Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The m-isomer showed specific target organ effects after oral repeated application at a dose level justifying a classification as STOT RE Cat 2. As similar effects are expected with the p-isomer substance, both substances are classified accordingly.

 

Preliminary and a main prenatal developmental studies have been conducted at doses of 25, 50, 100 mg/kg bw day and 5, 15 and 40 mg/kg bw day respectively with the p-isomer. Reductions in fetal, placental and litter weights and the type and incidence of fetal findings were evident in the prenatal developmental studies and consistent with a delay in growth these effects are considered to be a consequence of maternal toxicity rather than indicating any intrinsic toxicity to the developing conceptus. The No Observed Effect Level (NOEL) for the developing conceptus was considered to be 15 mg/kg bw/day. Treatment at 15 mg/kg bw/day was associated effects on body weight gain and food consumption. This is considered to be a reflection of irritation of the GI tract, which is frequently observed with test items of this nature, rather than systemic toxicity. The No Observed Effect Level (NOEL) for the pregnant dam was 5 mg/kg bw/day.

 

A two-generation reproduction study has been conducted at doses of 5, 15 and 25 mg/kg. The results of this test indicated that the 5 mg/kg bw/day was considered to be a No Observed Effect Level (NOEL) for the repeated administration of the test item on reproduction, pre-natal development and post-natal development of the rat when administered over two successive generations. As noted during the conduct of the prenatal developmental study irritation of the GI tract was observed at doses ≥15 mg/kg bw/day (15 and 25 mg/kg bw/d) and thereforeall the effects/changes in endpoints when collectively put together show that there is a spectrum of changes that have been brought about as a consequence of exposure to the test material which is having an effect on the adult gastrointenstinal mucosa (irritancy/corrosion) at doses>15 mg/kg bw/day and < 25 mg/kg bw/day. The altered physiology of the parent is likely to be an adaptation to the alterations in theirown physiology and a stress response at doses of 15 and 25 mg/kg bw d rather than intrinsic toxicity.It is therefore concluded based on expert opinion that 25 mg/kg bw dw should be considered the most appropriate value to be used for regulatory and risk assessment purposes.In addition, the substance should not be classified for reprotoxicity as effects only occurred at doses where animals were compromised by the test substances primary damaging effects on gut epithelia and subsequent stress /secondary responses on the physiology rather than intrinsic toxicity. This interpretation of the results is further supported by the STOT RE Cat 2. Classification that is applied based on the findings of the repeated dose study with the m-isomer.

 

According to the criteria of the CLP regulation, and based on the available study results, the two substances do not need to be classified for additional Human Health hazards beyond those described above.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 March 2016 to 8 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to requests within other regulatory jurisdictions (US-EPA) for provision of additional data (1), in response to the test rules “Testing of Certain High Production Volume Chemicals; Third Group of Chemical” (76 FR 65385, October 21, 2011), and its preference of a 2-generation reproductive study, a test was contracted to a testing laboratory and underway with the registered substance p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline prior to a request from ECHA to conduct an Extended One Generation Reproduction Study.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.35 (Two-Generation Reproduction Toxicity Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals and environmental conditions:
At the start of treatment the males weighed 175 to 226g and were approximately six to seven weeks old. The females weighed 135 to 171g, and were approximately six to seven weeks old.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
For both generations, adult animals were dosed once daily, by oral gavage, throughout the treatment period (with the exception of females during parturition, if applicable) using a stainless steel dosing cannula attached to a graduating syringe. The F1 offspring were dosed from Day 22 of age, prior to the formal start of that generation (this included spare offspring retained as contingency against deaths prior to the formal start or in the early stages of the F1generation). Wherever possible, dosing was performed at a similar time each day.

Dose levels were selected for the study based on available toxicity data including an Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat (Envigo Study Number 41502453) performed within this Test Facility.

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Propylene Glycol.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
Following at least 10 weeks of treatment, all animals were paired on a one male: one female basis within each dose group for a maximum of 14 days.

At the end of the pairing phase, males were returned to their original cages and females were transferred to individual cages with suitable nesting material. Pregnant females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter was performed during this period. This includes measurement of surface righting reflex on Day 1 post partum, air righting reflex on Day 17 post partum and pupillary reflex and auditory startle response on Day 21 post partum. At Day 21 post partum, offspring were randomly selected within each dose group to form the F1 generation.

Selected F1 males and F1 females were treated at the appropriate dose level throughout the treatment period. Treatment started at Day 22 of age and included spare offspring retained as contingency against deaths prior to the formal start or in the early stages of the F1 generation. All selected animals were observed for evidence of sexual maturation. These animals were also assessed for motor activity and sensory function at approximately ten weeks of age. F1 females had a vaginal smear prepared daily for 3 weeks prior to pairing. Males and females were paired for up to 14 days.

At the end of the mating phase, males were returned to their original cages and females were transferred to individual cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purpose of the study the test item was prepared at the appropriate concentrations in Propylene glycol. The stability and homogeneity of the test item formulations had previouslybeen determined as part of Rat Pre-Natal Development Toxicity Study (Envigo Research
Limited Report no.: 41502453). These analytical results showed that the formulations were stable for at least 16 days when stored at 4°C in the dark. Formulations for this study were therefore prepared in batches on an approximately weekly basis, with daily aliquots being
stored at approximately +4 °C in the dark prior to use.

Representative samples were taken of test item formulation at intervals during the study and were analyzed for concentration of p-(2,3-epoxypropoxy)-N,N-bis (2,3-epoxypropyl)aniline at Envigo Analytical Laboratory, Shardlow. The results indicated that the prepared formulations were within 90 to 103% of the nominal concentration indicating the suitability of the formulation procedure used on the study.
Duration of treatment / exposure:
Ten weeks for P0 and F1 animals
Frequency of treatment:
Daily
Details on study schedule:
i. All animals were treated at the appropriate dose level throughout the treatment period (except for females during parturition, if applicable).
ii. All females had a daily vaginal smear prepared for 3 weeks prior to mating.
iii. Following at least 10 weeks of treatment, all animals were paired on a one male: one female basis within each dose group for a maximum of 14 days.
iv. At the end of the pairing phase, males were returned to their original cages and females were transferred to individual cages with suitable nesting material.
v. Pregnant females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter was performed during this period. This includes measurement of surface righting reflex on Day 1 post partum, air righting reflex on Day 17 post partum and pupillary reflex and auditory startle response on Day 21 post partum.
vi. At Day 21 post partum, offspring were randomly selected within each dose group to form the F1 generation.
vii. At Day 21 post partum, all parental females were killed and given a detailed macroscopic examination including preservation of reproductive organs/tissues for histopathology. Suspected non-pregnant females were terminated around the time of other littering females.
viii. At Day 21 post partum, all offspring selected for organ weights were killed and given a detailed macroscopic examination, including preservation of selected tissues for possible histopathology. F1 offspring not otherwise selected to continue in the study were also killed and subjected to a detailed macroscopic examination with grossly abnormal tissues being retained.
ix. Following completion of the reproduction phase, all adult males were killed and given a detailed macroscopic examination including sperm analyses and preservation of reproductive organs/tissues for histopathology.
x. Selected F1 males and F1 females were treated at the appropriate dose level throughout the treatment period. Treatment started at Day 22 of age and included spare offspring retained as contingency against deaths prior to the formal start or in the early stages of the F1 generation.
xi. All selected animals were observed for evidence of sexual maturation. These animals were also assessed for motor activity and sensory function at approximately ten weeks of age.
xii. F1 females had a vaginal smear prepared daily for 3 weeks prior to pairing.
xiii. Males and females were paired for up to 14 days.
xiv. At the end of the mating phase, males were returned to their original cages and females were transferred to individual cages.
xv. F1 females were allowed to deliver their offspring and maintain them until Day 21 of lactation. Evaluation of each litter was performed during this period. This includes measurement of ano-genital distance on Day 1 post partum, surface righting reflex on Day 1 post partum, air righting reflex on Day 17 post partum and pupillary reflex and auditory startle response on Day 21 post partum.
xvi. At Day 21 post partum, all F1 parental females were killed and given a detailed macroscopic examination including preservation of reproductive organs/tissues for histopathology. Suspected non-pregnant females were terminated around the time of other littering females.
xvii. At Day 21 post partum, all F2 offspring selected for organ weights were killed and given a detailed macroscopic examination, including preservation of selected tissues for possible histopathology. Unselected offspring were also killed and subjected to a detailed macroscopic examination with grossly abnormal tissues being retained.
xviii. Following completion of the reproduction phase, all adult F1 males were killed and given a detailed macroscopic examination including sperm analysis and preservation of reproductive organs/tissues for histopathology.
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
No. of animals per sex per dose:
28 males and females for the P0 generation and 24 males and females for the F1 generation.
Control animals:
yes, concurrent vehicle
Details on study design:
The test item was administered once daily by oral gavage to three groups each of twentyeight male and twenty-eight female Wistar Han™:RccHan™:WIST strain rats, at dosages of 5, 15 and 25 mg/kg bw/day throughout the study. A further group of twenty-eight male and twenty-eight female F0 Generation rats were similarly dosed with the vehicle (Propylene Glycol) to serve as a control. Clinical signs, body weight development, food and water consumption were monitored during the study. After 10 weeks of treatment, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce the F1 litters. At weaning of offspring from the F0 mating phase, groups of twenty-four male and twenty-four female offspring from each dose group were selected to form the F1 generation. The remaining surviving F0 females and unselected offspring were terminated at Day 21 post partum, followed by the termination of all F0 male dose groups.

The offspring selected for the F1 generation (including control) were dosed at their respective dosage from Day 22 of age and then dosed for at least 10 weeks of the formal F1 generation before being paired within each dose group to produce the F2 litters. At weaning of the F2 litters all surviving adults and their offspring were killed, followed by the termination of all F1 male dose groups. Oestrous cycle assessment was performed daily for three weeks prior to mating for both the F0 and F1 generations. Observations for positive evidence of mating were recorded together with the start and completion of parturition. During the maturation phase of the F1 generation offspring, males and females were evaluated for sexual maturation. The ano-genital distance was recorded for all F2 generation offspring on Day 1 post partum. During the lactation phases daily clinical observations were performed on all surviving offspring, together with litter size.

Litter weight, individual offspring weights and landmark developmental signs were also recorded on specific days post partum.

All animals at termination were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.
Positive control:
Not applicable
Parental animals: Observations and examinations:
For each generation, during the treatment period, individual clinical observations were performed immediately before dosing, up to 30 minutes after dosing and one hour after dosing. All observations were recorded. For selected F1 offspring this included the period
between selection and the formal start of the F1 generation.

Body Weight
Individual body weights were recorded for F0 and F1 males prior to dosing on Day 1 and then weekly within each generation until scheduled termination. Individual body weights were also recorded at terminal kill.

