyroid weight. All of the individual values were within the normal range for rats of the strain and age used and the effect on male thyroid weight also did not show a true dose related response. However with the associated microscopic thyroid changes observed in this study the intergroup differences detected in thyroid weights can not be excluded as an effect of treatment.

Females treated with 1000 mg/kg bw/day showed a reduction in pituitary weight and an increase in kidney weight both absolute and relative to terminal body weight. In the absence of any associated histopathological changes detected in the kidneys or pituitary of females the intergroup differences were considered not to be of toxicological importance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Nine males treated with 1000 mg/kg bw/day had enlarged kidneys at necropsy, eight of which had pale kidneys; four showed a mottled appearance to the kidneys and five of these males also had a dark liver at necropsy. One male treated with 300 mg/kg bw/day had enlarged kidneys at necropsy.

No toxicologically significant effects were detected in females from any treatment group or in males treated with 100 mg/kg bw/day.

One male treated with 100 mg/kg bw/day had small testes and epididymides. One female treated with 300 mg/kg bw/day and two females treated with 100 mg/kg bw/day had reddened lungs at necropsy. Histopathological examination of these tissues did reveal associated microscopic findings to the macroscopic abnormalities however in the absence of a similar effect seen at 1000 mg/kg bw/day, the intergroup differences were considered to be incidental and of no toxicological importance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment related microscopic findings were detected:

Liver:hepatocyte hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Kidneys:proximal tubular basophilia and hyaline droplets were evident in males from all treatment groups. Tubular degeneration/debris was evident in males treated with 1000 and 300 mg/kg bw/day. These findings were demonstrated by immunohistochemical staining to be
due to alpha-2-microglobulin nephropathy. No such effects were detected in any treated female.

Stomach:epithelial hyperplasia was evident in the fore stomach of animals of either sex treated with 1000 mg/kg bw/day and in males treated with 300 mg/kg bw/day. No such effects were detected in females treated with 300 mg/kg bw/day or animals of either sex treated with 100 mg/kg bw/day.

Thyroid:follicular cell hypertrophy and hyperplasia was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Pituitary:increased incidence and severity of hypertrophic/vacuolated cells in pars anterior was evident in males treated with 1000 and 300 mg/kg bw/day. No such effects were detected in any treated female or in males treated with 100 mg/kg bw/day.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating
There were no treatment-related effects on mating performance.

Fertility
No treatment-related effects on fertility were detected for treated animals, when compared to controls.

One control female and one female treated 100 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female or male reproductive organs which could have been the cause of the infertility. One female treated with 1000 mg/kg bw/day failed to show any positive signs of mating and was non pregnant.

Gestation Length
There were no differences in gestation lengths. The distribution for treated females was comparable to controls. All animals showed gestation lengths of 22 to 24½ days.

Details on results (P0)

Clinical Signs:
1000 and 300 mg/kg bw/day: Increasing salivation in males and females. No effects in animals of either sex treated with 100 mg/kg bw/day.

Body Weight and Weight Changes:
Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3 of treatment. Overall body weight gain was reduced for these males.

No such effects were detected in treated females or in males treated with 300 or 100 mg/kg bw/day.

Females treated with 300 mg/kg bw/day showed a statistically significant increase in cumulative body weight gain during the final week of gestation. An increase in body weight gain is considered not to represent an adverse effect oftreatment therefore the intergroup difference was considered not to be of toxicological importance.

Food consumption and Compound Intake:
No adverse effect on food consumption was detected in treated animals when compared to control animals.

Females treated with 1000 mg/kg bw/day showed a statistically significant increase in food consumption during the second week of gestation. An increase in food consumption is considered not to represent an adverse effect of treatment therefore the intergroup difference was considered not to be of toxicological importance.

Water Consumption:
Daily measurements of water bottles revealed a marked increase in water consumption in males treated with 1000 mg/kg bw/day and a slight increase in males treated with 300 or 100 mg/kg bw/day throughout the treatment period. Females from all treatment groups also showed an increase in water consumption throughout the treatment period with statistical significance being achieved throughout gestation and lactation at 1000 mg/kg bw/day.

