Fatty acids, tallow, compds. with triethanolamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 June 2016 to 31 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Fatty acids, tallow, compds. with triethanolamine

EC Number:

263-158-1

EC Name:

Fatty acids, tallow, compds. with triethanolamine

Cas Number:

61790-67-8

IUPAC Name:

Fatty acids, tallow, esters of triethanolamine

Test material form:

liquid

Details on test material:

Appearance: Brown liquid
Storage: At ambient temperature, protected from light

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar [RccHan™;WIST] strain was used because of the historical control data available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: The males were 69 to 75 days old and the females 62 to 68 days old.
- Weight at study initiation: Males animals weighed 271 g to 302 g and female animals weighed 170 g to 193 g.
- Housing: Animals were housed in cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups. Solid bottom cages contained softwood bark-free fibre betting, which was changed at appropriate intervals.
- Number of animals per cage
Pre-pairing: Up to five animals of one sex.
Pairing: One male and one female.
Males after mating: Up to five animals.
Gestation: One female.
Lactation: One female plus litter.
- Diet: Ad libitum. The diet contained no added antibiotic or other themotherapeutic or prophylactic agent.
- Water: Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Five days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): 40-70 %.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated. Minimum 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Starting with the lowest concentration, the required amount of test item was weighed into a suitable container. Vehicle was added to the mixture (to achieve approximately 75 % of the final volume) and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous.
A series of suspensions at the required concentrations was prepared by dilution of individual weightings of the test item.
- Frequency of preparation: Weekly.
- Storage of formulation: Refrigerated (nominally 2-8 °C).

VEHICLE
- Justification for use and choice of vehicle: Not specified
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks. Male/female separation occurred the day mating was detected.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in vaginal smear.
- After successful mating each pregnant female was caged: Individually

Pairing commenced after a minimum of two weeks treatment. Day 0 of gestation was taken as when positive evidence of mating was detected. The pre-coital interval was calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures were determined as part of separate study conducted. In this study formulations in the range 2 to 200 mg/mL were shown to be stable for two days under ambient (nominally 21 °C) conditions and 15 days under refrigerated (nominally 2 - 8 °C) conditions.

- Achieved concentration: Samples of each formulation prepared for administration for the first and last formulation occasions were analyzed for achieved concentration of the test item.

ANALYTICAL PROCEDURE
The samples were analyzed in accordance to a validated method. The analytical method involved extraction and dilution in Ethanol followed by liquid chromatographic analysis with mass spectrometric detection (LC-MS/MS). Sample concentrations were determined with reference to single matrix matched bracketing standards. Procedural recovery samples were prepared concurrently with samples and results were corrected for the mean recovery value at analysis.

CONCENTRATION OF DOSE FORMULATIONS
On the First and Last Formulation preparation occasions, samples of the formulations were collected. Formulations (4 × 1 mL, accurately weighed) were sampled from the middle of the formulation. Duplicate samples were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency.
On the First Formulation occasion, contingency samples for Group 2 were analyzed as a precaution as the initial results were showing a coefficient of variation >± 5 % from the mean result. Following re-dilution of the original samples the cause of the high coefficient of variation was shown to be an analytical error in the first sample. Samples were disposed of once satisfactory results were achieved.

RESULTS AND CONCLUSION
No test material was detected in control samples. The mean concentrations of the test material in test formulations were between -4 % to +2.5 % of the nominal concentrations, confirming the accuracy of formulation. Difference from the mean of individual samples (including coefficient of variation for Group 2 on the First Formulation occasion) were <5 %, confirming precise analysis. Procedural recoveries analyzed concurrently with samples were within acceptance criteria, 94.8 % to 100.5 % indicating the continued accuracy of the method.

Duration of treatment / exposure:

- Males: Daily for a minimum of four weeks.
- Females: Daily for 15 days before pairing and until Day 6 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day.

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 12 to 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of the two-week preliminary toxicity study. In the 14-day repeat-dose dose range finding study, doses of 100, 300 and 1000 mg/kg/day were well tolerated and did not result in any mortality or signs of systemic toxicity.
In the 4-week repeat dose toxicity study, doses of 100, 300 and 1000 mg/kg/day were well tolerated and did not result in any mortality or signs of other adverse systemic toxicity based on the study investigations performed up to the end of the 4-week treatment period and excluding results of the histopathological assessment.
Based on this information, it was considered appropriate to investigate a high dose level of 1000 mg/kg/day (the limit dose for this study type). The intermediate and low dose levels, 300 and 100 mg/kg/day, respectively, were selected to assess any dose-responsiveness of any test substance-related finding.

- Rationale for animal assignment: On arrival using non-selective allocation to cages. On Day 1 of study all animals were weighed and bodyweights were reviewed before dosing commenced by Study Management. Body weight of animals did not exceed ± 20 % of the mean for each sex.
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch. Two males and one female were replaced before treatment commenced due to body weight range extremes.