For females, individual body weights were recorded prior to dosing on Day 1 and then weekly until pairing for both the F0 and F1 generations. During the mating phases, females were weighed daily until mating was confirmed. Mated females were weighed on Day 0, 7, 14 and 21 post coitum and body weights for littering females were recorded on Days 1, 4, 7, 14 and 21 post partum.

Food Consumption
For each generation, dietary intake was recorded for both sexes weekly from the formal commencemnt of each generation until the pairing phase and, for males only, weekly following the pairing phase. For each generation, food consumptions were not performed during the pairing phase.
For each generation, dietary intake for mated females was recorded between Days 0-7, 7-14 and 14-21 post coitum. Food consumptions for littering females was recorded between Days 1-4, 4-7, 7-14 and 14-21 post partum. Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for both sexes during the pre-pairing phases and for males during the postpairing phases. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

Mating
For each generation, where possible, animals were paired on a one male: one female basis within each dose group for up to fourteen days. Sibling pairings were avoided when F1 animals were paired. The stage of the estrous cycle was recorded during this period for all females via a vaginal smear. Smearing of individual females was discontinued when sperm were found. On confirmation of mating (Gestation Day 0), males were re-housed in their original cages and females were individually housed in solid floor cages. Uncertain mated females continued to be housed with the male until either mating was confirmed by the presence of sperm in a vaginal smear, or the pairing period for that generation was completed.

Pregnancy and Parturition
For each generation, each mated female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays if this schedule could not be followed. The following was recorded for each female:
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

Litter Data
For each generation, on completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was assessed:
i) Number of offspring born
ii) Number of live offspring from birth to weaning
iii) Sex of offspring on Days 1, 4, 7, 14 and 21
iv) Clinical condition of offspring from birth to weaning
v) Individual offspring weights on Days 1, 4, 7, 14 and 21 post partum (litter weights were calculated retrospectively from offspring weights)
vi) Surface righting reflex on Day 1 post partum. Air righting reflex on Day 17 post partum and pupillary reflex and auditory startle response on Day 21 post partum
vii) Necropsy findings of any offspring found dead. Particular attention was applied to those offspring found dead at, or shortly after parturition

Additionally for the F1 generation (F2 offspring) the following was assessed:
i. Ano-genital distance for each sex at Day 1 post partum

Oestrous cyclicity (parental animals):
For each generation, where possible, vaginal smears for females were taken daily for three weeks prior to mating for each generation. The stage of the estrous cycle was recorded for each day. One control F1 female showed no open vagina during the study and therefore the estrous cycle assessments could not be performed for this animal. All selected F1 offspring and offspring selected for post-weaning neurobehavioural assessments were observed for sexual development. For females, the day of appearance of vaginal opening (separation of the labia) was recorded; for males the day of separation of the
prepuce from the glans penis was recorded. The bodyweight for each individual at the time of sexual maturation was also recorded.
Litter observations:
For each litter the following was assessed:
i) Number of offspring born
ii) Number of live offspring from birth to weaning
iii) Sex of offspring on Days 1, 4, 7, 14 and 21
iv) Clinical condition of offspring from birth to weaning
v) Individual offspring weights on Days 1, 4, 7, 14 and 21 post partum (litter weights were calculated retrospectively from offspring weights)
vi) Surface righting reflex on Day 1 post partum. Air righting reflex on Day 17 post partum and pupillary reflex and auditory startle response on Day 21 post partum
vii) Necropsy findings of any offspring found dead. Particular attention was applied to those offspring found dead at, or shortly after parturition

Additionally for the F1 generation (F2 offspring) the following was assessed:
Postmortem examinations (parental animals):
See table presented below
Postmortem examinations (offspring):
For all F0 and F1 adult males, the following procedures were performed at necropsy:

i. The left testis and epididymis was removed, dissected from connective tissue and weighed separately.
ii. For the testis, the tunica albuginea was removed and the testicular tissue stored frozen at approximately -20 °C. At an appropriate later date, the tissues required (Groups 1 and 4 only) were thawed and homogenized in a suitable saline/detergent Ye mixture. Samples of the homogenate were examined microscopically to determine the numbers of homogenization resistant spermatids present.
iii. For the epididymis the distal region was incised and a sample of the luminal fluid is collected and transferred to a buffer solution for analysis of sperm motility and sperm morphology. Individual sperm was assessed using an automated semen analyzer to determine the numbers of motile, progressively motile and non motile sperm.
iv. A sample of sperm was preserved in fixative, transferred to a glass slide and then stained with eosin stain. For Groups 1 and 4 the slides were examined under a microscope to determine the number with apparent structural anomalies.

The cauda epididymis was separated from the body of the epididymis, and then weighed. The cauda epididymis will then be frozen at approximately -20 °C. At an appropriate later date the required tissues (Groups 1 and 4 only) were thawed and homogenized in an appropriate saline/detergent solution to determine the numbers of homogenization resistant spermatids.
Statistics:
Quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption, Pre- Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio,Implantation Sites, Post-Implantation Loss, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Developmental Parameters, sperm analysis (excluding morphology), Absolute Organ Weights, Body Weight-Relative Organ Weights. Where possible, data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:

Data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametricdata. If no dose response was found but the data shows nonhomogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel
(non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t test (parametric) or the Mann-Whitney U test (non-parametric).

Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing.
Reproductive indices:
Reproductive indices were calculated for all females/litters as appropriate. Non-pregnant animals are defined as having no implantations; Data from selected F1 offspring between weaning and the formal commencement of the F1 generation is retained with the raw data but is not presented in the report as this data was considered useful in assessing the effect of treatment between the treatment groups.

Mating Performance and Fertility
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 paired
Number of animals mated x 100
Pregnancy Index (%) = (Number of animals mated/ Number of pregnant females) x 100

Gestation and Parturition Data
The following parameters were calculated from 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 pregnant females/Number of females delivering live offspring) x 100
Offspring viability indices:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to weaning (Day 21 of age).

i. Implantation Losses (%) Group mean percentile post-implantation loss was calculated for each female/litter as follows:
Post–implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) x100

ii. Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index = (number of offspring alive on Day 1 / number of offspring born) x 100

Viability Index 1 = (number of offspring alive on Day 4 / number of offspring alive on Day 1) x 100

Viability Index 2 = (number of offspring alive on Day 7 / number of offspring alive on Day 4) x 100

Viability Index 3 = (number of offspring alive on Day 14 / number of offspring alive on Day 7) x 100

Viability Index 4 = (number of offspring alive on Day 21 / number of offspring alive on Day 14) x 100

Viability Index 5 = (number of offspring alive on Day 21 / number of offspring alive on Day 1) x 100

Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1, 4, 7, 14 and 21 post partum, using the following formula:

% Male offspring (Sex Ratio) = (number of male offspring / total number of offspring) x 100

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Isolated incidences of increased post-dosing salivation were observed for five males and seventeen females receiving 25 mg/kg bw/day during the F0 generation. Similar increased post-dosing salivation was also observed for one female receiving 15 mg/kg bw/day. Increased post-dosing salivation is often observed when animals are dosed via the oral gavage route and, at the incidence observed, most probably represent isolated difficulties in dosing occasional animals during the study, in combination with distaste/ slight irritancy of the dosing formulations, rather than any systemic effect of exposure to the test item.

One female at 15 and another female at 25 mg/kg bw/day showed a mass or masses during the course of the F0 generation but, these isolated findings were considered to be incidental and unrelated to exposure to the test item.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were three unscheduled adult deaths amongst F0 generation animals affecting one female receiving 5 mg/kg bw/day and two males receiving 15 mg/kg bw/day.

Female number 141 (5 mg/kg bw/day) was found dead around the time of parturition on Day 101 of the study and this death may reflect difficulties during the parturition process. No clinical signs had been apparent for this animal prior to this event. Macroscopic necropsy
revealed the stomach to be filled with red colored fluid, however these stomach contents probably reflect cleaning of the offspring and consumption of the placenta at parturition. Microscopic examination of the tissues did not indicate any obvious cause for this death.

Male number 72 (15 mg/kg bw/day) was found dead on Day 100 of the study. No clinical signs had been apparent for this animal prior to this event and no macroscopic abnormalities were detected during necropsy examination. Microscopic examination of the tissues did not
indicate any obvious cause for this death.

Male number 76 (15 mg/kg bw/day) showed staining around the snout, lethargy, piloerection and pallor of the extremities on Day 106 of the study and was killed in extremis for animal welfare considerations. Macroscopic necropsy revealed an enlarged and distended stomach filled with dark coloured contents and the liver, kidneys and prostate were all observed to be pale. Microscopic examination of the tissues did not indicate any obvious cause for this death.

Although the aetiology of these deaths were not determined, in the absence of any similar death amongst animals at the high dosage of 25 mg/kg bw/day in either generation, they were considered to be incidental and unrelated to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 25 mg/kg bw/day, body weight gain of males was lower than control throughout the ten week pre-pairing phase (to Day 71), with differences from control frequently attaining statistical significance. Subsequent body weight gain to termination also tended to be lower than control, although differences were not as marked and only occasionally attained statistical significance. This may reflect the lower body weight gain expected for the maturing animals at this later stage of the study. The lower body weight gain observed at this dosage resulted in statistically significant lower mean body weight compared with control on Day 15 which persisted until termination. Overall body weight gain was also statistically significantly lower than control.

At 15 mg/kg bw/day, body weight gain of males was statistically significantly lower than control for the first five weeks of the study (to Day 36) and again during Week 7 (Days 43 to 50). Subsequent body weight gain for the remainder of the study tended to be slightly lower than control but differences were slight, only attained statistical significance on one occasion and probably reflect normal biological variation. The lower initial body weight gain observed at this dosage resulted in statistically significant lower mean body weight compared with control on Day 22 which persisted until termination and overall body weight gain was also statistically significantly lower than control.

For males at 5 mg/kg bw/day, mean body weight and body weight gains, including overall body weight gain, were considered to be unaffected by treatment. Occasional statistically significant difference in body weight gain, compared to control, were observed but these were considered to be incidental and reflect normal biological variation.

For females, mean body weight and body weight gains, including overall body weight gain, during the ten week pre-pairing period was unaffected by treatment at 5, 15 or 25 mg/kg bw/day.

At 25 mg/kg bw/day, body weight gain was statistically significantly lower than control during the last week (Days 14-21) of gestation, resulting in statistically significant lower mean body weight and overall body weight gain at the end of gestation. Body weight
gain during the later period of gestation appeared to be influenced by a lower contribution by the developing litter (based on litter weight at birth) and mean body weight at the start of lactation was not significantly different from control. The magnitude of the differences between this dosage and control was similar at the start of gestation and at the start of lactation indicating that there was no underlying effect on maternal body weight during gestation. The pattern of body weight gain during lactation was similar to control, although higher body weight gain during the last week of lactation attained statistical significance when compared to control. This higher gain may reflect lower demand due to smaller litter size at this dosage, and only resulted in parity of mean body weight with control at the end of lactation period.