Hematology:
Males treated with 1000 mg/kg bw/day showed a reduction in hemoglobin. Three out of the five individual values were outside of the normal background range for this parameter.

No toxicologically significant effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 100 mg/kg bw/day.

Males treated with 1000 mg/kg bw/day showed a reduction in mean corpuscular hemoglobin. Males treated with 1000 and 300 mg/kg bw/day showed a reduction in mean corpuscular hemoglobin concentration. Males from all treatment groups also showed a reduction in
prothrombin time. All of the individual values were within the normal background range for these parameters, therefore the intergroup differences were considered not to be of toxicological importance. Females from all treatment groups showed a statistically significant increase in neutrophil count. The majority of individual values were within the normal background range for these parameters and a true dose related response was not evident, therefore the intergroup differences were considered not to be of toxicological significance.

Clinical Biochemistry:
No toxicologically significant effects were detected in the blood chemical parameters examined.

Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in calcium concentration and a statistically significant reduction in alkaline phosphatase. Males treated with 1000 mg/kg bw/day also showed a reduction in cholesterol. All of the individual values were within the normal ranges for rats ofthe strain and age used and in the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological importance.

Behaviour:
Functional Performance Tests - There were no toxicologically significant changes in functional performance. Males from all treatment groups showed a statistically significant increase in fore limb grip strength. The statistically significant difference was confined to one out of the three tests and in the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Organ Weight:
Animals of either sex treated with 1000 and 300 mg/kg bw/day and females treated with 100 mg/kg bw/day showed an increase in absolute and relative liver weight. Males treated with 1000 mg/kg bw/day also showed an increase in kidney weight both absolute and relative to terminal body weight. Males from all treatment groups showed an increase in absolute and relative thyroid weight whilst females treated with 1000 mg/kg bw/day showed a reduction in absolute and relative thyroid weight. All of the individual values were within the normal range for rats of the strain and age used and the effect on male thyroid weight also did not show a true dose related response. However with the associated microscopic thyroid changes observed in this study the intergroup differences detected in thyroid weights can not be excluded as an effect of treatment.

Females treated with 1000 mg/kg bw/day showed a reduction in pituitary weight and an increase in kidney weight both absolute and relative to terminal body weight. In the absence of any associated histopathological changes detected in the kidneys or pituitary of females the intergroup differences were considered not to be of toxicological importance.

Necropsy:
Nine males treated with 1000 mg/kg bw/day had enlarged kidneys at necropsy, eight of which had pale kidneys; four showed a mottled appearance to the kidneys and five of these males also had a dark liver at necropsy. One male treated with 300 mg/kg bw/day had enlarged kidneys at necropsy.

No toxicologically significant effects were detected in females from any treatment group or in males treated with 100 mg/kg bw/day.

One male treated with 100 mg/kg bw/day had small testes and epididymides. One female treated with 300 mg/kg bw/day and two females treated with 100 mg/kg bw/day had reddened lungs at necropsy. Histopathological examination of these tissues did reveal associated microscopic findings to the macroscopic abnormalities however in the absence of a similar effect seen at 1000 mg/kg bw/day, the intergroup differences were considered to be incidental and of no toxicological importance.

Histopathology:
Liver:hepatocyte hypertrophy was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Kidneys:proximal tubular basophilia and hyaline droplets were evident in males from all treatment groups. Tubular degeneration/debris was evident in males treated with 1000 and 300 mg/kg bw/day. These findings were demonstrated by immunohistochemical staining to be
due to alpha-2-microglobulin nephropathy. No such effects were detected in any treated female.

Stomach:epithelial hyperplasia was evident in the fore stomach of animals of either sex treated with 1000 mg/kg bw/day and in males treated with 300 mg/kg bw/day. No such effects were detected in females treated with 300 mg/kg bw/day or animals of either sex treated with 100 mg/kg bw/day.