- Other: Dosing was restricted to the F0 generation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk.

Positive control:

Not examined

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs associated with dosing were checked daily during the first week of treatment, weekly thereafter and for females on Days 0, 7, 14 and 20 after mating and Days 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration:
- Pre-dose.
- After completion of dosing the group.
- One to two hours after dosing.
- Three to four hours after dosing.
- As late as possible in the working day.
A detailed physical examination was performed weekly for males and for females weekly before pairing, on Days 0, 7, 14 and 20 after mating and Days 1 and 7 of lactation to monitor general health.
Clinical observations are presented for each animal that showed signs, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

BODY WEIGHT: Yes.
The weight of animals was recorded as follows:
F0 males
- During acclimatization.
- Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
- On the day of necropsy.
F0 females During acclimatization.
- Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
- Days 0, 3, 7, 10, 14, 17 and 20 after mating.
- Day 1, 4, and 7 of lactation.
- On the day of necropsy.
Group mean values and SD were calculated from individual body weight data on each recorded occasion. For the offspring, litter mean body weight (and SD) was calculated separately for males and females; the group mean values were derived from the individual litter values.
Group mean weight changes were calculated from the weight changes of individual animals.
Offspring body weight change was calculated relative to Day 1 of age.
Body weights were plotted graphically with respect to the start of dosing or the start of the relevant period.

FOOD CONSUMPTION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males
- Weekly before pairing (from first day of treatment until pairing).
F0 females
- Weekly before pairing (from first day of treatment until pairing).
- Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating
- Days 1-3 and 4-6 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.
Group mean food consumptions and standard deviations were derived from unrounded cage values.

OTHER:
VAGINAL SMEARS
Wet smears were taken daily after pairing until mating using pipette lavage.

PARTURITION OBSERVATIONS AND GESTATION LENGTH
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

PRE-COITAL INTERVAL
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals of durations of 1-4, 5-8, 9-12 and 13-14 days of pairing was calculated.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Individual litter values were tabulated for the number of corpora lutea, implantation sites, total offspring at Day 1 and live at Days 1, 4 and 7 of age. Group mean litter size and SD were calculated from the individual litter values. Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
- Individual offspring body weights: Body weights determined on days 1, 4 and 7 of age.
- Sex ratio of each litter: Recorded on Days 1, 4 and 7.
Individual offspring body weights: Recorded on Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS: Yes
- Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues were retained in an appropriate fixative.
- Offspring at scheduled termination: Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals During Week 5 of respective treatment.
- Maternal animals:
Females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 7 of lactation.
- Method of Kill: Carbon dioxide asphyxiation for all adult animals.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The organs weighed, tissue samples fixed and sections examined microscopically for F0 animals are detailed in Table 1.
For females, the number of corpora lutea and uterine implantation sites were recorded for each ovary.
The number of uterine implantation sites were checked after staining with ammonium sulphide, only for females failing to produce a litter.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals. Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.

Histology:
Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes (initially preserved in modified Davidson’s fluid).
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
- Full list: All animals in Groups 1 and 4 at scheduled termination.
- Abnormalities only: All F0 animals.
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light microscopy:
Tissues preserved for examination were examined as follows:
- All adult animals in groups 1 and 4: All tissues specified in Table 1.
- All F0 animals: Abnormalities only.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 7 days of age.
- Method of Kill: Intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
- Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed. Abnormal tissues were retained in an appropriate fixative.

- Offspring at scheduled termination: Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
At scheduled kill, only abnormalities were preserved for examination by light microscopy.

Statistics:

See below.

Reproductive indices:

MATING PERFORMANCE AND FERTILITY
Individual data was tabulated. Group values were calculated for males and females separately for the following:

Percentage mating (%) = (Number of animals mating / Animals paired) x 100

Conception rate (%) = (Number of animals achieving pregnancy/ Animals mated) x 100

Fertility index (%) = (Number of animals achieving pregnancy / Animals pairing) x 100


GESTATION LENGTH AND INDEX
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

SURVIVAL INDICES
The following indices were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number if offspring born) x 100

Viability index (%) = (Number of live offspring on Day 7/ Number live offspring on Day 1 after littering) x 100

Group mean values were calculated from individual litter values.


SEX RATIO
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 and 7 of age.

Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs observed were minor and not attributed to treatment.
There were no treatment related changes observed in association with the time of dosing. Salivation was observed in one male at 100 mg/kg/day and chin rubbing in one female at each of 300 or 1000 mg/kg/day. This low incidence of signs was considered to relate to a general distaste of the formulation and is not considered a toxic effect of treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no clear effect of treatment upon male body weight at any of the dose levels investigated.
Although without any dose-relationship, bodyweight gain for females before pairing was slightly higher than Controls in all treated groups. This trend of higher female body weight and body weight gain in treated groups continued throughout gestation, and most markedly, during lactation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no clear effect on food consumption before pairing in males or females.
During gestation female food consumption was marginally higher than Controls in all treated groups; during lactation this difference was more pronounced, except for the 1000 mg/kg/day group during Days 1-3 of lactation where intake was similar to Controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic findings in adult males or females at necropsy that would infer any treatment related changes.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Mean pre-coital interval was unaffected by treatment. All pairings on study mated within the first four days of pairing. Treatment had no effect upon mating performance or fertility.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Treatment had no effect upon mating performance or fertility and there was no effect of treatment upon mean gestation length or the gestation index.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There was no change in the clinical condition of offspring that was considered related to maternal treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was not effect on post-implantation survival, live birth or viability indices.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight of offspring on Day 1 of age was considered similar to Controls across all treatment groups, however, there was a tendency towards slightly greater offspring body weight gain at 300 or 1000 mg/kg/day in males and females during Days 1-7 of age, when compared to Control values.
Body weight gain of male and female offspring at 100 mg/kg/day during Days 1-7 of age was similar to Controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy findings observed in offspring dying prematurely or in those killed at scheduled termination revealed no findings that were considered to relate to parental treatment.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Litter Size, Sex Ratio and Survival Indices
One female (number 51 receiving 100 mg/kg/day) was found to be not pregnant. All other females reared a live litter to Day 7 of lactation.
There was no effect on post-implantation survival, live birth or viability indices. Mean litter size was similar to Controls across all dose levels on Day 1 of lactation, and subsequent litter size to Day 7 of lactation was unaffected by treatment. There was no effect of the test material on sex ratio. There was no effect of treatment on the mean number of corpora lutea.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Formulation Analysis Results

No test material was detected in control samples. The mean concentrations of the test material in test formulations were between -4 % to +2.5 % of the nominal concentrations, confirming the accuracy of formulation. Difference from the mean of individual samples were <5 % confirming precise analysis. Procedural recoveries analysed concurrently with samples were within acceptance criteria, 94.8 % to 100.5 % indicating the continued accuracy of the method.

Discussion

The oral (gavage) administration of the test material to Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 100, 300 or 1000 mg/kg/day was well tolerated with no adverse effect of treatment apparent.

There was no effect of treatment upon body weight or food consumption of the adult males at any of the dose levels investigated. Amongst all groups of treated females, there was a trend of greater body weight gain, and increased food consumption during gestation and lactation, compared with Controls.

Treatment had no effect upon mating performance or fertility and there was no effect of treatment upon mean gestation length or the gestation index.

The number of offspring born, their subsequent survival to Day 7 of age and the clinical condition of the offspring were unaffected by treatment at dose levels up to 1000 mg/kg/day.

Offspring growth to Day 7 of age at 300 or 1000 mg/kg/day was slightly greater than in Controls, with a dose response evident. This was concluded not to represent an adverse effect of treatment on post natal development as it did not affect the post natal survival, clinical condition or macropathology findings of the offspring.

There were no macroscopic findings in offspring that were considered to relate to parental treatment.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, there were no adverse effects of treatment upon reproductive or developmental parameters up to the high dose level of 1000 mg/kg/day.

Executive summary:

The potential for the test material to cause reproductive/developmental effects, was investigated in a GLP study conducted in accordance to the standardised guideline OECD 421.

Three groups of ten male and ten female rats received test material at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration at a dose volume of 5 mL/kg. Males were treated daily for 15 days before pairing, during the pairing period and then up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing and gestation, and until Day 6 of lactation. Females were allowed to litter and rear their offspring before they were killed on Day 7 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same dose volume as the treated groups.

During the study, clinical condition, body weight, food consumption, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations of organs of the reproductive system were undertaken. The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed.

The oral (gavage) administration of the test material to Han Wistar (RccHan™;WIST) rats for at least 4 weeks at dose levels of 100, 300 or 1000 mg/kg/day was well tolerated, with no deaths and no effect of treatment upon the clinical condition of animals. There was no effect of treatment on body weight gain or food consumption of males at any of the dose levels investigated. In treated females there was a trend towards greater body weight gain and increased food consumption during gestation and lactation, compared with Controls.

Pre-coital interval, mating performance and fertility and gestation length were unaffected by treatment. The number of offspring born, offspring sex ratio and offspring survival were unaffected by parental treatment and there were no clinical observations or macroscopic necropsy findings amongst offspring that were considered to relate to parental treatment. Offspring body weight gain to Day 7 of age at 300 or 1000 mg/kg/day was slightly greater than in Controls, with a dose response evident, but unaffected at 100 mg/kg/day.

There were no treatment related macroscopic or microscopic findings amongst the adult animals, and no differences in organ weights which were attributed to treatment.

Under the conditions of the study, it is concluded that oral administration of the test material to male and female Han Wistar (RccHan™;WIST) for at least 4 weeks at dose levels of 100, 300 or 1000 mg/kg/day was well tolerated, with no adverse effect of treatment upon reproductive or developmental parameters up to the high dose level of 1000 mg/kg/day.