Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A preliminary reproductive performance study in Sprague Dawley rats by oral gavage administration of B-TEGME was conducted. The purpose of this study was to assess the general systemic toxic potential in rats, including reproductive/developmental effects, with administration of B-TEGME by oral gavage administration for at least seven weeks. The study was designed to provide sufficient data on offspring survival and development to permit selection of dose levels for an OECD 443 extended one-generation reproductive toxicity study (EOGRTS). For the F0 generation, three groups of eight male and eight female rats received B-TEGME at dose levels of 100, 300 or 1000 mg/kg/day by oral gavage administration. F0 animals were treated daily for 15 days before pairing until termination either after all litters had been born for the males or at weaning of F1 generation for the females. A similarly constituted control group received the vehicle, corn oil, over the same treatment period and at the same volume dose as the treated groups. The F1 generation comprised of ten male and ten female progeny from each group, and they continued to receive the relevant dose, as per the F0 generation, from weaning on Day 21 to Day 28 of age. Similarly constituted control groups received the vehicle, corn oil, over the same treatment period and at the same volume dose as the treated groups. During the study, clinical observations, body weight, food consumption and macroscopic pathology investigations were undertaken on each adult generation. Organ weights, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance were undertaken on F0 adult generation only. The clinical condition of offspring, litter size and survival, sex ratio, body weight and macroscopic pathology investigations were undertaken for F1 offspring.

There were three premature decedents in the F0 generation at 1000 mg/kg/day, one female was killed for welfare reasons after dosing on Day 7 of treatment, one male and one female were found dead after dosing on Day 8 and 14, respectively. These premature deaths reflected trauma during the dosing period and were considered unrelated to treatment. One female receiving 100 mg/kg/day and two females receiving 1000 mg/kg/day failed to achieve pregnancy and were therefore terminated early. There were no test item-related signs observed during the detailed physical examination, no post-dosing observations and no effects on food consumption. Overall mean body weight was slightly higher for males at 1000 mg/kg/day for the treatment period and for all treated females during the pre-pairing phase. Females at 300 or 1000 mg/kg/day showed slightly higher overall body weight gain during gestation but body weight on Day 1 of lactation indicated no underlying effect on maternal body weight. At the start of lactation (Days 1-4 ) females given 1000 mg/kg/day showed a mean body weight loss but overall weight gain during lactation was similar to control. Estrous cyclicity, pre-coital interval, mating performance, gestation length and gestation index were unaffected by treatment with B-TEGME. One female receiving 100 mg/kg/day and two females receiving 1000 mg/kg/day were found to be not pregnant and therefore conception/fertility rates for females at 1000 mg/kg/day were low. Changes in organ weights consisted of slightly lower absolute and body weight adjusted mean epididymides and ovary weights for males and females at 1000 mg/kg/day when compared to control. There were no adverse test-item related macroscopic findings observed for the F0 generation at all dose levels investigated.

There was no effect of B-TEGME administration on body weight, sex ratio, clinical condition, or birth weight of the F1 litters. In the 1000 mg/kg/day group, lower live birth and Day 4 viability indices resulted in slightly lower litter size on Day 4 of lactation prior to culls when compared with the controls. There were no test item-related macroscopic abnormalities detected among offspring dying prematurely or among offspring killed at scheduled termination on Day 21 of age. Direct B-TEGME administration to the selected F1 generation animals from Day 21 of age at dose levels of 100, 300 or 1000 mg/kg/day was generally well tolerated. Two males receiving 100 mg/kg/day were dispatched for welfare reasons on Day 23 and Day 25 of age, both deaths were considered to reflect accidental trauma during the dosing procedure and were therefore unrelated to treatment. The general condition, food consumption and macropathology of the F1 animals showed no adverse effect of treatment. Group mean body weight gain for the treatment period was slightly lower for males and females receiving 1000 mg/kg/day compared to controls. Body weight performance for males and females receiving 100 or 300 mg/kg/day was unaffected by test item administration.