At 15 mg/kg bw/day, body weight gain was statistically significantly lower than control during the last week (Days 14-21) of gestation, resulting in statistically significant lower mean body weight and overall body weight gain at the end of gestation. Body weight gain during the later period of gestation appeared to be influenced by a lower contribution by the developing litter (based on litter weight at birth). Mean body weight at the start of lactation was not significantly different from control but the magnitude of the differences between this dosage and control increased slightly from the start of gestation to the start of lactation.
However, the difference was considered insufficient to indicate an underlying effect on maternal body weight during gestation, particularly in the absence of any similar effect at the high dosage of 25 mg/kg bw/day. Mean body weights were slightly lower than control throughout lactation, with differences attaining statistical significance during Days 7-14, however, the pattern of body weight gain during lactation was similar to control. Statistically significant body weight gain during the last week of lactation attained statistical significance when compared to control. This higher gain may reflect lower demand due to smaller litter size at this dosage, but was insufficient to give parity of mean body weight with control at the end of lactation period.

At 5 mg/kg bw/day, there was no obvious effect of treatment on body weight or body weight gain during gestation or lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg bw/day, there was no obvious adverse effect of treatment on food consumption for males during the pre-mating phases of the F0 generation, however slightly lower food consumption was apparent for these animals during the post-pairing phase of the study. The differences from control for mean food intake were slight and may reflect normal biological variation and the smaller relative size of these animals, compared to their control counterparts, at this stage of the study.
There was no obvious effect of treatment on food consumption for males during the pre- and post-mating phases of the F0 generation at 5 and 15 mg/kg bw/day.

There was no obvious effect of treatment on food consumption for females during the pre mating phase of the F0 generation at 5, 15 or 25 mg/kg bw/day.
At 25 mg/kg bw/day, food consumption of females during the last week of gestation (Days 14-21) was slightly, but statistically significantly, lower than control. This difference may reflect the lower litter size at this dosage compared with control. Food intake was also statistically significantly lower than control from Day 4 of lactation, with statistical significance being reached during the last two weeks (Days 7-21) of lactation. Food consumption during lactation at this dosage may have been influenced by the lower demand on the lactating female from the smaller litter size, compared to control. At 15 mg/kg bw/day, there was no obvious effect of treatment on food consumption during gestation. For females during lactation however, food intake during lactation was lower than control from Day 4 of lactation, with statistical significance being reached during the second week (Days 7-14) of lactation. Food consumption during lactation at this dosage may have been influenced by the lower demand on the lactating female from the smaller litter size, compared to control.

At 5 mg/kg bw/day, there was no obvious effect of treatment on food consumption during gestation and lactation
Food efficiency:
no effects observed
Description (incidence and severity):
There were no consistent inter-group differences in food conversion efficiency values that indicated any obvious effect of treatment on the efficiency of food utilisation for either sex at 5, 15 or 25 mg/kg bw/day. As anticipated, the values for food efficiency decreased as the
animals matured and were no longer in a steep growth curve.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Assessment of the F1 animals in a standard arena did not reveal any obvious effects of treatment for either sex at 5, 15 or 25 mg/kg bw/day.
One female at 15 mg/kg bw/day showed noisy respiration during the standard arena assessment but, as discussed during the evaluation of clinical signs, the incidence of noisy respiration observed during the study was considered to be incidental and unrelated to exposure to the test item.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Stomach
Ulceration in the antral area of the stomach (mild or moderate) was noted in 8/28 males and 1 female at 25 mg/kg bw/day. Degeneration/atrophy of the glandular mucosa (minimal to moderate) was noted in 16/28 males at 25 mg/kg bw/day and one male at 15 mg/kg bw/day (minimal).
No changes related to administration of the test item were present in both sexes at 5 mg/kg bw/day or females at 15 mg/kg bw/day.
Duodenum
Degeneration/atrophy of the mucosa (minimal to moderate) was present in 26/28 males and 13/28 females at 25 mg/kg bw/day. At 15 mg/kg bw/day, it was present in 9/27 males and 2/28 females (minimal or mild). Regenerative hyperplasia of the mucosa (minimal to moderate) was present in 24/28 males and all females at 25 mg/kg bw/day. At 15 mg/kg bw/day, it was present in 13/27 males and 11/28 females (minimal or mild). No changes were present in both sexes at 5 mg/kg bw/day.
Jejunum
Degeneration/atrophy of the mucosa (minimal or mild) was present in 5/28 males and 1/28 females at 25 mg/kg bw/day. Regenerative hyperplasia of the mucosa (minimal or mild) was present in 13/28 males and 11/28 females at 25 mg/kg bw/day. At 15 mg/kg bw/day, it was present at a minimal level in 3/28 females. No changes were present in males at 15 mg/kg bw/day or any animals at 5 mg/kg bw/day.

Lymph Nodes
Mesenteric Lymph Node
In males receiving 25 mg/kg bw/day, erythrocytosis/erythrophagocytosis was present in 26/28, graded from minimal to moderate in severity, with sinusoidal ectasia present in 5/28.
Neither finding was present in control males. In females receiving 25 mg/kg bw/day, erythrocytosis/erythrophagocytosis was present in 12/27 control (minimal) and 18/28 25 mg/kg bw/day females (minimal to mild). There was a minor increase in severity as well as incidence in females receiving 25 mg/kg bw/day. No changes related to administration of the test item were present in both sexes at 5 or 15 mg/kg bw/day.
Mandibular Lymph Node
Cortical atrophy, minimal or mild was present in 13/28 males and 7/27 females at 25 mg/kg bw/day.
Pancreatic Lymph Node
In females, erythrocytosis/erythrophagocytosis was increased in incidence and severity at 25 mg/kg bw/day. In males, sinusoidal ectasia was present in 2/27 animals (minimal) at 5 mg/kg bw/day, 9/28 animals (minimal or mild) at 15 mg/kg bw/day and 7/23 animals (mild or moderate) at
25 mg/kg bw/day.
Spleen
White pulp depletion, minimal, was present in 5/28 males at 25 mg/kg bw/day, 2/28 control females and 7/28 females at 25 mg/kg bw/day. In addition mild hematopoiesis was present in 5/28 females at 25 mg/kg bw/day compared to one control.
No other findings that were present at histopathology correlated with in-life changes noted, all other findings were considered to be incidental.

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of estrous cycles of F0 females during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 5, 15 and 25 mg/kg bw/day. Within this generation there was a relatively high incidence of females that showed irregular estrous cycling or evidence of extended di-estrous (the incidence of extended di-estrous may reflect pseudo-pregnancy from the initiation of smearing). However, the distribution of these non-regular cycles showed no association with treatment and similar high incidences were not apparent in the subsequent F1 generation.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There was no obvious effect of treatment on sperm concentration and sperm motility, homogenisation resistant spermatid counts or sperm morphology for males receiving 5, 15 or 25 mg/kg bw/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
Sexual Maturation

Sexual maturation for adult animals were restricted to the F1 generation.
For males at 15 and 25 mg/kg bw/day, the age of attainment of balano preputial separation was statistically significantly later than control, however mean body weight at the age of attainment was similar to control. The similarity in body weight indicated the apparent delay in sexual maturation reflects lower growth/maturity for males at these dosages rather than any underlying disturbance of sexual development.

For males at 5 mg/kg bw/day, age and body weight on the day of attainment of balano preputial separation was similar to control and unaffected by treatment. There was no obvious effect of treatment on age and body weight on the day of attainment of vagina opening for females receiving 5, 15 and 25 mg/kg bw/day.

One control female allocated to the F1 generation showed no vaginal opening during the study and had to be excluded from the assessment of sexual maturation.

Assessment of estrous cycles of F0 females during the pre-pairing phase of the study did not indicate any obvious effect of treatment at 5, 15 and 25 mg/kg bw/day.

Within this generation there was a relatively high incidence of females that showed irregular estrous cycling or evidence of extended di-estrous (the incidence of extended di-estrous may reflect pseudo-pregnancy from the initiation of smearing). However, the distribution of these
non-regular cycles showed no association with treatment and similar high incidences were not apparent in the subsequent F1 generation.

Mating performance as assessed by the number of paired animals that mated and pre-coital interval was unaffected by treatment at 5, 15 and 25 mg/kg bw/day. The majority of paired animals showed evidence of mating within the first four days of pairing indicating mating at the first estrous opportunity.

At 25 mg/kg bw/day, four mated females subsequently failed to achieve pregnancy; this incidence was slightly higher than observed for the control and other treatment groups within this generation. However, the incidence of non-pregnancy at this dosage was better than control during the F1 generation, therefore this finding may reflect normal biological variation and no association with treatment was considered proven.

There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at 5 and 15 mg/kg bw/day.

The intergroup distribution of gestation lengths observed during F0 generation did not indicate any obvious effect of treatment at 5, 15 and 25 mg/kg bw/day.

Two, one and four F0 females were found to be non-pregnant at 0 (control), 5 and 25 mg/kg bw/day respectively. Additionally, one pregnant female at 5 mg/kg bw/day was found dead around the time of parturition (and the litter was therefore killed) and one female at 15 mg/kg bw/day showed evidence of pregnancy but no litter was observed to be born. Among littering females, one control female, one female at 5 mg/kg bw/ay and five females at 25 mg/kg bw/day showed total litter loss post partum. The following assessment is generally based on the 25, 25, 27 and 19 females successfully rearing young to weaning (Day 21 of age) at 0 (Control), 5, 15 and 25 mg/kg bw/day respectively.

At 25 mg/kg bw/day, the number of implantation sites was slightly lower than control, although differences from control failed to attain statistical significance. Post-implantation loss at this dosage was only slightly higher than control and also failed to attained statistical significance. However the combination of implantation count and post-implantation loss, resulted in statistically significant lower litter size at birth/Day 1 compared to control, which persisted to weaning (Day 21 of age). Post-natal offspring survival for litters surviving to weaning appear to be unaffected by maternal treatment, however a total of five females failed to maintain their litter to weaning. Many of these offspring losses occurred on or shortly after birth with all five total litter losses being evident by Day 3 of age.

At 15 mg/kg bw/day, the number of implantation sites was slightly lower than control, but differences from control failed to attain statistical significance. Post-implantation loss at this dosage was also slightly, but not statistically significantly, higher than control. The combination of implantation count and post-implantation loss subsequently resulted in statistically significant lower litter size at birth/Day 1 compared to control, which persisted to weaning (Day 21 of age). Post-natal offspring survival for litters surviving to weaning appear to be unaffected by maternal treatment, with all litters surviving to weaning.

