Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 January 2010 to 24 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Date of inspection: 15 September 2009 Date of Signature: 26 November 2009)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TIPX
- Substance type: Clear colourless liquid.
- Physical state: Liquid.
- Lot/batch No.: 09Z162.
- Storage condition of test material: Room temperature in the dark, under nitrogen.

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Han™:HsdRccHan™:WIST strain, obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, United Kingdom.

- Age at study initiation: 12 weeks of age.

- Weight at study initiation: The males weighed 301 to 363 g; the females weighed 199 to 230 g.

- Fasting period before study: Not reported.

- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages.
Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided.

- Diet: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, United Kingdom) ad libitum.

- Water: Mains drinking water ad libitum.

- Acclimation period: 11 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%.
- Air changes (per hr): At least 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

IN-LIFE DATES: From: Day 1 To: Day 43 (males); Day 1 To: Day 5 post partum (females). The first day of dosing was designated as Day 1 of the study.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was prepared at 30, 150 and 750 mg/kg/day as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were determined at the test laboratory. The formulations were shown to be stable for at least 25 days. Formulations were therefore prepared twice monthly and stored at approximately 4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was stable for at least 25 days in arachis oil BP and the prepared formulations were within ±6% of the nominal concentration.

- Amount of vehicle (if gavage): 4 ml/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary; The concentration of TIPX in the test material formulations was determined by gas chromatography (GC) using an external standard technique.

Samples; The test material formulations were extracted with methanol with final theoretical concentration of approximately 0.1 mg/ml.

Standards; Standard solutions of test material were prepared in methanol at a nominal concentration of 0.1 mg/ml.

Procedure; The sample and standard solutions were analysed by GC.

Homogeneity determinations; The test material formulations were assessed visually.

Stability determinations; The test material formulations were sampled and analysed initially and then after storage at approximately 4ºC in the dark for 25 days.

Verification of test material formulation concentrations; The test material formulations were sampled and analysed within 2 days of preparation.

Results; Mean measured concentration found was in the range of 99 – 104% of nominal concentrations. The results showed the formulation to be stable for at least 25 days.
Details on mating procedure:
- Impregnation procedure: cohoused.

- If cohoused: On Day 15 of the exposure, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of 14 days. 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 (completion of mating is during Week 6).

- M/F ratio per cage: 1:1.

- Length of cohabitation: For a maximum of 14 days.

- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No. As all animals mated within 4 days of pairing.

- Further matings after two unsuccessful attempts: Yes.

- Verification of same strain and source of both sexes: Yes.

- Proof of pregnancy: During mating period for up to 14 days, cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Duration of treatment / exposure:
From Day 1 to Day 42 (males); Day 1 To: Day 4 post partum (females).
Frequency of treatment:
Once daily.
Duration of test:
(IN-LIFE DATES)
From: Day 1 To: Day 43 (males); Day 1 To: Day 5 post partum (females). The first day of dosing was designed as Day 1 of the study.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
other: nominal in vehicle
Remarks:
Doses / Concentrations:
750 mg/kg/day
Basis:
other: nominal in vehicle
No. of animals per sex per dose:
10 animals per sex per dose and the control group.
Control animals:
yes, concurrent vehicle
Details on study design:
i) Groups of ten male and ten 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) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.

iii) 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.

iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.

v) The male dose groups were killed and examined macroscopically on Day 43.

vi) At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.

- Dose selection rationale: The dose levels were chosen based on the results of the oral repeted dose toxicity in rats (see Section 7.5.1).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, soon after dosing, and 1 and 5 hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing, and 1 hour after dosing at weekends (except for females during parturition where applicable).
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION: Yes
During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).

FOOD EFFICIENCY: Yes
- Weekly food efficiency (bodyweight gain/food intake) was calculated for males and for females during the pre-mating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes. A possible treatment-related effect was detected, therefore gravimetric measurements were initiated from Day 24 for males and during the the gestation and lactation phases of the study for females.

MATING: Yes
During mating period for up to 14 days, cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day
0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

PREGNANCY AND PARTURITION: Yes
Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and around the period of expected parturition. Observations were carried out at approximately 08:30 and 12:30 hours at weekends and public holidays. The following was recorded for each female:
1) 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

SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- Maternal animals: Adult females were killed by intravenous overdose of a sodium pentobarbitone followed by exsanguination on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.

GROSS NECROPSY: Yes
All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution.

ORGAN WEIGHTS: Yes
The epididymides and testes are removed from terminal kill males, dissected free from fat and weighed before fixation.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin.
coagulating gland, epididymides, ovaries, mammary tissue (female only), pituitary, prostate, seminal vesicles, testes, uterus/cervix and vagina.