Conclusion:

Oral administration of B-TEGME to F0 animals at dose levels of 100, 300 or 1000 mg/kg/day was generally well tolerated with no adverse effects on clinical condition, body weight performance, food consumption or macropathology. At 1000 mg/kg/day, two out of the six mated females failed to achieve pregnancy and live birth index and Day 4 viability index were lower, although a clear relationship to treatment was not established. While these effects have the potential to compromise reproductive assessment at this dose level, there was considered to be insufficient evidence from this preliminary study, that they would be repeated in the planned OECD 443 study for this test item. The clinical condition and body weight gain of the offspring was unaffected by maternal treatment at 1000 mg/kg/day.

Oral administration of B-TEGME to F1 animals at dose levels of 100, 300 or 1000 mg/kg/day was also well tolerated, with no adverse effects on clinical condition, body weight performance or macropathology. Body weight gain of directly dose selected F1 offspring was slightly lower than control at 1000 mg/kg/day but not at a level precluding further investigation of toxicity at this dose level.

Based on the results obtained in this preliminary study of reproductive performance; it was concluded that the high dose level for the main extended one-generation (OECD 443) study should be set at 1000 mg/kg/day.

 

Subsequently, an extended one-generation reproductive toxicity study (OECD 443) in Sprague Dawley rats by oral gavage administration of B-TEGME was conducted. The purpose of this study was to assess the influence of B-TEGME on reproductive performance when administered by oral gavage to Sprague-Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation, estrous cycles, and reproductive performance. In addition, cohorts of F1 animals were used to assess developmental neurotoxicity and developmental immunotoxicity.

B-TEGME administration at dose levels up to and including 1000 mg/kg/day was generally well tolerated by F0 and F1 males throughout the study, by the F1 Cohorts 1A, 2A and 3 animals throughout the study, and by the F0 and F1 Cohort 1B females up to Day 20 of gestation. During these study phases there were no test item-related premature deaths or test item‑related changes in general clinical condition, or observations related to dose administration, no effects on food consumption, food conversion efficiency or estrous cycle regularity and pre-coital interval. Similarly, there was no effect of treatment on the attainment of sexual maturation of the F1 animals, the duration between vaginal opening and first estrus in the F1 Cohort 1A females or the stage of estrus at termination of the F0 or the F1 Cohort 1A and 1B females. Macroscopic examination of the F0 animals and F1 Cohort 1A, 1B, 2A, 2B and 3 animals at scheduled termination did not reveal any test item-related abnormalities. In F0 generation only at all study phases, there were no significant reductions in body weight gain, mating performance, conception rate and fertility index were also unaffected. There were also no observable effects on the immunophenotyping parameters measured in spleen leukocytes of the F1 Cohort 1A animals.

Detailed histopathological evaluation of the tissues retained from the F0, F1 Cohort 1A and 1B animals revealed the epididymis (F0, F1A, F1B males), testes (F1B males only), thymus (F0 males only) and kidneys (F0 females only) to be target organs.

There was cellular debris at a minimal level present in the epididymis of males given 1000 mg/kg/day in the F0 generation and F1 generation cohorts 1A and 1B. This correlated with a slightly lower absolute organ weight in the F1 Cohort 1B males only and numerous changes in the sperm analysis. Whilst no accompanying histopathological change was present in the testes in the F0 and F1 Cohort 1A males there was an accompanying minimal degeneration of occasional seminiferous tubules in the testes in the F1B males perhaps associated with the longer dosing period in these animals. Although, organ weight changes in testes were noted for F0 males at 1000 mg/kg/day only. As the changes in the sperm analysis were significant in F0 and F1 Cohort 1A males at 1000 mg/kg/day, coupled with the reduced fertility observed in F1 Cohort 1B animals, the histopathological changes at 1000 mg/kg/day in the epididymides and testes were considered adverse.

There were a slightly higher incidence of lower lymphocyte counts in the cortex of the thymus in the F0 generation males and the incidences observed were outside the Historical Control Data (HCD) range. The finding also correlated with a lower thymus weight in F0 males given 1000 mg/kg/day. The lower weight was also notable in F1 Cohort 1A males given 1000 mg/kg/day although without histopathological correlation in this generation. As this finding was minor and of low incidence, it was therefore of equivocal significance but it was considered to be related to test item administration.