At 5 mg/kg bw/day, litter size at birth/Day 1 and subsequent offspring survival to weaning appeared unaffected by maternal treatment. Post-implantation loss at this dosage was slightly higher than control but, in the absence of any adverse effect on litter size at birth/Day 1 this finding was considered to reflect normal biological variation rather than any effect of treatment.

There was no obvious effect of treatment on sex ratio at 5, 15 and 25 mg/kg bw/day. Occasional differences in sex ratio from control did attain statistical significance on Days 14 and 21 at 5 mg/kg bw/day and on Days 7, 14 and 21 at 25 mg/kg bw/day. As these valueswere only marginally different from those observed at birth/Day 1, they were regarded as incidental and not to indicate any selective effect of maternal treatment on offspring survival for either sex.
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Critical effects observed:
no
Lowest effective dose / conc.:
25 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the type, incidence nor distribution of clinical signs among surviving animals indicated any obvious adverse effect of treatment at 5, 15 or 25 mg/kg bw/day. Isolated incidences of increased post-dosing salivation were observed for all males and nineteen females receiving 25 mg/kg bw/day during the F1 generation. Similar increased post-dosing salivation was also observed for thirteen males and one female receiving 15 mg/kg bw/day. Increased post-dosing salivation was only observed for one male on one occasion at 5 mg/kg bw/day. As previously discussed, increased post-dosing salivation is often observed when animals are dosed via the oral gavage route and, at the incidence observed, most probably represent isolated difficulties in dosing some of the animals during the study, in combination with distaste/ slight irritancy of the dosing formulations, rather than any systemic effect of exposure to the test item. Isolated incidences of noisy respiration were observed for two males receiving 5 mg/kg bw/day, three males and two females receiving 15 mg/kg bw/day and five males receiving 25 mg/kg bw/day during the F1 generation, however similar noisy respiration was also apparent for two control males and one control female. Although, the incidence of this finding was slightly higher amongst F1 animals than that observed for animals of the F0 generation there was no indication that this was related to the relatively earlier exposure to the test item in the F1 generation and, this finding was considered to be incidental and
unrelated to treatment.
One female at 5 and one male at 15 mg/kg bw/day showed a mass during the course of the F1 generation but, these isolated findings were considered to be incidental and unrelated to exposure to the test item.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was one unscheduled adult death amongst F1 generation animals affecting one male receiving 5 mg/kg bw/day.
Male number 272 (5 mg/kg bw/day) showed staining around the snout during Days 93-95, noisy respiration during Days 93-95 and 98, piloerection during Days 93-94, hunched posture during Days 93-98 and chromodacryorrhea during Day 93 of the F1 generation. No clinical signs were apparent during Day 99 but the animal was subsequently found dead on Day 100 of the F1 generation. Macroscopic necropsy revealed a gross lesion in the oesophagus, enlarged stomach with thin appearance and dark red contents, red contents within jejunum and ileum, enlarged spleen with dark areas and enlarged liver with pale and mottled appearance. The necropsy findings indicated that death was most probably the results of accidental trauma at some stage during the dosing procedure and as such, this death was clearly unrelated to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg bw/day, body weight gain of males was statistically significantly lower than control throughout the ten week pre-pairing phase (to Day 71) and subsequent body weight gain also tended to be statistically significantly lower than control to termination. Initial mean body weight at the formal start of the F1 generation was slightly, but not statistically significantly, lower than control, however the lower body weight gain at this dosage resulted in statistically significant lower mean body weight after the first week (Day 8) compared with control which persisted until termination. Overall body weight gain was also statistically significantly lower than control.
At 15 mg/kg bw/day, body weight gain of males tended to be lower than control during the ten week pre-pairing phase (to Day 71), with differences attaining statistical significance during Week 2 (Day 8-15), Weeks 4-7 (Days 22-50) and Week 9 (Days 57-64). Subsequent body weight gain to termination tended to be similar control, although lower body weight gains during Week 14 (Days 92-99) and 17 (Days 113-120) and higher body weight gain during Week 18 attained statistical significance. Initial mean body weight at the formal start of the F1 generation was slightly, but not statistically significantly, lower than control, however the lower body weight gain at this dosage resulted in statistically significant lower mean body weight after five weeks (Day 36) compared with control which persisted until termination. Overall body weight gain was also statistically significantly lower than control.

For males at 5 mg/kg bw/day, mean body weight and body weight gains, including overall body weight gain, were considered to be unaffected by treatment. Occasional statistically significant difference in body weight gain, compared to control, were observed but these were considered to be incidental and reflect normal biological variation. For females, mean body weight and body weight gains, including overall body weight gain, during the ten week pre-pairing period was unaffected by treatment at 5, 15 or 25 mg/kg bw/day.

At 25 mg/kg bw/day, body weight gain was statistically significantly lower than control during the last week (Days 14-21) of gestation, resulting in statistically significant lower mean body weight and overall body weight gain at the end of gestation. Body weight gain during the later period of gestation appeared to be influenced by a lower contribution by the developing litter (based on litter weight at birth). However, there was no increase in mean body weight from the start of gestation to the start of lactation which may indicate a slight underlying effect on maternal body weight during gestation. Mean body weight was lower than control throughout lactation with statistical significance being attained from Day 4 to termination (Day 21), despite a statistically significant higher body weight gain during the last week of lactation. This higher body weight gain during the final week of lactation may reflect lower demand due to smaller litter size at this dosage however the pattern of body weight gain during lactation was similar to control. At 15 mg/kg bw/day, body weight gain was statistically significantly lower than control during the last week (Days 14-21) of gestation, resulting in statistically significant lower mean body weight and overall body weight gain at the end of gestation. Body weight gain during the later period of gestation appeared to be influenced by a lower contribution by the developing litter (based on litter weight at birth). Mean body weight at the start of lactation was not significantly different from control but the magnitude of the differences between this dosage and control increased slightly from the start of gestation to the start of lactation, although the difference was probably insufficient to indicate a clear underlying effect on maternal body weight during gestation. Mean body weight was lower than control throughout lactation with statistical significance being attained from Day 7 to termination (Day 21), despite a statistically significant higher body weight gain during the last week of
lactation. This higher body weight gain during the final week of lactation may reflect lower demand due to smaller litter size at this dosage however the pattern of body weight gain during lactation was similar to control.
At 5 mg/kg bw/day, there was no obvious effect of treatment on body weight or body weight gain during gestation or lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At 25 mg/kg bw/day, slightly lower food consumption was apparent during the later stages of the pre-pairing phase (from Day 64) and most of the post-pairing phase of the F1 generation.
The differences from control for mean food intake were slight and may reflect normal biological variation and the smaller relative size of these animals, compared to their control counterparts.
There was no obvious effect of treatment on food consumption for males during the pre- and post-mating phases of the F1 generation at 5 and 15 mg/kg bw/day.

There was no obvious effect of treatment on food consumption for females during the pre mating phase or gestation phase of the F1 generation at 5, 15 or 25 mg/kg bw/day. At 15 and 25 mg/kg bw/day, food consumption was statistically significantly lower than control through lactation. Food consumption during lactation at these dosages may have been influenced by the lower demand on the lactating female from the smaller litter size, compared to control. At 5 mg/kg bw/day, there was no obvious effect of treatment on food consumption during gestation and lactation.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no consistent inter-group differences in food conversion efficiency values that indicated any obvious effect of treatment on the efficiency of food utilisation for either sex at 5, 15 or 25 mg/kg bw/day. As anticipated, the values for food efficiency decreased as the
animals matured and were no longer in a steep growth curve.
Sexual maturation:
no effects observed
Description (incidence and severity):
For males at 15 and 25 mg/kg bw/day, the age of attainment of balano preputial separation was statistically significantly later than control, however mean body weight at the age of attainment was similar to control. The similarity in body weight indicated the apparent delay
in sexual maturation reflects lower growth/maturity for males at these dosages rather than any underlying disturbance of sexual development.
For males at 5 mg/kg bw/day, age and body weight on the day of attainment of balano preputial separation was similar to control and unaffected by treatment.
There was no obvious effect of treatment on age and body weight on the day of attainment of vagina opening for females receiving 5, 15 and 25 mg/kg bw/day.
One control female allocated to the F1 generation showed no vaginal opening during the study and had to be excluded from the assessment of sexual maturation.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 25 mg/kg bw/day, absolute and body weight relative spleen weights for males were statistically significantly lower than control. At 25 mg/kg bw/day, absolute and body weight relative uterus weights for females were statistically significantly lower than control. At 25 mg/kg bw/day, absolute epididymal (including left cauda), testes, prostate and seminal vesicles weights were statistically significantly lower than control. Body weight relative epididymal (including left cauda) and testes weights were statistically significantly higher than control and body weight relative prostate and seminal vesicles weights were statistically significantly lower than control. Similar changes in male reproductive organ weights were apparent at 15 mg/kg bw/day, with absolute epididymal (including left cauda), testes, prostate and seminal vesicles weights being statistically significantly lower than control. Body weight relative testes weights were statistically significantly higher than control and body weight relative epididymal (including left cauda), prostate and seminal vesicles weights were statistically significantly lower than control. These statistical significance differences in male reproductive organ weights were not associated with adverse effect on measured sperm parameters or histopathological change.
At 25 mg/kg bw/day, lower absolute and higher body weight relative kidney weights for males attained statistically significance when compared with control. For females, both absolute and body weight relative kidneys values were statistically significantly higher than control. In the absence of any histopathological change these findings were considered to be of no toxicological significance.
For males at all dosages, absolute thyroid weights were statistically significantly lower than control; however mean values showed no dosage relationship. Although differences from control continued to show statistical significance, the lack of dosage relationship was more apparent when body weight relative thyroid weights were considered, with values at 5 and 15 mg/kg bw/day being lower than control and values at 25 mg/kg bw/day being higher.
Additionally lower absolute and body weight relative thyroid weights for females at 25 mg/kg bw/day were statistically significantly lower than control, although no statistically significant differences were apparent for females receiving 25 mg/kg bw/day. In the absence of any supporting histopathological change, the observed differences in thyroids weights were considered to be of no toxicological significance.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Stomach
Ulceration in the antral area of the stomach (mild or moderate) was noted in 6/24 males and 2 females at 25 mg/kg bw/day. Erosion was present in 8/24 males at 15 mg/kg bw/day along with 2 males and 1 female at 25 mg/kg bw/day. Degeneration/atrophy of the glandular mucosa (minimal or mild) was noted in 11/24 males and 1 female at 25 mg/kg bw/day. It was present at a minimal level in 4/24 males at 15 mg/kg bw/day. No changes were present in the stomach of both sexes at 5 mg/kg bw/day and females at 15 mg/kg bw/day.