Thyroid:follicular cell hypertrophy and hyperplasia was evident in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day. No such effects were detected in females treated with 100 mg/kg bw/day.

Pituitary:increased incidence and severity of hypertrophic/vacuolated cells in pars anterior was evident in males treated with 1000 and 300 mg/kg bw/day. No such effects were detected in any treated female or in males treated with 100 mg/kg bw/day.

Reproductive Performance:
Fertility - No treatment-related effects on fertility were detected for treated animals, when compared to controls.

One control female and one female treated 100 mg/kg bw/day did not achieve pregnancy following evidence of mating. No histopathological correlates were evident in the female or male reproductive organs which could have been the cause of the infertility. One female treated with 1000 mg/kg bw/day failed to show any positive signs of mating and was non pregnant.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive Toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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, cold, weak, pale, no milk in stomach, physical injury, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Litter weights on Day 4 post partum from females treated with 1000 mg/kg bw/day were reduced when compared to control litters however statistical significance was not achieved. Offspring body weight gain at 1000 mg/kg bw/day was also reduced between Days 1 and 4 post partum. Statistical significance was achieved for male off spring only. No such effects were detected in litters from females treated with 300 or 100 mg/kg bw/day.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related macroscopic abnormalities were detected for interim death or terminal kill offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Statistical analysis of surface righting reflex data did not reveal any significant intergroup differences.

Details on results (F1)

Offspring Litter Size, Sex Ratio and Viability
No significant differences were detected for corpora lutea, implantation counts or implantation losses for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Of the litters born, litter size at birth and subsequently on Day 1 post partumwere comparable to controls. Litter size on Day 4 post partumhowever was reduced in litters from females treated with 1000 mg/kg bw/day although statistical significance was not achieved. Offspring viability on Day 4 post partumwas statistically significantly reduced in these litters. A total litter loss was also observed for one female treated with 1000 mg/kg bw/day. No such effects were detected in litters from females treated with 300 or 100 mg/kg bw/day.

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
Litter weights on Day 4 post partumfrom females treated with 1000 mg/kg bw/day were reduced when compared to control litters however statistical significance was not achieved. Offspring body weight gain at 1000 mg/kg bw/day was also reduced between Days 1 and 4 post partum. Statistical significance was achieved for male offspring only.

No such effects were detected in litters from females treated with 300 or 100 mg/kg bw/day. Statistical analysis of surface righting reflex 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, cold, weak, pale, no milk in stomach, physical injury, found dead or missing, were considered to be low incidence findings observed in offspring in studies of this type and were considered unrelated to test item toxicity.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reduced post natal offspring viability, offspring body weight gain and litter size at 1000 mg/kg bw/day.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Table 2. Summary of Reproductive Performance - Groups Values

 

Dose Group (mg/Kg bw/day

0 (Control)

100

300

1000

Males

 

Initial group size

12

12

12

12

Paired

12

12

12

12

Failed to mate

0

0

0

1

Failed to induce pregnancy in female partner

1

1

0

0

Induced pregnancy in female partner

11

11

12

11

Surviving to terminal necropsy

12

12

12

12

 

Females

 

Initial group size

12

12

12

12

Paired

12

12

12

12

Failed to mate

0

0

0

1

Non-pregnant

1

1

0

0

Rearing young to Day 5 of age

11

11

12

10

Total Litter Loss

0

0

0

1

 

Table 3. Group Mean Functional Test Values and Standard Deviations - Males

Group

(sex)

 

Test 1 Forelimb

(g)

Test 1 Hindlimb

(g)

Test 2 Forelimb

(g)

Test 2 Hindlimb

(g)

Test 3 Forelimb

(g)

Test 3 Hindlimb

(g)

Overall

Activity

Overall Mobile

Last 20%

Activity

Last 20% Mobile

1 (M)

Mean

1143.2

422.6

1004.2

524.4

985.2

445.0

377.2

0.6

3.4

0.0n

S.D.