In females in the F0 generation only, there was an increase in basophilic tubules in the kidneys of females given 1000 mg/kg/day with more multifocal distribution which correlated with a higher organ weight and changes in the urinalysis and thus was considered to be adverse. Changes in urinary parameters included high total protein concentration and low urinary pH in males and females given 1000 mg/kg/day, and total chloride concentration was high in females at 1000 mg/kg/day. Organ weight changes were also noted in both sexes receiving 1000 mg/kg/day. After examination of the F0 kidneys of females given 100 or 300 mg/kg/day they showed a marginally higher incidence of minimal basophilic tubules. The multifocal distribution at 300 mg/kg/day was similar to females given 1000 mg/kg/day, but any significance of this change in females given 100 or 300 mg/kg/day is equivocal as no clear evidence of relationship to dose was established, changes occurred in one sex only and at 100 mg/kg/day the severity and distribution was similar to controls. The higher weight of the kidneys was however consistent in both the F0 and F1A generations and may be indicative of a minor functional change and slight changes were also observed in the urinalysis of F1A animals.

Following the sperm assessment on the F0 and F1 Cohort 1A males, sperm motility and morphology for animals that received 1000 mg/kg/day were affected by test item administration. The motility of the sperm was reduced and there was a higher percentage of abnormal sperm observed in F0 and F1A males given 1000 mg/kg/day. Total epididymal sperm count was low in F1A males only. Changes in the sperm analysis correlated with histopathological changes in the epididymis in F0 and F1A males.

An increase in test item-related premature termination of F1B Cohort females was necessary due to fertility issues with the majority of females failing to litter or suffering total litter loss at 1000 mg/kg/day. At 1000 mg/kg/day, eleven animals did not produce litters (ten of which were confirmed non-pregnant), one failed to mate and another four suffered a total litter loss. Whilst there is clearly an adverse fertility/reproductive effect in F1B Cohort females it is not clear if it is accounted for entirely by the reproductive tract issues in the corresponding males or if there was also an effect in the females. Further examination of the histopathological changes in the epididymides and testes showed no correlation between individual males with epididymal and testicular changes being paired with the females affected by fertility issues. Whilst no histopathological changes were noted in the pituitary of F0  and F1 males and females given 1000 mg/kg/day, there was also a consistently low pituitary weight, particularly in females, which may have had a functional effect on reproductive activity, although individual data suggests that there is no relationship between males or females with the lowest pituitary weights and the females with fertility issues. The reduced performance in F1B Cohort females resulted in lower conception rate and fertility index for females receiving 1000 mg/kg/day. In addition, mean body weight gain for the F1 Cohort 1B females that received 1000 mg/kg/day was statistically significantly lower compared to control during the gestation period. At 300 mg/kg/day, three females that mated failed to conceive: the male partner of one of these females had notable tubular atrophy in the testes which was unrelated to treatment and the other two pairs had no histopathology findings to explain the failure to conceive but at this low incidence it was considered incidental and not related to treatment. 

From Day 20 of gestation, the F0 and F1 Cohort 1B females were assessed three times per day for the progress of parturition. F0 and F1 Cohort 1B females in the 1000 mg/kg/day group showed a trend towards slightly extended gestation length. In the F0 females, there was no effect on gestation index and all females were pregnant and therefore this slight extension was considered non-adverse. Whilst in the F1B females, the extension to gestation length was more severe with six females having a 24 or 24.5 day gestation length, and this seemed to correlate with small litter sizes in these females. In the F1 Cohort 1B females at 300 mg/kg/day a slight shift in gestation length was also observed but this difference was very slight and therefore considered non-adverse.

There was no conclusive effect of B-TEGME administration on F1 or F2 offspring sex ratio, clinical condition, and subsequent growth of the litters. The initial litter size for the F1 offspring was similar for all dose levels but the Day 4 viability index was low in the F1 litters in the 1000 mg/kg/day group, due to an increased number of pup deaths between birth and Day 4 of lactation although subsequent offspring survival to weaning on Day 21 of age was unaffected. For F2 litters in the 1000 mg/kg/day group, number of implantation sites, litter size on Day 1 and 4 of lactation, post-implantation survival and the number of live offspring were lower when compared to controls, reflecting an increase in the number of conceptus deaths between implantation and Day 4 of lactation; subsequent offspring survival of the F2 offspring to weaning was unaffected.