All tissues were despatched to the test site in Switzerland for processing. The tissues from control and 1000 mg/kg/day dose group animals, any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with Haematoxylin and Eosin for subsequent microscopic examination. In addition, sections of testes and epididymides from all control and 750 mg/kg/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined. Microscopic examination was conducted by the Study Pathologist.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Number of offspring born, number of offspring alive recorded daily and reported on Day 1 and 4 post partum, sex of offspring on Day 1 and 4 post partum, clinical condition of offspring from birth to Day 5 post partum, individual offspring weights on Day 1 and 4 post partum (litter weights were calculated retrospectively from this data), surface righting reflex as physical development.
Statistics:
For males and females during the pre-mating phase, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Bartletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed nonhomogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Data for females during gestation and lactation, and offspring data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed
using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test. Non-parametric methods were also used to analyse implantation loss, offspring sex ratio and landmark developmental markers.
Probability values (P) are presented as follows:
P < 0.001 ***
P < 0.01 **
P < 0.05 *
p ≥ 0.05 (not significant)
Indices:
Reproductive indices
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:

Mating Index (%) = (Number of animals mated÷Number of animals paired) x 100

Pregnancy Index (%) = (Number of pregnant females÷Number of animals mated) x 100

Gestation and Parturition Data

The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length

Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index

The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring÷Number of pregnant females) x 100

(See 'any other information on materials and methods incl. tables for 'offspring viability indices')
Historical control data:
Data in the study report. They were used to assess the toxicological importance of the effects observed in the tested animals.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study.
Increased salivation was detected soon after and one hour after dosing for animals of either sex treated with 750 mg/kg/day, with the effect extending into the 150 mg/kg/day dose group. This was also occasionally observed up to five hours after dosing in animals of either sex treated with 750 mg/kg/day, and isolated instances of increased salivation prior to dosing were also observed for males treated with 750 mg/kg/day and for one female treated with 150 mg/kg/day. See Table 1, as attached, for the results.
One female treated with 750 mg/kg/day showed red fluid around the snout and mouth, lachrymation and chromodacryorrhea on Day 29. Complete regression was evident thereafter, and as such, this isolated finding was considered to have arisen incidentally and was not considered to represent systemic toxicity. Remaining clinical signs were confined to incidents of generalised fur loss observed for one female treated with 750 mg/kg/day and two females treated with 150 mg/kg/day. This observation was also noted for one control female. Red/brown staining around the mouth was also evident for one female treated with 750 mg/kg/day. These observations are occasionally observed in laboratory maintained animals and are unrelated to
treatment with the test material.

BODY WEIGHT AND BODY WEIGHT GAIN
See tables 2 and 3, as attached, for the results. Males treated with 750 mg/kg/day showed actual bodyweight losses during the premating phase of the study and statistically significant reductions in bodyweight gains when compared to controls during the treatment period (P<0.05 - P<0.01). The reduction in bodyweight gains during the treatment period extended into the male 150 mg/kg/day dose group, with a statistically significant reduction observed during Week 3 (P<0.01).
Bodyweight losses were also evident for females treated with 750 mg/kg/day during the first week of treatment, resulting in statistically significant reductions in bodyweight gains observed during the first week of treatment when compared to controls (P<0.05). Females treated with 150 mg/kg/day also showed statistically significant reductions in bodyweight gains in comparison to control values during the first week of treatment (P<0.05). Reduced bodyweight gains were also evident during Week 2 for females treated with 750 and 150 mg/kg/day. Females treated with 750 mg/kg/day showed statistically significant reductions in bodyweight gains in comparison to control values during the final week of the gestation phase (P<0.01). Cumulative bodyweight gains during the overall gestation phase were therefore lower for high dose females when compared to controls (P<0.01). During the lactation phase, females treated with 750 mg/kg/day showed a statistically significant increase in bodyweight gains compared to controls (P<0.01). Similar increases in bodyweight gains were also evident for females treated with 150 mg/kg/day (P<0.05).

FOOD CONSUMPTION
See tables 4 and 5, as attached, for the results. A reduction in dietary intake was evident for animals of either sex treated with 750 mg/kg/day during the first two weeks of the study. Food conversion efficiency (the ratio of bodyweight gain to dietary intake) was also reduced for females treated with 750 mg/kg/day during the pre-mating phase, and for males treated at the highest dose level, throughout the treatment period (with the exception of the mating phase when food consumption was not recorded). Slight reductions in dietary intake were also evident for males treated with 150 mg/kg/day during the treatment period, when compared to controls, and for females treated with 150 mg/kg/day during the pre-mating phase. Food conversion efficiencies were similarly affected. Slight reductions in dietary intake were also evident for females treated with 750 and 150 mg/kg/day during the first week of the gestation period, and for females treated with 750 mg/kg/day during the lactation period, although statistical analysis of the data did not reveal any significant intergroup differences.