234.7

131.5

113.9

66.2

91.6

76.7

122.9

0.9

4.0

0.0

N

5

5

5

5

5

5

5

5

5

5

 

2 (M)

Mean

1176.8

510.2

1303.0*

581.4

1084.8

628.2

384.0

0.6

8.2

0.0n

S.D.

237.0

140.4

313.6

105.2

146.7

157.4

214.5

0.5

9.5

0.0

N

5

5

5

5

5

5

5

5

5

5

 

3 (M)

Mean

1353.0

593.8

1450.4*

472.0

1378.2

528.2

334.6

0.2

4.2

0.0n

S.D.

297.1

121.5

200.0

147.6

442.9

177.5

141.7

0.4

5.2

0.0

N

5

5

5

5

5

5

5

5

5

5

 

4 (M)

Mean

1071.2

620.8

1271.0*

473.4

1118.6

555.0

250.8

0.2

36.2

0.0n

S.D.

257.2

184.4

250.8

230.9

185.2

151.8

118.6

0.4

58.3

0.0

N

5

5

5

5

5

5

5

5

5

5

General Footnote: Unit = Time (seconds) for Motor Activity Assessments

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

* Significantly different from control group p<0.05

n Data not appropriate for statistical analysis

 

Table 4. Group Mean Body Weight Values - Males

 

Group

(sex)

 

Increase in Body Weight (g)

 

Day Numbers Relative to Start Date

Abs Gain

% Gain

From:

1

8

15

22

29

36

1

1

To:

8

15

22

29

36

43

43

43

1 (M)

Mean

14.4

13.0

9.7

14.6

10.2

6.8

68.6

20.9

S.D.

5,8

6.5

6.6

6.0

4.5

3.7

21.7

5.9

N

12

12

12

12

12

12

12

12

 

 

 

2 (M)

Mean

13.9

12.5

6.9

13.3

12.0

4.3

63.0

19.0

S.D.

3.8

3.9

6.3

4.4

3.4

5.1

16.9

4.8

N

12

12

12

12

12

12

12

12

 

 

 

3 (M)

Mean

13.4

15.3

7.3

14.1

11.9

8.1

70.2

21.3

S.D.

5.5

5.0

3.9

4.6

5.6

3.4

18.8

5.3

N

12

12

12

12

12

12

12

12

 

 

 

4 (M)

Mean

5.5**

10.0

1.7**

12.4

8.1

5.1

42.8

13.0

S.D.

9.8

7.5

6.8

3.8

4.8

4.8

18.0

5.3

N

12

12

12

12

12

12

12

12

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

** Significantly different from control group p<0.01

 

Table 5. Group Mean Body Weight Gains - Females

 

Group

(sex)

 

Increase in Body Weight (g)

Cumulative Body Weight Change (g)

 

Days

Days

 

Gestation

Lactation

Gestation

From:

0

7

14

1

0

0

To:

7

14

20

4

14

20

1 (F)

Mean

29.8

28.9

60.4

7.9

58.7

119.1

S.D.

4.4

3.9

13.0

5.4

7.3

16.3

N

11

11

11

11

11

11

 

2 (F)

Mean

27.4

28.3

60.8

11.8

55.6

116.5

S.D.

4.8

5.0

12.5

10.3

6.1

14.9

N

11

11

11

11

11

11

 

3 (F)

Mean

33.3

33.1

69.1

12.8

66.4

135.5*

S.D.

5.2

5.2

10.9

7.1

7.8

15.2

N

12

12

12

12

12

12

 

4 (F)

Mean

29.8

27.9

57.3

6.4

57.7

115.0

S.D.

6.8

6.5

11.7

12.2

11.4

17.1

N

11

11

11

10

11

11

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

* Significantly different from control group p<0.05

 

Table 6. Group Mean Food Consumptions - Females

Group

(sex)

 

Day Numbers

Gestation

Lactation

From

0

7

14

1

To

7

14

20

4

1 (F)

Mean

19.7

22.3

25.1

26.5

S.D.