Assessment of ano-genital distance of the F2 offspring on Day 1 of age revealed a slight but statistically significantly higher values in males and females in the 1000 mg/kg/day group; similar differences were not apparent in the F1 offspring. All F1 and F2 male offspring were assessed on Day 13 of age for the presence of nipples; no nipples were observed. In the absence of evidence of nipples in the F2 male offspring, any changes in ano‑genital distance in the F1 offspring, no effects on the age at attainment of sexual maturation of the F1 animals, the duration between vaginal opening and first estrus in the F1 Cohort 1A females, or the stage of estrus at termination of the F1 Cohort 1A and 1B females, the slight differences in ano‑genital distance of the F2 offspring were considered to be of no biological or toxicological relevance.

Assessment of the F1 Cohort 2A and 2B animals did not reveal any evidence of developmental neurobehavioural or neuropathology changes. Auditory startle response habituation, locomotor activity and the performance of the F1 Cohort 2A animals in the Functional Observational Battery (FOB) tests were unaffected by treatment with the expectation of a slightly lower hind limb grip strength in males given 1000 mg/kg/day which is likely to be a result of the lower mean body weight for this group. Brain morphometry of the F1 Cohort 2A and 2B animals and detailed histopathological evaluation of the neurological tissues of the F1 Cohort 2A animals were unaffected. Brain weights in both sexes given 1000 mg/kg/day were marginally lower but this was without histopathological or morphometric correlation and therefore the significance was unclear.

Assessment of the F1 Cohort 3 animals did not reveal any evidence of developmental immunotoxicity. Spleen weights at scheduled termination were unaffected, and there was no impact on the T-Cell Dependent Antibody Response (TDAR) following immunization with 300 µg of Keyhole Limpet Hemocyanin (KLH) on Days 47 and 54 of age.

Hematological examination conducted at scheduled termination of the F0 animals revealed slightly lower lymphocyte, eosinophil and basophil counts in males receiving 1000 mg/kg/day and this could be associated with the lower lymphocytes observed histopathologically in the cortex of the thymus of F0 males given 1000 mg/kg/day, but as these changes had no impact on the clinical condition of the animals these slight changes were considered non-adverse. Further differences in the F0 generation, when compared to controls, included lower haematocrit, reticulocyte counts, and mean cell volume predominately apparent in males at 1000 mg/kg/day. These changes were not corroborated with any histopathological findings and therefore these differences were considered to reflect normal biological variation and are considered to be of no toxicological relevance. In the F1 Cohort 1A animals, when compared to controls, some slight differences in red blood cell concentrations, mean cell hemoglobin concentrations, mean cell volume and red cell distribution width were apparent, predominantly in the 1000 mg/kg/day group; as there was no associated histopathological changes associated with these differences, they were considered to reflect normal biological variation and of no toxicological significance.

Biochemical analysis of the plasma at scheduled termination of the F0 animals revealed, when compared to controls, slightly high mean urea concentrations in males receiving 1000 mg/kg/day. Mean plasma urea concentrations were also slightly higher in F1A males and females at 1000 mg/kg/day, and bile acids were high at all treatment groups for F1A females. Histopathological changes were observed in the kidneys of F0 females that received 1000 mg/kg/day but these changes were not observed in F0 males or F1A animals, there was, however, an increase in kidney weights observed in these animals. Given the minimal nature of these biochemical changes, they were considered not adverse. 

Analysis of the urine of F0 animals indicated higher urinary protein values in both sexes at 1000 mg/kg/day, urinary pH was also lower and total chloride concentration was low in females at 1000 mg/kg/day only. These minor differences observed could be associated with the increased kidney weights observed in F0 males and females at 1000 mg/kg/day and the histopathological changes observed in the kidneys for F0 females that received 1000 mg/kg/day, but as no similar histopathological changes were observed in F0 males, these differences were considered to be non-adverse. There were no histopathological correlates observed for the slightly low urinary pH in F1 cohort 1A males and females or the low urinary protein, sodium, potassium, and chloride concentrations in the F1 Cohort 1A males given 1000 mg/kg/day; however, slightly higher kidney weights were recorded for these animals.