WATER CONSUMPTION
Daily visual inspection of water bottles revealed an increase in water intake at the highest dose level. Daily measurements were therefore initiated. Higher water intake was clearly evident for males treated with 750 mg/kg/day when compared to controls thereafter. Increased water intake was also evident for females treated with 750 mg/kg/day during the gestation period, although statistical significance was only achieved on Day 9 (P<0.01). Increases were also observed on Day 2 of lactation, however statistical significance was not achieved.

MATING
No treatment-related effects were detected in mating performance for treated animals when compared to controls.

FERTILITY
One female treated with the highest dose level did not produce a pregnancy following successful mating. All remaining females from the treated and control groups achieved pregnancy and delivered offspring.

GESTATION LENGTH
No treatment-related effects were detected in the length of gestation between control and treated groups. Statistical analysis of the gestation length data did not reveal any significant intergroup differences.
One female treated with 30 mg/kg/day showed a gestation length of 25 days. A total litter loss was subsequently observed for this animal. This is a low incidence finding occasionally observed in reproductive studies and in isolation, was considered not to be related to treatment.

ORGAN WEIGHTS
No significant differences were detected in testes or epididymide weights for treated males when compared to controls.
Statistically significant increases in absolute and bodyweight-relative testes weights were evident for 150 mg/kg/day males when compared to controls. The significance achieved was minimal (P<0.05) and in the absence of a dose-related response or any histopathogical correlates, these increases were considered to have arisen incidentally and were considered to be unrelated to treatment with the test material.

GROSS PATHOLOGY
No treatment-related macroscopic abnormalities were detected.
Findings were confined to sloughing of the glandular region of the stomach for one control female, one control female showed generalised fur loss and one female treated with 750 mg/kg/day showed ovaries encased in a fluid-filled sac. These findings were isolated considered to have arisen incidentally and were considered to be unrelated to treatment.

HISTOPATHOLOGY
Histopathological examinations did not reveal any treatment-related effects.
The few microscopic findings recorded, were the stages of oestrous cycle in the vagina, the functional status in the mammary gland, and exfoliation of mucosal epithelium in the stomach of one control female. The findings in the vagina and mammary gland corresponded to the physiological reproductive status. The origin of exfoliation of gastric mucosa, accompanied by aggregates of pollen from the diet, was not clear from this study but was considered to be of no toxicological importance.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Remarks:
(parents and offspring)
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
(In total, ten females from control and 150 mg/kg/day dose group, nine females treated with 30 mg/kg/day and seven females treated with 750 mg/kg/day gave birth to a live litter and successfully reared young to Day 5 of age. One female treated with 750 mg/kg/day failed to achieve pregnancy following evidence of mating and two females treated as this dose level showed a total litter loss after parturition. A total litter loss was also evident for one female treated with 30 mg/kg/day. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation, where applicable.)

LITTER SIZE AND VIABILITY
See tables 6 - 8, as attached. Slightly lower numbers of corpora lutea, implantation sites and litter sizes at birth were evident at 750 mg/kg/day when compared to controls. Slightly higher post-implantation losses were also observed at 750 mg/kg/day when compared to controls and two litters showed a total litter loss at 750 mg/kg/day. Statistical analysis of the data did not reveal any significant intergroup differences. The number of offspring deaths on the day of parturition at 750 mg/kg/day was higher than interim deaths from the other treatment levels and the controls.
A total litter loss was observed at 30 mg/kg/day but this is occasionally observed in reproductive studies and was considered to be incidental.

CLINICAL SIGNS
No treatment-related clinical signs were observed. The number of decedents was higher at 750 mg/kg/day compared to the remaining dose groups. This was due to the two litter losses observed at this dose level.

BODY WEIGHT
See tables 6, 9 and 10, as attached. Reduced total litter weights were evident at 750 mg/kg/day when compared to controls, with a statistically significant reduction observed on Day 4 when compared to controls (P < 0.001). Mean bodyweights for offspring were comparable to controls on Day 1 of lactation, although reduced mean bodyweight gains were observed for male and female offspring from the 750 mg/kg/day litters compared to those from the control litters (P < 0.001 and P < 0.01 respectively).