1.8

1.9

2.4

4.6

N

11

11

11

11

 

2 (F)

Mean

20.2

22.5

24.6

29.5

S.D.

2.0

2.3

1.2

8.3

N

11

11

11

11

 

3 (F)

Mean

20.7

23.6

25.5

28.2

S.D.

1.2

1.2

1.1

3.3

N

12

12

12

12

 

4 (F)

Mean

20.9

24.2*

26.8

25.4

S.D.

1.6

2.2

2.3

6.2

N

11

11

11

10

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

* Significantly different from control group p<0.05

 

Table 7. Group Mean Water Consumption - Females

Group

(sex)

 

Day Numbers

Gestation

Lactation

From

0

7

14

1

To

7

14

20

4

1 (F)

Mean

27.6

31.0

36.0

39.9

S.D.

3.8

5.1

6.8

4.3

N

11

11

11

11

 

2 (F)

Mean

32.1

36.9

39.9

44.2

S.D.

5.3

5.9

4.8

10.6

N

11

11

11

11

 

3 (F)

Mean

36.5

42.9

44.8

45.8

S.D.

2.2

13.9

10.7

7.1

N

12

12

12

12

 

4 (F)

Mean

52.6***

67.8***

76.4***

54.1**

S.D.

20.4

22.5

34.1

12.5

N

11

11

11

11

Dose Levels: Group 1 - 0(Control); Group 2 - 100 mg/kg bw/day; Group 3 - 300 mg/kg bw/day; Group 4 - 1000 mg/kg bw/day

** Significantly different from control group p<0.01

*** Significantly different from control group p<0.001

 

Table 8. Group Mean Hematological Values - Males

Group

 

Hb (g/dL)

MCH (pg)

MCHC (g/dL)

CT (seconds)

Group 1 (0 – Control)

Mean

16.92

18.86

35.66

9.92

S.D.

0.44

0.44

1.49

0.47

N

5

5

5

5

 

Group 2 (100 mg/Kg bw/day)

Mean

16.86

18.64

35.18

9.40*

S.D.

0.74

0.11

0.67

0.23

N

5

5

5

5

 

Group 3 (300 mg/Kg bw/day)

Mean

16.08

18.36

34.12**

9.34*

S.D.

0.31

0.48

0.53

0.26

N

5

5

5

5

 

Group 4 (1000 mg/Kg bw/day)

Mean

14.94**

17.58**

33.72**

9.34*

S.D.

1.56

0.63

0.28

0.36

N

5

5

5

5

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 9. Group Mean Hematological Values - Females

Group

 

Neut (109/L)

Group 1 (0 – Control)

Mean

1.360

S.D.

0.417

N

5

 

Group 2 (100 mg/Kg bw/day)

Mean

2.500*

S.D.

0.568

N

5

 

Group 3 (300 mg/Kg bw/day)

Mean

2.146*

S.D.

0.463

N

5

 

Group 4 (1000 mg/Kg bw/day)

Mean

1.872*

S.D.

0.509

N

5

* Significantly different from control group p<0.05

 

Table 10. Group Mean Blood Chemical Values - Males

Group

 

Ca++(mmol/L)

AP (IU/L)

Chol (mg/dL)

Group 1 (0 – Control)

Mean

2.448

234.0

95.2

S.D.

0.258

52.1

5.3

N

5

5

5

 

Group 2 (100 mg/Kg bw/day)

Mean

2.522

201.6

102.6

S.D.

0.146

26.1

14.4

N

5

5

5

 

Group 3 (300 mg/Kg bw/day)

Mean

2.730*

150.6**

99.4

S.D.

0.077

10.8

10.7

N

5

5

5

 

Group 4 (1000 mg/Kg bw/day)

Mean

2.644*

157.6**

78.0*

S.D.