Lower T4 serum concentrations were observed in F0 males given 1000 mg/kg/day and F1 generation males that received 300 or 1000 mg/kg/day, with the difference from control attaining statistical significance, and mean serum TSH concentrations were higher compared to control for F0 males only. As the majority of values were within the historical control range and no histological changes were observed in the thyroid for either generation, these findings are considered to be of no toxicological relevance.

At scheduled termination uterus/cervix/oviduct weights of the F1B females given 300 and 1000 mg/kg/day were slightly higher, and the higher weight at 1000 mg/kg/day is likely to be related to cyclical activity with an increased proportion of females in estrus compared to control animals, however, the low number of surviving females given 1000 mg/kg/day makes interpretation complex and thus relationship to treatment was equivocal.

The qualitative evaluation of the ovaries of F1 Cohort 1A females given 1000 mg/kg/day revealed a slightly lower number of follicles. In the absence of an effect on the number of corpora lutea or any histopathological changes in the ovary, this finding was considered to be of no toxicological relevance.

Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 300 mg/kg/day due to the high incidences of reduced fertility in females of F1 Cohort 1B receiving 1000 mg/kg/day and the increased incidences of minimal epididymal cellular debris coupled with sperm motility and morphology changes in both F0 and F1 Cohort 1B males given 1000 mg/kg/day, accompanied with degeneration in the testes for F1 Cohort 1B males at 1000 mg/kg/day only.

Aside from the above-mentioned instances of reduced fertility and male reproductive system changes at 1000 mg/kg/day, increased incidences of basophilic tubules in the kidneys of F0 females and increased incidence of decreased lymphocytes in the cortex of the thymus in the F0 generation males were observed at 1000 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 300 mg/kg/day.

The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 300 mg/kg/day due to reduced early post-partum survival at 1000 mg/kg/day in both generations and low litter size in F2 litters.

There was no evidence of developmental neurotoxicity or developmental immunotoxicity on this study, therefore the NOAEL for these endpoints was concluded to be 1000 mg/kg/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

reproductive toxicity, other

Remarks:

Range-finding study for EOGRTS

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was designed to provide sufficient data on offspring survival and development to permit selection of dose levels for an OECD 443 extended one-generation reproductive toxicity study.
For the F0 generation, three groups of eight male and eight female rats received B-TEGME at dose levels of 100, 300 or 1000 mg/kg/day by oral gavage administration. F0 animals were treated daily for 15 days before pairing until termination either after all litters had been born for the males or at weaning of F1 generation for the females. A similarly constituted control group received the vehicle, corn oil, over the same treatment period and at the same volume dose as the treated groups. The F1 generation comprised of ten male and ten female progeny from each group, and they continued to receive the relevant dose, as per the F0 generation, from weaning on Day 21 to Day 28 of age. Similarly constituted control groups received the vehicle, corn oil, over the same treatment period and at the same volume dose as the treated groups.
During the study, clinical observations, body weight, food consumption and macroscopic pathology investigations were undertaken on each adult generation. Organ weights, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance were undertaken on F0 adult generation only. The clinical condition of offspring, litter size and survival, sex ratio, body weight and macroscopic pathology investigations were undertaken for F1 offspring.

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.

Limit test:

no

Justification for study design:

The purpose of this study was to assess the general systemic toxic potential in rats, including reproductive/developmental effects, with administration of B-TEGME (an industrial chemical) by oral gavage administration for at least seven weeks. The study was designed to provide sufficient data on offspring survival and development to permit selection of dose levels for an OECD 443 extended one-generation reproductive toxicity study.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age of the F0 animals at the start of the treatment: Males: 82 to 88 days old; Females: 75 to 81 days old
- Weight range of the F0 animals at the start of the treatment: Males 364 to 446 g; Females 215 to 274 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage: Pre-pairing: Up to four animals of one sex; Pairing: One male and one female; Males after mating: Up to four animals; Gestation: One female; Lactation: One female + litter; F1 Generation: Up to four animals of one sex.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet ad libitum. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply ad libitum via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Five days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Correction factor: A correction factor of 1.14 was used when calculating quantities of test item used during dose preparation. This calculation was used to correct for the test item purity.
- Method of preparation: The required amount of test item was weighed and 50% of the final vehicle was added. The formulation was magnetically stirred until the test item was uniformly mixed. Vehicle was added to achieve the final volume and the formulation was mixed using a magnetic stirrer until homogenous. The formulation was transferred to the final containers, via syringe, whilst magnetically stirring.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
- Frequency of preparation: Weekly, may have been prepared in advance of the first day of dosing.
- Storage of formulation: Refrigerated (2 to 8ºC).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): The test item is instable in water (hydrolyzes to 3x methyltriglycol + boric acid).
- Amount of vehicle (if gavage): 4 mL/kg

ADMINISTRATION:
- Route: Oral gavage using a suitably graduated syringe and a semi-ridged cannula inserted via the mouth.
- Treated at: Constant doses in mg/kg/day.
- Volume dose: 4 mL/kg body weight.
- Individual dose volume: Calculated from the most recently recorded scheduled body weight.
- Control (Group 1): Vehicle at the same volume dose as treated groups.
- Frequency: Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
- Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. Following three incidents of mis-dosing, the following procedure was introduced to improve the dosing process: After filling the dose equipment for each animal, the semi-ridged cannula was wiped dry and dipped in corn oil before administration.

Details on mating procedure:

- F0 pairing commenced: After two weeks of treatment.
- Male/female ratio: 1:1 from within the same treatment groups.
- Duration of pairing: Up to two weeks.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of formulations during storage was determined as part of another study (Covance Study Number MQ60WK). Stability in the concentration range of 2 to 250 mg/mL was established for one day at ambient temperature (15 to 25°C) or 15 days when stored refrigerated (2 to 8°C).

Duration of treatment / exposure:

F0 animals: For a minimum of 15 days before pairing until Day 20 of lactation for females. For a minimum of 7 weeks.
F1 animals: Day 21 to Day 28 of age.

Frequency of treatment:

Once daily.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

F0: 8 animals per sex per dose;
F1: 10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the low toxicity of the test compound, 1000 mg/kg bw/day was selected as the high dose level. The other dose levels were selected in order to generate a graduated dose-response in case of toxicity at 1000 mg/kg bw/day. The dose level of 1000 mg/kg body weight was well tolerated in a previous 90-day oral toxicity study in rats.
- Rationale for animal assignment (if not random):
F0: On arrival and non-selective allocation to cages. On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before provision of formulation. The groups were adjusted to reduce inter-/intra-group variation.
F1: Allocation prior to Day 21 of age. The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals. Where possible, one male and one female were selected from each selected litter; where more than one were required up to two males and two females were selected from each selected litter. Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortalities. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

Parental animals: Observations and examinations:

MORTALITY:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS:
Signs Associated with Dosing:
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males: Week 1 - daily. Week 2 onwards - weekly.
F0 females: Week 1 - daily. Week 2 - once. Gestation phase - Days 0, 7, 14 and 20. Lactation phase - Days 1, 7, 14 and 20.
Detailed observations were recorded at the following times in relation to dose administration:
- Pre-dose observation.
- One to two hours after completion of dosing.
- As late as possible in the working day.

Clinical signs:
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
F0 males: Once each week.
F0 females: Once each week until mating. Gestation phase - Days 0, 5, 12, 18 and 20. Lactation phase - Days 1, 7, 14 and 21.

Clinical observations were reported for each animal, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

BODY WEIGHT:
The weight of animals was recorded as follows:
F0 males: Twice weekly. Day of necropsy.
F0 females: Twice weekly until mating was detected. Days 0, 3, 7, 10, 14, 17 and 20 after mating. Days 1, 4, 7, 11, 14, 18 and 21 of lactation.