Duodenum
Degeneration/atrophy of the mucosa (minimal to moderate) was present in all males and 15/24 females at 25 mg/kg bw/day. It was also present in 10/24 males (minimal or mild) at 15 mg/kg bw/day. Regenerative hyperplasia of the mucosa (minimal or mild) was present in 15/24 males and all
females at 25 mg/kg bw/day. At 15 mg/kg bw/day, it was present in 13/24 males and 4/24 females. No changes were present in the duodenum of animals at 5 mg/kg bw/day.
Jejunum
Degeneration/atrophy of the mucosa (minimal) was present in 3/24 males at 15 mg/kg bw/day and 9/24 males and 10/24 females at 25 mg/kg bw/day. Regenerative hyperplasia of the mucosa (minimal or mild) was present in 3/24 males and one female at 15 mg/kg bw/day and 16/24 males and 22/24 females at 25 mg/kg bw/day.
Ileum
Regenerative hyperplasia of the mucosa (mild) was present in 3/24 females at 25 mg/kg bw/day.
Caecum
Mucosal hypertrophy (minimal) was present in 1/24 males at 5 mg/kg bw/day, 2/24 males at 15 mg/kg bw/day and 5/24 males at 25 mg/kg bw/day. Mucosal hypertrophy (minimal or mild) was present in one female at 5 mg/kg bw/day, 4/24 females at 15 mg/kg bw/day and 7/24 females at 25 mg/kg bw/day. There was an increase in mucosal inflammation in females at 25 mg/kg bw/day. The incidence in both sexes at 5 mg/kg bw/day and males at 15 mg/kg bw/day was considered to be incidental.
Lymph Nodes
Mesenteric Lymph Node In males, erythrocytosis/erythrophagocytosis was present in 7/24 animals at 15 mg/kg bw/day and 20/24 at 25 mg/kg bw/day. It showed a dose related increase in severity
Sinusoidal ectasia was present in 7/24 males at 25 mg/kg bw/day compared with 1/24 Control males and 2/24 males at 15 mg/kg bw/day.
In females, erythrocytosis/erythrophagocytosis (minimal) was present in 6/24 control animals, 2/24 animals at 5 mg/kg bw/day and 4/24 animal at 15 mg/kg bw/day.
Erythrocytosis/erythrophagocytosis (minimal or mild) was present in 19/24 Group at 25 mg/kg bw/day; there was an increase in severity as well as incidence at this dosage.
Mandibular Lymph Node Cortical atrophy, minimal or mild was present in 2/24 males receiving 25 mg/kg bw/day.
Pancreatic Lymph Node
In females, erythrocytosis/erythrophagocytosis was marginally increased in incidence at 25 mg/kg bw/day.
Sinusoidal ectasia was present in one control male (minimal), 11/24 males and 2/24 females at 15 mg/kg bw/day and 14/24 males and 12/24 females in at 25 mg/kg bw/day (minimal to moderate).
Spleen
White pulp depletion, minimal, was present in 7/24 males and 2/24 females at 25 mg/kg bw/day.
Hematopoiesis was present in a few animals from treated groups at a low incidence and severity, showing no dose dependent trend and was therefore considered to be incidental.
No other findings that were present at histopathology correlated with in-life changes noted, all other findings were considered to be incidental.
Female Reproductive Tract
There were 23/24 pregnant females at 25 mg/kg bw/day compared with 19/24 pregnant females in the concurrent control. One of the females at 25 mg/kg bw/day suffered total litter loss.
Of the lactating animals, vaginal mucification (pregnancy/lactation morphology) was present in 10/19 control females and 19/22 females at 25 mg/kg bw/day. This indicates fewer females at 25 mg/kg bw/day had returned to normal cycling patterns. Levels of mucification
in females receiving 5 or 15 mg/kg bw/day were similar to control animals.
Qualitative examination of the ovaries did not show any consistent differences and in general the appearance of the ovaries and uterus was as expected for the cycle stage.
Follicle counting carried out in the pathology laboratory indicated a statistically significant reduction in primordial follicles at 25 mg/kg bw/day compared to the concurrent control. No statistically significant differences were apparent for females at 5 or 15 mg/kg bw/day
compared with controls.
Other effects:
no effects observed
Description (incidence and severity):
For male offspring at all dosages, ano-genital distance was higher than control, attaining statistical significance at 5 and 25 mg/kg bw/day, and remained higher than control when normalised for body weight, attaining statistical significance at all dosages. However the differences in ano-genital distance showed no dosage relationship and were therefore considered to reflect normal biological variation and to be unrelated to maternal treatment.
No statistically significant differences in ano-genital distance were apparent for female offspring at 5, 15 or 25 mg/kg bw/day.
There was no obvious effect of maternal treatment on offspring surface righting, air righting, startle response and pupil reflex abilities, with the percentage of successful offspring being similar to control at 5, 15 and 25 mg/kg bw/day.
At 25 mg/kg bw/day offspring clinical signs to Day 15 of age were typical for the age observed and the incidence and type of clinical signs observed did not indicate any obvious effect of maternal treatment. The incidence of clinical signs such as cold, pale, no milk in stomach between Days 1-4 of age was marginally higher than control at this dosage but these clinical signs are not unusual and were restricted to isolated litters. However, during the third week of lactation (Days 15-21 of age), a small number of offspring (five litters affected) were noted to be pale; this finding is considered to be unusual for animals at this age.
At 5 or 15 mg/kg bw/day, offspring clinical signs were typical for the age observed and the incidence and type of clinical signs observed did not indicate any obvious effect of maternal treatment on development. At 15 mg/kg bw/day, the incidence of clinical signs such as cold
or weak between Days 1-4 of age was marginally higher than control but these clinical signs are not unusual and were restricted to only a few litters
Behaviour (functional findings):
no effects observed
There was no obvious effect of treatment on sperm concentration and sperm motility, homogenisation resistant spermatid counts or sperm morphology for males receiving 5, 15 or 25 mg/kg bw/day.
F1 Necropsy
At 25 mg/kg bw/day, two decedent males and six decedent females (one litter affected) showed gaseous distension of the stomach and gastro-intestinal tract. At 15 mg/kg bw/day, similar gaseous distension of the stomach was apparent for four decedent males and nine
decedent females (two litters affected), with one male and five females also showing gaseous distension of the gastro-intestinal tract.
At 25 mg/kg bw/day, amongst animals surviving to termination showed an increase incidence of small and/or pale offspring, compared to control. Necropsy revealed an increased incidence of findings for the lungs (pale or grey discoloration), liver (including pale, mottled
appearance, white/grey/green discoloration), heart (enlarged) and kidneys (including pale and increased pelvic space/pelvis dilated) and thymus (small), compared to control.
At 15 mg/kg bw/day, although isolated offspring at termination showed some similar findings for the lungs, liver and kidneys to that seen at 25 mg/kg bw/day, the incidence of these findings were very low and were considered insufficient to indicate an effect of maternal
treatment on the offspring.
At 5 mg/kg bw/day, neither the incidence, type nor distribution of macroscopic necropsy findings for decedent offspring or offspring killed at termination (Day 21 of age) indicated any obvious effect of maternal treatment on the offspring.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
Dose descriptor:
NOEL
Generation:
F1
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
histopathology: neoplastic
other: Offspring necropsy
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
duodenum
ileum
jejunum
lymph node
mesenteric lymph node
spleen
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Reproductive effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
Based on the results of this study, a dosage of 5 mg/kg bw/day is considered to be a No Observed Effect Level (NOEL) for the repeated administration of the test item on reproduction, pre-natal development and post-natal development of the rat when administered over two successive generations.
Executive summary:

The study was performed to establish the effects of the repeated administration of the test item on reproduction, pre-natal development and post-natal development of the rat when administered over two successive generations. The results of the study are believed to be of value in predicting the reproductive toxicity of the test item to man and may allow the estimation of the ‘No Observed Effect Level’ (NOEL) for adults and their subsequent progeny.

The study was designed to be in compliance with the following regulations or guidelines:

· US EPA Health Effects Test Guideline OPPTS 870.3800 (August 1998).

· OECD Guidelines for the Testing of Chemicals, No. 416 “Two-Generation Reproduction Toxicity Study” (adopted 22 January 2001).

· Japanese Ministry of Agriculture Forestry and Fisheries (JMAFF), Testing Guidelines for Toxicology Studies (2-1-17), 12 Nohsan No. 8147, November 24, 2000 (partially revised on June 26, 2001).

The study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The effects of exposure to the test item was determined by daily observation of general condition for clinical signs of toxicity, measurement of body weight and food consumption, assessment of mating, fertility and littering, including the growth and survival of litters to weaning, macroscopic examination at necropsy for pathological changes together with selected adult organ weight assessment and histopathology over two generations.

Methods

The test item was administered once daily by oral gavage to three groups each of twenty-eight male and twenty-eight female Wistar Han™:RccHan™:WIST strain rats, at dosages of 5, 15 and 25 mg/kg bw/day throughout the study. A further group of twenty-eight male and twenty-eight female F0 Generation rats were similarly dosed with the vehicle (Propylene Glycol) to serve as a control.

Clinical signs, body weight development, food and water consumption were monitored during the study. After 10 weeks of treatment, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce the F1 litters. At weaning of offspring from the F0 mating phase, groups of twenty-four male and twenty-four female offspring from each dose group were selected to form the F1 generation. The remaining surviving F0 females and unselected offspring were terminated at Day 21 post partum, followed by the termination of all F0 male dose groups.

The effects of exposure to the test item was determined by daily observation of general condition for clinical signs of toxicity, measurement of body weight and food consumption,assessment of mating, fertility and littering, including the growth and survival of litters to weaning, macroscopic examination at necropsy for pathological changes together with selected adult organ weight assessment and histopathology over two generations.

The offspring selected for the F1 generation (including control) were dosed at their respective dosage from Day 22 of age and then dosed for at least 10 weeks of the formal F1 generation before being paired within each dose group to produce the F2 litters. At weaning of the F2 litters all surviving adults and their offspring were killed, followed by the termination of all F1 male dose groups. Oestrous cycle assessment was performed daily for three weeks prior to mating for both the

F0 and F1 generations. Observations for positive evidence of mating were recorded together with the start and completion of parturition. During the maturation phase of the F1 generation offspring, males and females were evaluated for sexual maturation. The ano-genital distance was recorded for all F2 generation offspring on Day 1 post partum. During the lactation phases daily clinical observations were performed on all surviving offspring, together with litter size. Litter weight, individual offspring weights and landmark developmental signs were also recorded on specific days post partum. All animals at termination were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Adult Responses

Mortality

There were three unscheduled adult deaths amongst F0 generation adult animals and one unscheduled death amongst F1 generation adult animals during the study.