0.129

55.2

13.3

N

5

5

5

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 11. Summary Incidence of Necropsy Findings - Males

 

Males

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Number of animals examined

12

12

12

12

Epididymides

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

11

12

12

Small; Left

0

1

0

0

 

Kidneys

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

12

11

3

Enlarged

0

0

1

9

Mottled Appearance

0

0

0

4

Pallor

0

0

0

3

Pale

0

0

0

6

 

Liver

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

12

12

7

Dark

0

0

0

5

 

Testes

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

11

12

12

Small; Left

0

1

0

0

 

Table 12. Summary Incidence of Necropsy Findings - Females

 

Females

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Number of animals examined

12

12

12

12

Liver

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

12

12

11

Dark

0

0

0

1

 

Lungs (With Bronchi)

 

Submitted

(12)

(12)

(12)

(12)

No Visible Lesions

12

10

11

12

Reddened

0

2

1

0

  

Table 13. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

 

 

Males

Females

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

0

Control

100

mg/Kg bw/day

300

mg/Kg bw/day

1000

mg/Kg bw/day

Kidneys

Mean (g)

2.29348

2.31240

2.38234

3.16702**

1.54460

1.65510

1.66136

1.97002**

S.D.

0.32697

0.14310

0.16218

0.44018

0.14343

0.11476

0.20712

0.11782

N

5

5

5

5

5

5

5

5

 

Mean (%)

0.574

0.585

0.616

0.830**

0.565

0.610

0.602

0.701**

S.D.

0.046

0.013

0.051

0.102

0.047

0.035

0.055

0.041

N

5

5

5

5

5

5

5

5

 

Liver

Mean (g)

12.7830

13.2647

14.3898**

19.3412**

10.5429

11.7936*

12.7876**

16.1219**

S.D.

1.51792

0.98038

1.33119

1.23327

0.51191

0.52983

1.29755

1.61466

N

5

5

5

5

5

5

5

5

 

Mean (%)

3.203

3.355

3.710**

5.070**

3.857

4.353*

4.638**

5.742**

S.D.

0.125

0.111

0.164

0.230

0.149

0.194

0.424

0.639

N

5

5

5

5

5

5

5

5

 

Pituitary

Mean (g)

 

 

 

 

0.01793

0.01529

0.01783

0.01467**

S.D.

 

 

 

 

0.00277

0.00241

0.00264

0.00180

N

 

 

 

 

11

11

12

10

 

 

Mean (%)

 

 

 

 

0.006

0.006

0.006

0.005**

S.D.

 

 

 

 

0.001

0.001

0.001

0.001

N

 

 

 

 

11

11

12

10

 

Thyroid/Parathyroid

Mean (g)

0.01552

0.02172**

0.02162**

0.02112**

0.002238

0.01972

0.02046

0.01776*

S.D.

0.00377

0.00176

0.00140

0.00207

0.00395

0.00339

0.00202

0.00259

N

5

5

5

5

5

5

5

5

 

Mean (%)

0.004

0.005**

0.006**

0.006**

0.008

0.007

0.007

0.006*

S.D.

0.001

0.000

0.000

0.000

0.002

0.001

0.001

0.001

N

5

5

5

5

5

5

5

5

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

 

Table 14. Histopathology – Treatment-related Findings

 

Male

Female

Dosage

(mg/Kg bw/day)

0

100

300

1000

0

100

300

1000

 

Liver

Number Examined

5

5

5

9

5

5

5

6

- Hypertrophy; hepatoc.