Group mean values and SD were calculated from individual body weight data on each recorded occasion. Group mean weight changes were calculated from the weight changes of individual animals surviving the specified period.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males and females until paired for mating: Weekly.
Females F0: Days 0-3, 3-7, 7-10, 10-14, 14-17 and 17-20 after mating. Days 1-4, 4-7, 7-11, 11-14, 14-18 and 18-21 of lactation.

From these records the mean daily consumption per animal (g/animal/day) was calculated for each relevant phase.
Group mean food consumptions and standard deviations for each period were derived from unrounded cage values. Where animals were gang housed, values were presented for the average amount of food consumed per animal in each cage. Values presented allow for any animal that died or was killed during the period. After Day 14 of lactation, food consumption is increasingly influenced by the offspring and is no longer an accurate reflection of maternal consumption.

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
Duration of gestation: Time that elapsed between mating and commencement of parturition.
Parturition observations: From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded and any difficulties observed were noted.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears: For 15 days before pairing, using cotton swabs.
Wet smears: After pairing until mating, using pipette lavage.

The incidence and percentage females showing the following classifications of estrous cycles before treatment commenced were reported:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days).
Irregular: At least one cycle of 2, 3 or 6 to 10 days.
Acyclic: At least 10 days without estrus.

Litter observations:

RECORDS MADE DURING LITTERING PHASE:
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age. On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.
- Sex ratio of each litter: Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
- Individual offspring body weights: Unselected offspring: Recorded on Days 1, 4, 7, 14, 17 and 21 of age. Selected offspring: Recorded on Days 1, 4, 7, 14, 17, 21, 25 and 28 of age and on the day of necropsy.

LITTER SIZE:
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after culling), 7, 14 and 21 of age. Group mean litter size and SD were calculated from the individual litter values.

SEX RATIO:
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after culling) and 21 of age.
Percentage males = (# of males in litter / Total # of offspring in litter) x 100
Group mean values were calculated from individual litter values.

MORTALITY:
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

DETAILED CLINICAL OBSERVATIONS:
Signs Associated with Dosing:
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F1 offspring: Days 21 to 28 of age - daily.
Detailed observations were recorded at the following times in relation to dose administration:
- Pre-dose observation.
- One to two hours after completion of dosing.
- As late as possible in the working day.

Clinical signs:
A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
Selected F1 generation after weaning: Once each week.

Clinical observations were reported for each animal, providing detail of the type of sign, day of occurrence and information on the duration of the sign applicable.

BODY WEIGHT:
The weight of animals was recorded as follows:
F1 selected animals: Days 21, 25 and 28 of age. On the day of necropsy.

Litter mean body weight (+SD) was calculated separately for males and females and the group mean values derived from the individual litter values. Offspring body weight change was calculated relative to Day 1 of age. Body weights were plotted graphically with respect to the first day of the relevant period.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F1 selected animals: Days 21 to 25 and 25 to 29 of age.

Postmortem examinations (parental animals):

All F0 animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

TIME OF NECROPSY:
F0 males: Scheduled kill - following successful littering by females.
F0 females: Scheduled kill - Day 21 of lactation. Failing to litter - Day 25 after mating.

METHOD OF KILL:
F0 animals: Carbon dioxide asphyxiation.

Males and females: Subject to complete macroscopic examination. Abnormal tissues were retained in an appropriate fixative. Females - each uterine horn Number of implantation sites. The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski 1964)], for females failing to produce a viable litter only.

Tissue and regions examined:
Abnormalities - fixed; Epididymides (caput, corpus and cauda) - weighed and fixed; Ovaries - weighed and fixed; Pituitary - weighed and fixed for suspect fertility males and females only; Prostate - weighed and fixed for suspect fertility males only; Seminal vesicles (with coagulation gland) - weighed and fixed for suspect fertility males only; Testes - weighed and fixed; Uterus with cervix and oviducts - fixed for suspect fertility females only; Vagina - fixed for suspect fertility females only.

Organ Weights: For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals. For adults organ weights, group mean values and SD were calculated for absolute weights and also for weights expressed relative to body weight (%) using the body weight recorded on the day of necropsy.

Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of testes that were preserved in modified Davidson’s fluid.