At 5 mg/kg bw/day, Female 141 was found dead around the time of parturition on Day 101 of the F0 generation; this death was considered to reflect difficulties during the parturition process. At 5 mg/kg bw/day, Male 272 was found dead on Day 100 of the F1 generation. Macroscopic necropsy included a gross lesion in the oesophagus indicating that this death was the result of accidental trauma at some stage during the dosing procedure. At 15 mg/kg bw/day, Male 72 was found dead on Day 100 of the F0 generation. Clinical signs, macroscopic necropsy findings and subsequent histopathology findings did not indicate any obvious cause for this death.

At 15 mg/kg bw/day, Male 76 was killed in extremis on Day 106 of the F0 generation after showing adverse clinical signs including lethargy, piloerection, pallor of the extremities and staining around the snout. Necropsy revealed an enlarged and distended stomach filled with dark coloured contents and pale liver, kidneys and prostate, however histopathological assessment did not indicate any obvious cause of death.

These deaths at 5 and 15 mg/kg bw/day, in the absence of mortalities at 25 mg/kg bw/day, were considered to be incidental and unrelated to treatment.

Clinical Observations

Clinical signs were generally restricted to isolated incidences of increased post-dosing salivation for one F1 male at 5 mg/kg bw/day and F1 males and occasional F0 and F1 females at 15 mg/kg bw/day and both sexes during the F0 and F1 generations at 25 mg/kg bw/day, and did not indicate any obvious systemic effect of treatment.

Behavioral Assessments

Assessment of the F1 animals in a standard arena did not reveal any obvious effects of treatment for either sex at 5, 15 or 25 mg/kg bw/day.

Functional Performance Tests

Intergroup differences in motor activity and grip strength for F1 animals did not reveal any obvious effects of treatment for either sex at 5, 15 or 25 mg/kg bw/day.

Sensory Reactivity Assessments

Intergroup differences observed in the scores for sensory reactivity for F1 animals did not reveal any obvious effects of treatment for either sex at 5, 15 or 25 mg/kg bw/day.

Body Weight

At 25 mg/kg bw/day, body weight gain of males was lower than control throughout the ten week pre-pairing phase (to Day 71), with subsequent body weight gain to termination also tending to be lower than control for both generations. The lower body weight gain observed resulted in lower mean body weight compared with concurrent control on Day 15 for F0 males and Day 8 for F1 males which persisted until termination. Overall body weight gain of males was also lower than concurrent control across both generations.

At 15 mg/kg bw/day, body weight gain of males was generally lower than concurrent control for the first seven weeks of the ten week F0 pre-pairing phase and throughout the ten week F1 pre-pairing phase. The lower body weight gains resulted in lower mean body weight, compared with concurrent control, on Day 22 for F0 males and Day 36 for F1 males which persisted until termination. Overall body weight gain of males was also statistically significantly lower than concurrent control across both generations.

At 5 mg/kg bw/day, mean body weight and body weight gains, including overall body weight gain, for males were considered to be unaffected by treatment across both generations. For females, mean body weight and body weight gains, including overall body weight gain, during the ten week pre-pairing period was unaffected by treatment across both generations at 5, 15 or 25 mg/kg bw/day.

At 25 mg/kg bw/day, lower body weight gain, compared to concurrent control, was apparent during the last week of gestation, resulting in lower mean body weight and overall body weight gain at the end of gestation across both generations. For both generations, body weight gain during this period of gestation was influenced by a lower contribution by the developing litter (based on litter weight at birth). For F1 females, there was no increase in mean body weight from the start of gestation to the start of lactation which may indicate a slight underlying effect on body weight during gestation. The pattern of body weight gain during lactation was similar to concurrent control in both generations, however higher body

weight gains were apparent for F0 females during last week of lactation and for F1 females from Day 4 of lactation. The higher gain may reflect lower demand due to smaller litter size at this dosage compared to concurrent control and recovery from lower gain observed during late gestation.

At 15 mg/kg bw/day, body weight gain lower than control during the last week (Days 14-21) of gestation, resulting in lower mean body weight and overall body weight gain at the end of gestation across both generations. For both generations, body weight gain during the later period of gestation was influenced by a lower contribution by the developing litter (based on litter weight at birth). The pattern of body weight gain during lactation was similar to concurrent control in both generations, however statistically significant higher body weight gains were apparent during last week of lactation in both generations. This higher gain may reflect lower demand due to smaller litter size at this dosage compared to control.

At 5 mg/kg bw/day, there was no obvious effect of treatment on body weight or body weight gain during gestation or lactation across both generations.

Food Consumption

At 25 mg/kg bw/day, pre-pairing food consumption for F1 males was slightly lower than concurrent control from Day 64 of the pre-pairing phase and slightly lower food intake was also apparent, compared to concurrent control, during the post-pairing phase of both generations. These differences may reflect the relatively smaller relative size of these animals, compared to their control counterparts.

There was no obvious effect of treatment on male food consumption at 5 and 15 mg/kg bw/day across both generations.

There was no obvious effect of treatment on pre-mating food consumption for females at 5, 15 or 25 mg/kg bw/day across both generations.

At 25 mg/kg bw/day, food consumption of F0 females was lower than concurrent control during the last week of gestation; however, there was no obvious effect on gestation food consumption for F1 females at this dosage. Subsequent food consumption during lactation at 25 mg/kg bw/day was lower than concurrent control from Day 4 for F0 females and throughout lactation for F1 females. This lower food consumption during lactation may have been influenced by lower demand on the lactating female from the smaller litter size, compared to control, across both generations.

At 15 mg/kg bw/day, there was no obvious effect of treatment on gestation food consumption across both generations. Subsequent food consumption during lactation at 15 mg/kg bw/day was lower than concurrent control from Day 4 for F0 females and throughout lactation for F1 females. This lower food consumption during lactation may have been influenced by lower demand on the lactating female from the smaller litter size, compared to control, across both generations.

At 5 mg/kg bw/day, there was no obvious effect of treatment on food consumption during gestation and lactation across both generations.

Food Conversion Efficiency

There were no obvious effect of treatment on the efficiency of food utilisation for either sex during the pre-pairing phase of the study or for males during the post-pairing phase of the study in either generation.

Reproductive Performance

Sexual Maturation

At 15 and 25 mg/kg bw/day, the age of attainment of balano preputial separation for F1 males was later than control, but mean body weight at attainment was similar to control indicating a probable association with delays in growth/maturity rather than any underlying disturbance of sexual development. At 5 mg/kg bw/day, the age and body weight on the day of attainment of balano preputial separation of F1 males was unaffected by treatment. There was no obvious effect of treatment on age and body weight on the day of attainment of vagina opening for F1 females receiving 5, 15 and 25 mg/kg bw/day.

Estrous Cycle

Estrous cycles for females during the pre-pairing phases of the study appeared unaffected by treatment at 5, 15 and 25 mg/kg bw/day in either generation.

Mating

Mating performance appeared unaffected by treatment at 5, 15 and 25 mg/kg bw/day in either generation.

Fertility

At 25 mg/kg bw/day four mated F0 females failed to achieve pregnancy but only one female was non-pregnant in the F1 generation and an association with treatment appeared unlikely.

There was no obvious effect on fertility, as assessed by the number of females that achieved pregnancy, at 5 and 15 mg/kg bw/day across both generations.

Gestation Length

Gestation lengths appeared unaffected by treatment at 5, 15 and 25 mg/kg bw/day in either generation.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

At 25 mg/kg bw/day, although differences from control were only slight, lower number of implantation sites and higher post-implantation loss resulted in lower litter size at birth/Day 1 compared to control across both generations. In both generations, subsequent post-natal offspring survival for litters surviving to weaning appeared unaffected by maternal treatment, however a total of five F0 females failed to maintain their litter to weaning. This high incidence of total litter loss was not apparent amongst F1 females with only one female failing to rear offspring to weaning. At 15 mg/kg bw/day, although differences from control were only slight, lower number of implantation sites and higher post-implantation loss resulted in lower litter size at birth/Day 1 compared to control across both generations. In both generations, subsequent post-natal offspring survival for litters surviving to weaning appeared unaffected by maternal treatment, however a total of six F1 females failed to maintain their litter to weaning. No instances of total litter loss had been apparent for F0 females. At 5 mg/kg bw/day, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to weaning was considered to be unaffected by maternal treatment across both generations.

There was no obvious effect of treatment on sex ratio at 5, 15 and 25 mg/kg bw/day across both generations.

Offspring Growth

At 25 mg/kg bw/day, offspring body weight of the F1-F2 offspring on Day 1 of age and body weight gains from Day 4 to weaning (Day 21 of age), were lower than control.

Litter weights

were lower than control from Day 1 to weaning reflecting both lower litter size and the inferior offspring body weight gain at this dosage. For F0-F1 offspring, body weight on Day 1 of age was similar to control and body weight gains from Day 4 were only marginally lower than control, although this may have been influenced by the five litter losses removing a number of offspring that failed to thrive. F0-F1 litter weights were lower than control from Day 1 to weaning but principally as a consequence of the lower litter size at this dosage. At 15 mg/kg bw/day, body weight of the offspring on Day 1 of age and subsequent body weight gain of the offspring to weaning (Day 21 of age) appeared unaffected by treatment across both generations. However, for F1-F2 offspring, body weight gains may have been influenced by the six litter losses removing a number of offspring that failed to thrive. Litter weight was lower than control from Day 1 to weaning as a consequence of lower litter size in both generations. At 5 mg/kg bw/day, body weight of the offspring on Day 1 of age, subsequent body weight gain to weaning and litter weights appeared unaffected by maternal treatment across both generations.

Offspring Development

At 25 mg/kg bw/day, a slightly lower percentage of F0-F1 offspring successfully showed surface righting reflex on Day 1 of age compared to control, but this finding was not repeated for F1-F2 offspring. The success rate for offspring air righting reflex, startle response and pupil reflex appeared unaffected by maternal treatment across both generations. At 5 and 15 mg/kg bw/day, there was no obvious effect of maternal treatment on offspring surface righting, air righting, startle response and pupil reflex abilities, across both generations. There was no obvious effect of maternal treatment on ano-genital distance for either sex at 5, 15 and 25 mg/kg bw/day across both generations. At 15 and 25 mg/kg bw/day, the incidence of clinical signs such as cold, weak, no milk in stomach between Days 1-4 of age was marginally higher than control across both generations although these clinical signs are not unusual and findings were generally restricted to a few litters. However at 25 mg/kg bw/day, a small number of offspring were noted to be pale during the third week of lactation, a finding considered to be unusual for animals at this age. At 5 mg/kg bw/day, offspring clinical signs did not indicate any obvious effect of maternal treatment across both generations.