GROSS PATHOLOGY
No treatment-related macroscopic abnormalities were detected for offspring dying during lactation or at termination on Day 5 post partum. The number of offspring deaths on the day of parturition at 750 mg/kg/day was higher than interim deaths from the other treatment levels and the controls.
The findings observed in the interim death offspring were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test material.
No treatment-related macroscopic abnormalities were detected for offspring at terminal kill. Macroscopic findings observed at termination were considered to be low incidence findings occasionally observed in reproductive studies of this type, and not related to test material toxicity.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of TIPX to rats by gavage, at dose levels of 750, 150 and 30 mg/kg/day, resulted in treatment-related effects at 750 and 150 mg/kg/day and therefore a clear ‘No Observed Effect Level’ (NOEL) was established at 30 mg/kg/day. The effects detected in the adults were considered not to represent an adverse effect. The ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 750 mg/kg/day. Treatment-related effects were detected in litters from the 750 mg/kg/day, therefore, the ‘No Observed Effect Level’ (NOEL) was considered to be 150 mg/kg/day for reproductive toxicity.
The test material does not meet the criteria for classification according to EU classification system (Council Directive 67/548/EEC and Regulation (EC) No 1272/2008).
Executive summary:

Introduction.The study was performed to screen for potential adverse effects of the testmaterial on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

Methods.The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to eight weeks (including a two week maturation phase, pairing, gestation and early lactation for females), at dose levels of 30, 150 and 750 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, bodyweight change, dietary intake 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.

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

Results.

Adult Responses:

Mortality. There were no unscheduled deaths.

Clinical Observations. Increased salivation was detected after dosing and also occasionally prior to dosing for animals of either sex treated with 750 and 150 mg/kg/day. No such observations were evident for animals of either sex treated with 30 mg/kg/day.

Bodyweight. Initial bodyweight losses and reduced bodyweight gains were evident for 750 mg/kg/day males, and lower bodyweight gains were observed for 150 mg/kg/day males when compared to controls.

Initial bodyweight losses and reduced bodyweight gains were also observed for 750 mg/kg/day females during the pre-mating phase, with the effect extending into the 150 mg/kg/day female dose group. Reduced bodyweight gains were also evident for 750 mg/kg/day females during the final week of gestation when compared to controls. Increases in bodyweight gains in comparison to controls were observed for females

treated with 750 and 150 mg/kg/day during lactation.

No adverse effects on bodyweight change were detected for animals of either sex treated with 30 mg/kg/day.

Food Consumption and Food Efficiency. Reduced dietary intake and food efficiency were evident for males treated with 750 and 150 mg/kg/day when compared to controls.

Reduced dietary intake and food conversion efficiency was also evident for females treated with 750 and 150 mg/kg/day when compared to controls, during the pre-mating phase. A reduction in dietary intake was also evident during early gestation for 750 and 150 mg/kg/day female dose groups, with the effect also observed for females treated with 750 mg/kg/day during lactation.

No adverse effects of dietary intake or food conversion efficiency were evident for animals of either sex treated with 30 mg/kg/day.

Water Consumption. An increase in water intake was observed for animals of either sex treated with 750 mg/kg/day.

No adverse effects on water intake were observed for animals of either sex treated with 150 or 30 mg/kg/day.

Reproductive Screening:

Mating. No treatment-related effects were detected in mating performance.

Fertility. One female treated with 750 mg/kg/day failed to produce a pregnancy. All remaining females from all treatment and control groups achieved pregnancy.

Gestation Lengths. No treatment-related effects were detected in the length of gestation.

Litter Responses:

Offspring Litter Size, Sex Ratio and Viability. Slightly lower corpora lutea and implantation site numbers and smaller litter sizes at birth and two total litter losses were evident at 750 mg/kg/day when compared to the control group.

Offspring Growth and Development. Reduced litter weights were evident at 750 mg/kg/day when compared to controls on Day 4 of lactation. Bodyweight gains for offspring of either sex from the 750 mg/kg/day litters were lower than those from control litters.

Offspring Observations. No treatment-related clinical signs were observed.

Pathology:

Organ Weights. No differences in testis or epididymis weights were detected for treated males when compared to controls.

Necropsy. No treatment-related macroscopic abnormalities were detected for offspring or adults.

Histopathology. No treatment-related microscopic effects were detected.

Conclusion. The oral administration of TIPX to rats by gavage, at dose levels of 750, 150 and 30 mg/kg/day, resulted in treatment-related effects at 750 and 150 mg/kg/day and therefore a clear ‘No Observed Effect Level’ (NOEL) was established at 30 mg/kg/day. The effects detected in the adults were considered not to represent an adverse effect. The ‘No Observed Adverse Effect Level’ (NOAEL) was considered to be 750 mg/kg/day. Treatment-related effects were detected in litters from the 750 mg/kg/day, therefore, the ‘No Observed Effect Level’ (NOEL) was considered to be 150 mg/kg/day for reproductive toxicity.

The test material does not meet the criteria for classification according to EU classification system (Council Directive 67/548/EEC and Regulation (EC) No 1272/2008).