0

5 (1.0)

5 (1.0)

9 (1.7)

0

0

3 (1.0)

6 (1.0)

- Congestion

0

0

0

5 (1.0)

0

0

0

1(3.0)

 

Thyroid

Number Examined

5

5

5

5

5

5

5

5

- Hypertrophy / Hyperplasia;

Follicular Cell

2 (1.0)

5 (1.4)

5 (1.4)

5 (1.8)

2 (1.0)

2 (1.0)

3 (1.3)

5 (1.2)

 

Pituitary Gland

Number Examined

12

12

12

12

12

12

12

10

-Hypertrophic / Vacuolated cells in

Pars Anterior

10 (1.0)

9 (1.2)

12 (1.4)

9 (1.6)

0

0

3 (1.0)

0

 

Kidney

Number Examined

5

5

5

9

5

5

5

5

- Tubular Basophilia

0

3 (1.0)

4 (1.3)

9 (2.0)

0

0

0

1 (1.0)

- Tubular Degeneration

/ Debris

0

0

4 (1.0)

7 (2.0)

0

0

0

0

- Hyaline Droplets

0

5 (1.0)

5 (1.8)

9 (2.1)

0

0

0

0

 

Kidney Immunohistochemistry

Number Examined

3

3

3

3

0

0

0

0

- alpha-2-microglobulin

3 (1.0)

3 (2.0)

3 (2.3)

3 (2.7)

0

0

0

0

 

Stomach - Forestomach (microscopic changes recorded in the stomach)

Number Examined

5

5

5

5

5

5

5

5

- Hyperplasia, epithelial

0

0

2 (1.0)

2 (1.0)

0

0

0

3 (1.0)

(): Mean Severity

Findings in Bold were considered treatment-related

Table 15. Group Mean offspring Weights

Group

 

Offspring Body Weight Change (g) – Days 1-4

Males

Females

Control (0 mg/Kg bw/day)

Mean (g)

3.18

3.07

S.D.

0.90

0.82

N

10

11

 

100 mg/Kg bw/day

Mean (g)

3.07

3.02

S.D.

0.67

0.61

N

11

11

 

300 mg/Kg bw/day

Mean (g)

2.60

2.54

S.D.

0.62

0.48

N

12

12

 

1000 mg/Kg bw/day

Mean (g)

2.30*

2.42

S.D.

0.85

0.78

N

10

9

* Significantly different from control group p<0.05

 

Table 16. Group Mean Implantation Losses and Survival Indices Values

Group

 

Pre-Implantation

Loss (%)

Post-Implantation

Loss (%)

Live Birth

Index (%)

Viability

Index (%)

Control

(0 mg/Kg bw/day)

Mean

13.9

8.5

100.0

98.9

S.D.

13.7

9.2

0.0

3.8

N

11

11

11

11

 

100 mg/Kg bw/day

Mean

7.9

12.6

100.0

99.2

S.D.

16.2

15.3

0.0

2.5

N

11

11

11

11

 

300 mg/Kg bw/day

Mean

3.4

5.4

100.0

99.4

S.D.

4.9

6.0

0.0

1.9

N

12

12

12

12

 

1000 mg/Kg bw/day

Mean

12.2

12.1

97.1

91.9*

S.D.

18.6

8.2

7.2

9.5

N

11

11

11

10

* Significantly different from control group p<0.05

Applicant's summary and conclusion

Conclusions:
Oral administration of Nonene, branched to rats for 8 weeks, at levels of 100, 300 and 1000 mgk/kg/day, resulted in treatment related effects in both sexes and at all treatment levels. The effects seen were however either adaptive in nature or were not considered toxicologically relevant for man. The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day based on reduced offspring viability, offspring body weight gain, litter size and litter weigh at 1000 mg/kg bw/day.
Executive summary:

In a key Guideline (OECD 422) combined repeated dose reproductive/developmental toxicity study, the test material (Nonene, branched; CAS# 97280-95-0) was administered by gavage to three groups of Wistar Han™:RccHan™:WIST strain rats (12/sex/dose) 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 mg/Kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

 

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Pairing of animals within each dose group was undertaken on a one male: one 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 five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post-partum. Hematology and blood chemistry were evaluated prior to termination on five 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 5 post-partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. One female which did not show positive evidence of mating and did not produce a pregnancy was terminated on Day 57. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