Terminal Investigations

Adult Necropsy

At 25 mg/kg bw/day, two F0 males and five F1 males showed gaseous distension of the stomach (the stomach of the F0 males was filled with yellow coloured fluid) and a further three F0 males showed ulceration of the glandular region of the stomach. Additionally five F1 females showed gaseous distension of the stomach and/or parts of the intestinal tract. These findings suggest a local irritant effect of the test item rather than any systemic toxicity. At 15 mg/kg bw/day, gaseous distension of the stomach was observed for only one F1 male, there were no similar findings for F0 males or females from both generations. At 5 mg/kg bw/day, macroscopic findings at terminal necropsy for both sexes did not indicate

any obvious effect of treatment in either generation.

Sperm Analysis

At 5, 15 or 25 mg/kg bw/day, there was no obvious effect of treatment on sperm concentration and sperm motility, homogenisation resistant spermatid counts or sperm morphology for males across both generations.

Adult Organ Weights

At 25 mg/kg bw/day, lower absolute and body weight relative spleen weights, compared to control, were apparent for males of both generation and F0 females. Absolute and body weight relative spleen weights were also lower than control for F0 males at 15 mg/kg bw/day. At 25 mg/kg bw/day, absolute and body weight relative uterus weights for F1 females were lower than control.

Adult Histopathology

At 25 mg/kg bw/day, ulceration in the antral area of the stomach was apparent for some males and occasional females from both generations. Erosion of the stomach was also observed for a few further F1 males and one F1 female at this dosage. Degeneration/atrophy of the glandular mucosa was apparent for some males from both generations and one F1 female. At 15 mg/kg bw/day, microscopic changes observed for the stomach were restricted to

degeneration/atrophy of the glandular mucosa for one F0 male.

At 25 mg/kg bw/day, degeneration/atrophy of the mucosa of the jejunum was present for some males over both generations, one F0 female and some F1 females. Regenerative hyperplasia of the mucosa of the jejunum was present for some F0 animals (both sexes) and F1 males and the majority of F1 females. Degeneration/atrophy of the mucosa of the duodenum was evident for the majority of F0 males, all F1 males and some females across both generations. Regenerative hyperplasia of the mucosa of the duodenum was apparent for the majority of males over both generations, all F0 females and some F1 females. Additionally, regenerative hyperplasia of the mucosa of the ileum was observed for occasional F1 females and mucosal hyperplasia of the caecum was apparent for some animals (both sexes) in the F1 generation, an increase in mucosal inflammation also being observed for the F1 females.

At 25 mg/kg bw/day, erythrocytosis/erythrophagocytosis was present in the mesenteric lymph node of the majority of males across both generations, with sinusoidal ectasia also being present for some males in both generations. For females, although erythrocytosis/erythrophagocytosis within the mesenteric lymph node was apparent for some control females in both generations, there was an increase in severity and incidence for females receiving 25 mg/kg bw/day with the majority of females across both generations being affected. Cortical atrophy for the mandibular lymph node was observed for some F0 animals (both sexes) and occasional F1 males. For the pancreatic lymph node, erythrocytosis/erythrophagocytosis was increased in incidence and severity, compared to control, for females at 25 mg/kg bw/day across both generations. Sinusoidal ectasia was also present in the pancreatic lymph node for some F0 males and some F1 animals (both sexes). At 25 mg/kg bw/day, white pulp depletion in the spleen was present for some males across both generations, some F0 females and occasional F1 females. In addition, mild hematopoiesis was present for some females across both generations.

At 25 mg/kg bw/day, for lactating F1 females, there was a higher incidence of vaginal mucification (pregnancy/lactation morphology) compared to control, indicating fewer females at 25 mg/kg bw/day had returned to normal estrous cycling patterns. At 25 mg/kg bw/day, the number of primordial follicles counted in the ovaries of F1 females was lower than concurrent control. At 5 mg/kg bw/day, histopathological findings did not indicate any obvious effect of treatment in either generation.

Offspring Necropsy

At 25 mg/kg bw/day, F1-F2 offspring from one litter showed gaseous distension of the stomach and intestinal tract. Similar gaseous distension of the stomach and/or intestinal tract was observed for F1-F2 offspring from two litters at 15 mg/kg bw/day. At 25 mg/kg bw/day, amongst F1-F2 offspring surviving to termination there was an increase incidence of small and/or pale offspring at termination, compared to control. Necropsy revealed an increased incidence of findings for the lungs, liver, heart, kidneys and thymus compared to control. At 15 and 25 mg/kg bw/day, F0-F1 offspring necropsy findings did not indicate any obvious effect of maternal treatment. At 5 mg/kg bw/day, offspring necropsy findings did not indicate any obvious effect of maternal treatment in either generation.

Offspring Organ Weights

At 25 mg/kg bw/day, absolute and body weight relative thymus weights for offspring were lower than control across both generations. At 15 mg/kg bw/day, body weight relative thymus weight for F1-F2 offspring was lower than control.

At 25 mg/kg bw/day, absolute and body weight relative brain weights for F1-F2 offspring were lower than control. At 15 mg/kg bw/day, there were no obvious effects of treatment on offspring organ weights during the F0 generation.

At 5 mg/kg bw/day, there were no obvious effects of treatment on offspring organ weights during either generation.

Offspring Histopathology

At 25 mg/kg bw/day, there was an absence of fat vacuolation in the bone marrow of most F1 - F2 male and female offspring. At 25 mg/kg bw/day, extramedullary hematopoiesis in the liver was present for some F1-F2 male and female offspring. Additionally centrilobular rarefaction (probably due to glycogen deposition) was observed for occasional F1-F2 male and some F1-F2 female offspring. At 25 mg/kg bw/day, white pulp depletion in the spleen was recorded in some F1-F2 male and occasional F1-F2 female offspring, additionally there was a decrease in hematopoiesis in the spleen of male offspring. At 25 mg/kg bw/day, thymic atrophy was present in the thymus of some F1-F2 male and

female offspring.

Conclusion

Based on the results of this study, a dosage of 5 mg/kg bw/day is considered to be a No Observed Effect Level (NOEL) for the repeated administration of the test item on

reproduction, pre-natal development and post-natal development of the rat when administered over two successive generations.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Due to requests within other regulatory jurisdictions (US-EPA) for provision of additional data (1), in response to the test rules “Testing of Certain High Production Volume Chemicals; Third Group of Chemical” (76 FR 65385, October 21, 2011), and its preference of a 2-generation reproductive study, a test was contracted with the registered substance p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline. The results of this test identified NOEL and NOAEL levels of 5 and 25 mg/kg bw/d respectively.

Effects on developmental toxicity

Description of key information

Preliminary and a main prenatal developmental studies have been conducted at doses of 25, 50, 100 mg/kg bw day and 5, 15 and 40 mg/kg bw day respectively with the p-isomer. Reductions in fetal, placental and litter weights and the type and incidence of fetal findings were evident in the prenatal developmental studies and consistent with adelay in growth these effects are considered to be a consequence of maternal toxicityrather than indicating any intrinsic toxicity to the developing conceptus. The No Observed Effect Level (NOEL) for the developing conceptus was considered to be 15 mg/kg bw/day. Treatment at 15 mg/kg bw/day was associated effects on body weight gain and food consumption. This is considered to be a reflection of irritation of the GI tract, which is frequently observed with test items of this nature, rather than systemic toxicity. The No Observed Effect Level (NOEL) for the pregnant dam was 5 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25th November 2015 to 18th August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Animals from the first mating batch were weighed on Day 9 of gestation in error. The body weights were not used for adjustment of dosage volumes, so the error had no adverse impact on the study.
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
Source: 96 Sprague-Dawley Crl:CD (SD) IGS BR rats from Charles River (UK) Limited, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation.
Housing: individually housed in solid floor polypropylene cages with stainless steel mesh lids, furnished with softwood flakes.
Food and Water: Free access to pelleted diet (Rodent 2018C Teklad Global Certified Diet) and mains drinking water supplied from polycarbonate bottles.

ENVIRONMENT
Temperature and relative humidity were maintained at 22 ± 3 ºC and 50 ± 20% respectively. Photoperiod was controlled to give twelve hours continuous light and twelve hours darkness.

Diet, drinking water, bedding and environmental enrichment considered to not contain any contaminant at a level that might have affected the purpose of the study.
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
Dose levels were selected based on available toxicity data including a preliminary study in the Rat. Results from this preliminary study were used to select a high dosage of 40mg/kg bw/day and a dosage sequence of 0 (control), 5, 15, 40 mg/ kg bw/day as follows:

Treatment Group Dose Level (mg/kg bw/ day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Control 0 4 0 24
Low 5 4 1.25 24
Intermediate 15 4 3.75 24
High 40 4 10 24

The test item was administered daily from Day 5 to Day 19 of gestation, by gavage with propylene glycol as the vehicle. Control animals were treated in an identical manner with the vehicle alone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analyzed for concentration of p-(2,3-epoxypropoxy)-N,N-bis (2,3-epoxypropyl)aniline at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 101 to 109% of the nominal concentration indicating the suitability of the formulation procedure.

The test item concentration in the test samples was determined by high performance liquid chromatography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatographic profile consisting of a single peak.
Details on mating procedure:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from a recognised breeder. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On the first day of treatment (Day 5 of gestation) the females weighed 214 to 310g.
Frequency of treatment:
Daily from Day 5 to Day 19 of gestation.
Duration of test:
20 days of gestation.
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
40 mg/kg bw/day
No. of animals per sex per dose:
Only female animals used, 24 females per treatment group.
Control animals:
yes
Details on study design:
Animals were delivered in
two batches containing females prior to Day 3 of gestation. The day that positive evidence of
mating was observed was designated Day 0 of gestation.

The animals were randomly allocated to treatment groups using a randomization procedure
based on stratified body weight to ensure similarity between the treatment groups.
Maternal examinations:
CLINICAL OBSERVATIONS
Following arrival, all animals were examined for overt signs of toxicity, ill-health or
behavioral changes once daily during the gestation period. Additionally, during the dosing
period, observations were recorded immediately before and soon after dosing and one hour
post dosing. All observations were recorded.
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days
5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at
terminal kill (Day 20).

FOOD CONSUMPTION
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20
of gestation.
Water intake was observed daily by visual inspection of the water bottles for any overt
changes.

POST MORTEM EXAMINATION
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on
Day 20 of gestation. All animals were subjected to a full external and internal examination
and any macroscopic abnormalities were recorded.
Ovaries and uterine content:
The ovaries and uteri of pregnant females
were removed, examined and the following data recorded:
Number of corpora lutea
Number, position and type of intrauterine implantation
Fetal sex
External fetal appearance
Fetal weight
Placental weight
Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium
polysulphide solution to reveal evidence of implantation.
Fetal examinations:
Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test
methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk
normality test and Bartlett’s test for homogeneity of variance and one way analysis of
variance, followed by Dunnett’s multiple comparison test or, if unequal variances were
observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric
analysis of variance; and a subsequent pairwise analysis of control values against treated
values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 40 mg/kg bw/day, initial treatment on Day 5 of gestation was associated with slight,
statistically significant, mean body weight loss compared to control. Initial body weight loss and episodes of lower body weight gain resulted in statistically significant lower
cumulative mean body weight gain, compared to control, throughout the treatment period and
the mean overall weight gain remained statistically significantly lower than control, following
adjustment for the contribution of the gravid uterus. The lower body weight gain was
consistent with lower food intake for these animals throughout the treatment period.

At 15 mg/kg bw/day, initial treatment on Day 5 of gestation was associated with slightly
lower body weight gain, compared to control, but differences were not statistically significant
and may reflect normal biological variation. Subsequently body weight gain was similar to
control to Day 8 of gestation but statistically significant lower mean body weight gains were
apparent from Day 8 to Day 14 of gestation. Statistically significant lower mean body weight
gain, compared to control, was again apparent between Day 17 and Day 20 of gestation, but
this may have been influenced by a lower in-utero contribution from the fetuses/litters. The
episodes of lower body weight gain resulted in statistically significant lower cumulative mean
body weight gain, compared to control, from Day 11 of gestation and the mean overall weight gain remained statistically significantly lower than control.

At 5 mg/kg bw/day, there were no statistically significant differences in phased body weight
gains, compared to control, throughout gestation, although differences from control for
cumulative body weight attained statistical significance from Day 17 of gestation. These
differences were considered not to represent an adverse effect on maternal body weight gain,
as overall gain, when adjusted for the contribution of the gravid uterus, was similar to control
and showed no statistical significance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 40 mg/kg bw/day, statistically significant lower mean food consumption, compared to
control, was apparent throughout the treatment period. This lower food consumption was
consistent with lower maternal and fetal body weight observed at this dosage.

At 15 mg/kg bw/day, statistically significant lower mean food consumption, compared to
control, was apparent between Days 11 to 14 of gestation. Food consumption prior to and
after this period was considered to be similar to control.

At 5 mg/kg bw/day, food consumption was considered to be similar to control throughout
gestation.
Water consumption and compound intake (if drinking water study):
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 40 mg/kg bw/day, mean fetal weights, placental weight and litter weights were statistically
significantly lower than control. The lower litter weight observed was consistent with a
statistically significantly lower gravid uterus weight, compared to control, apparent at this
dosage. The lower fetal, litter and placental weights may be a consequence of reduced
nutrition in maternal animals as a result of lower food consumption at this dosage.
At 5 and 15 mg/kg bw/day, mean fetal and placental weight were similar to control and
appeared unaffected by maternal treatment. Litter weights (and gravid uterus weights) were
slightly lower than control, with differences for litter weights attaining statistical significance;
however these differences appeared to reflect the slight differences in litter size rather than
any obvious effect of maternal treatment.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 40 mg/kg bw/day, there was a higher incidence of fetuses/litters showing bipartite
ossification of the thoracic centrum, dumb-bell-shaped thoracic centrum or an ossification
centre for the rib associated with the 7th cervical vertebrae, with the litter incidence of these
findings attaining statistical significance. The skeletal effects are consistent with the low
fetal weights observed and are considered to reflect delayed fetal development as a
consequence of maternal toxicity (manifest as reduced body weight gain and, in particular,
food consumption) rather than intrinsic toxicity to the developing conceptus. Reduced
ossification of the thoracic centrum is a typical finding of reduced growth due to the
ossification of such thoracic regions last in-utero.

Neither the type, incidence nor distribution of findings apparent during detailed skeletal
examination of fetuses indicated any obvious effect of maternal treatment on fetal
development at 5, or 15 mg/kg bw/day.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Neither the type, incidence nor distribution of findings apparent during detailed visceral
examination of fetuses indicated any obvious effect of maternal treatment on fetal
development at 5, 15 or 40 mg/kg bw/day.
At 5 mg/kg bw/day, a lower incidence of fetuses/litters showed increases in renal pelvic
cavitation compared to control with the fetal incidence attaining statistical significance.
However, this finding, in the absence of any statistical significance differences from control
at higher dosages, was considered to be incidental and unrelated to maternal treatment.
Key result
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Key result
Developmental effects observed:
no
Conclusions:
Within this study, treatment at 40 mg/kg bw/day was associated with initial mean body
weight loss, lower body weight gain and reduced food consumption for parental females.

The NOEL for the developing conseptus was considered to be 15 mg/ kg bw/day.
Executive summary:

The study was performed to investigate the effects of the test item, p-(2,3-epoxypropoxy)-

N,N-bis (2,3-epoxypropyl)aniline, on embryonic and fetal development following repeated

administration by gavage to the pregnant female during gestation including the period of

organogenesis.

The study was designed to comply with the following guidelines:

 US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental

Toxicity Study’ (August 1998)

 Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for

Toxicology studies, 12 NohSan No 8147, (24 November 2000)

 OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental

Toxicity Study’ (adopted 22 January 2001)

 Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to

Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the

Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated

Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation

inclusive at dose levels 5, 15, and 40 mg/kg bw/day. A further group of twenty-four time

mated females was exposed to the vehicle only (Propylene glycol) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the

study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy

including examination of the uterine contents. The number of corpora lutea, number, position

and type of implantation, placental weights, fetal weight, sex and external and internal

macroscopic appearance were recorded. Half of each litter were examined for detailed

skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

There were no unscheduled deaths.

Clinical Observations

No clinical signs were apparent throughout the study at 5, 15 or 40 mg/kg bw/day.

Body Weight

At 40 mg/kg bw/day, initial treatment on Day 5 of gestation was associated with slight, but

statistically significant, mean body weight loss compared to control. Statistically significant

lower mean body weight gains, compared to control, were apparent between Day 8 to Day 14

and Day 17 to 20 of gestation, although the latter period may have been influenced by lower

in-utero contribution from gravid uterus. Cumulative mean body weight gain was

statistically significantly lower than control throughout the treatment period and mean overall

weight gain remained statistically significantly lower than control, after adjustment for the

contribution of the gravid uterus.

At 15 mg/kg bw/day, body weight gain on Day 5 of gestation was slightly, but not

statistically significantly, lower than control. Statistically significant lower mean body

weight gains, compared to control, were apparent between Day 8 to Day 14 of gestation and

Day 17 to Day 20 of gestation, although the latter period may have been influenced by lower

in-utero contribution from gravid uterus. Cumulative mean body weight gain was

statistically significantly lower than control from Day 11 of gestation and mean overall

weight gain remained statistically significantly lower than control, after adjustment for the

contribution of the gravid uterus.

At 5 mg/kg bw/day, there were no statistically significant differences in phased body weight

gains, compared to control, throughout gestation, although differences from control for

cumulative body weight attained statistical significance from Day 17 of gestation. These

differences were considered not to represent an adverse effect on maternal body weight gain,

as overall gain, when adjusted for the contribution of the gravid uterus, was similar to control

and showed no statistical significance.

Food Consumption

At 40 mg/kg bw/day, statistically significant lower mean food consumption, compared to

control, was apparent throughout the treatment period.

At 15 mg/kg bw/day, statistically significant lower mean food consumption, compared to

control, was apparent between Days 11 to 14 of gestation.

At 5 mg/kg bw/day, food consumption was considered to be similar to control throughout

gestation.

Water Consumption

No obvious effect on water consumption was observed at 5, 15 or 40 mg/kg bw/day.

Post Mortem Studies

No macroscopic findings were apparent for adult females at 5, 15 or 40 mg/kg bw/day.

Litter Data

There was no effect of maternal treatment on numbers of implantations, in-utero offspring

survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex

ratio and post-implantation losses at 5, 15 or 40 mg/kg bw/day.

Litter, Placental and Fetal Weights

At 40 mg/kg bw/day, mean fetal weights, placental weight and litter weights (and gravid

uterus weight) were statistically significantly lower than control.

At 5 and 15 mg/kg bw/day, litter weights (and gravid uterus weights) were lower than control

but only litter weights attained statistical significance. Differences appeared to reflect normal

variation in litter size as mean fetal and placental weight were similar to control.

Fetal Examination

External Examinations

At 40 mg/kg bw/day, there was an increase in the incidence of fetuses/litters with external

observations primarily due to a higher number of small fetuses.

Neither the type, incidence nor distribution of findings apparent during external examination

of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal

development at 5 or 15 mg/kg bw/day.

Visceral Examinations

Neither the type, incidence nor distribution of findings apparent during detailed visceral

examination of fetuses indicated any obvious effect of maternal treatment on fetal

development at 5, 15 or 40 mg/kg bw/day.

Skeletal Examinations

At 40 mg/kg bw/day, there was a statistically significant higher incidence of fetuses/litters

showing bipartite ossification of the thoracic centrum, dumb-bell-shaped thoracic centrum or

an ossification centre for the rib associated with the 7th cervical vertebrae. The skeletal

findings were consistent with the low fetal weights observed and were considered to reflect

delayed fetal development as a consequence of maternal toxicity, particularly lower food

consumption, rather than intrinsic toxicity to the developing conceptus.

Neither the type, incidence nor distribution of findings apparent during detailed skeletal

examination of fetuses indicated any obvious effect of maternal treatment on fetal

development at 5, or 15 mg/kg bw/day.

Conclusion

Within this study, treatment at 40 mg/kg bw/day was associated with initial mean body

weight loss, lower body weight gain and reduced food consumption for parental females.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Within this study, treatment at 40 mg/kg bw/day was associated with initial mean body weight loss, lower body weight gain and reduced food consumption for parental females.
The NOEL for the developing conseptus was considered to be 15 mg/ kg bw/day.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Following review and interpretation the substance should not be classified.

1) F1 sexual maturation - balano preputial separation, F1 Uterus Weight and F1 Number of primordial follicles effects at 15 and 25 mg/kg bw/d) are all correlated with effects on bodyweight (Hood, R. 2106) and therefore it is hypothesised that this delay in separation is due to the reduced body weight and food consumption rather thn an intrinsic toxicological response

.  

2) F1 Vaginal mucification (pregnancy/lactation morphology) Vaginal mucification is associated with progesterone excess, by prolactin-secreting pituitary neoplasm or a result of pseudopregnancy, all of which are also impacted by body weight and food consumption not intrinsic toxicity.

In conclusion, all the effects/changes in endpoints when collectively put together show that there is a spectrum of changes that have been brought about as a consequence of exposure to a test material that is having an effect on the adult (gastrointenstinal mucosal irritancy/corrosion). The altered physiology is liklely to be an adaptation to the alterations in their own physiology and a stress response rather the intrinsic toxicity. It is therefore concluded that 25 mg/kg bw dw is considered to be the most appropriate value to be used for risk assessment purposes. In addition, the substance should not be classified for reprotoxicity as effects only occurred at doses where animals were compromised by the test substances primary damaging effects on gut epithelia and subsequent stress /secondary responses on the physiology rather than intrinsic toxicity.