No test material related mortality was observed through the study period. Clinical signs were detected in animals of either sex treated with 300 and 1000 mg/Kg bw/day during the study. Episodes of increased salivation were reported from Day 8 onwards. The physical condition of males treated with 1000 mg/Kg bw/day was also affected with reductions in body weight development during Weeks 1 and 3 of treatment. Subsequently, a reduction in overall body weight gain in these males and a slight reduction in food efficiency during week 1, was evident. A reduction in haemoglobin was also observed in these males. Water consumption was also significantly increased for animals of either sex from all treatment groups throughout the treatment period. Observations of this nature are often reported when a test material formulation is unpalatable or irritant and can be associated with gastric irritancy rather than be attributable to systemic toxicity. This was supported microscopically with stomach changes identified as epithelial hyperplasia in the forestomach in animals of either sex treated with 1000 mg/Kg bw/day and in males treated with 300 mg/Kg bw/day. This finding was considered to be the result of local irritancy of the test material and therefore cannot be considered indicative of true systemic toxicity.

 

Although there were some statistically significant differences in treated animals from controls for the blood chemical parameters measured, these differences were considered not to be of toxicological significance. Macroscopic findings detected at necropsy were confined to enlarged, pale and mottled kidneys and a dark liver in a number of males treated with 1000 mg/Kg bw/day. One male treated with 300 mg/Kg bw/day also had enlarged kidneys.

 

Histopathological examination of the liver revealed hepatocellular hypertrophy in animals of either sex treated with 1000 and 300 mg/Kg bw/day and in males treated with 100 mg/Kg bw/day. Organ weight data supported this finding with increased absolute and relative liver weights observed in these animals. In the absence of any degenerative or inflammatory changes, this condition is considered to be adaptive in nature.

 

Microscopic examination of the thyroid revealed increased incidence and severity of follicular cell hypertrophy/hyperplasia in animals of either sex treated with 1000 and 300 mg/Kg bw/day and in males treated with 100 mg/Kg bw/day. Males treated with 1000 and 300 mg/Kg bw/day also showed hypertrophic/vacuolated cells in the pituitary. The thyroid, liver and pituitary changes are characteristic of a consequence of hepatocellular induction as a result of enhanced hepatic metabolism. As a side effect of hepatic induction an increased liver metabolism of thyroid hormones T3 and T4 can occur. This subsequently leads to an enhanced thyroid gland production of these hormones as a consequence of a negative feedback stimulation of TSH production. The appearance of thyroid follicular cell hypertrophy and hypertrophic/vacuolated cells in the pituitary are themselves considered to be a result of this process. The thyroid and pituitary changes were considered to be adaptive in nature.

 

Microscopic examination also revealed effects in the kidneys of males from all treatment groups. Tubular basophilia and hyaline droplets was present in males from all treated groups. These tubular findings were also accompanied by tubular degeneration/debris in males treated with 1000 and 300 mg/Kg bw/day. The hyaline droplets can be directly linked to accumulation of alpha 2u-globulin, which is unique to the male rat. This finding is commonly observed in male rats following treatment with some hydrocarbons and is not predictive of any adverse effect in humans. The remaining kidney findings consisting of tubular degeneration/debris may be considered to represent an adverse effect of the test item, however immunohistochemical staining demonstrated that these findings were correlated to the same condition as hyaline droplets.

 

Mating performance and fertility was unaffected by treatment. Offspring viability was however reduced in litters from females treated with 1000 mg/Kg bw/day on Day 4 post-partum. Subsequently reduced litter size and litter weights were evident in these litters on Day 4 post-partum when compared to controls. One female treated with 1000 mg/Kg bw/day also had a total litter loss between Days 2 and 4 post-partum. The mean offspring body weight gains for litters which survived to Day 5 post-partum were also reduced between Days 1 and 4 post-partum at this treatment level.

 

Based on the results observed, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was determined to be 1000 mg/Kg bw/day. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 300 mg/Kg bw/day, based on reduced offspring viability, offspring body weight gain, litter size and litter weights on Day 4 post-partum at 1000 mg/Kg bw/day.