Postmortem examinations (offspring):

All unselected F1 offspring and selected F1 generation animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

TIME OF NECROPSY:
Unselected F1 offspring: Culls - Day 4 of age. Scheduled kill - Day 21 of age.
Selected F1 generation: Scheduled kill - Day 29 of age.

METHOD OF KILL:
Offspring before Day 14 of age: Intraperitoneal injection of sodium pentobarbitone.
Offspring on or after Day 14 of age: Carbon dioxide asphyxiation.

Premature deaths (before weaning): Missing offspring and those grossly autolyzed or grossly cannibalized could not be examined. All other offspring dying before weaning were subject to complete macroscopic examination with assessment of stomach for presence of milk, where possible. Abnormal tissues retained in an appropriate fixative.
Culled offspring (Day 4): Culled offspring with clinical signs on Day 4 of age were subject to complete macroscopic examination with assessment of stomach for presence of milk, where possible. Abnormal tissues were retained in an appropriate fixative. Culled offspring with no clinical sign on Day 4 of age were killed and discarded without necropsy examination.
Unselected offspring at scheduled kill on Day 21 of age: Subject to complete macroscopic examination. Abnormal tissues were retained in an appropriate fixative.
Selected F1 animals: Subject to complete macroscopic examination. Abnormal tissues were retained in an appropriate fixative.

Findings from examination of offspring would have been reported on an individual basis for affected litters and offspring. There were no macroscopic findings at scheduled termination for F1 offspring and consequently no data were reported.

Statistics:

Summary statistics (e.g. means and standard deviations) were calculated from computer-stored individual raw data. The summary statistics and the individual data were stored in the computer to a certain number of decimal places, different for each parameter. For reporting purposes, however, they were usually rounded to fewer places.
Statistical analyses were performed on the majority of data and results of these tests were reported on the relevant tables. For pre-coital interval and gestation index, the similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis (except gestation and lactation phases). For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
- Body weight, using absolute weights and gains over appropriate study periods
- Food consumption, over appropriate study periods
- Estrous cycles
- Gestation length
- Mating performance and fertility
- Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before cull study periods
- Organ weights, absolute and relative to terminal body weight

Reproductive indices:

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

MATING PERFORMANCE & FERTILITY:
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (# of animals mating / Animals paired) x 100
Conception rate (%) = (# of animals achieving pregnancy / Animals mated) x 100
Fertility index (%) = (# of animals achieving pregnancy / Animals paired) x 100

GESTATION LENGTH & GESTATION INDEX:
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = (# of live litters born / # pregnant) x 100

Offspring viability indices:

The following were calculated for each litter:
- Post implantation survival index (%) = (Total # of offspring born / Total # of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
- Live birth index (%) = (# of offspring on Day 1 after littering / Total # of offspring born) x 100
- Viability index (%) = (# of live offspring on Day 4 before culling / # of live offspring on Day 1 after littering) x 100
- Lactation index (%) = (# of live offspring on Day 21 after littering / # of live offspring on Day 4 (after culling)) x 100
Group mean values were calculated from individual litter values.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test item-related signs observed at routine clinical examination and no post-dosing observations associated with test item administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were three premature deaths on this study, all of which were considered unrelated to test item administration.
On Day 7 of dosing, one female (4F No. 225) that received 1000 mg/kg/day was gasping following the dosing procedure, the animal then became unresponsive and was therefore euthanized in the animal unit for welfare reasons. Macroscopic examination of this animal revealed a perforated oesophagus with abnormal red fluid in the thoracic cavity. These findings indicated that this female was mal-dosed.
One male (4M No. 26) that received 1000 mg/kg/day was found dead after dosing on Day 8 of treatment. Macroscopic examination of this animal revealed a perforated oesophagus with abnormal red fluid and a pale material being found in the thoracic cavity and frothy pale fluid found in the trachea. These findings indicated that this male was mal-dosed.
On Day 14 of the study, prior to dosing, one female (4F No. 227) that received 1000 mg/kg/day was found dead. This female was previously observed as irritable on Day 4 of dosing but had no other clinical signs recorded since then. Necropsy findings suggested that this animal was mis-dosed as the oesophagus was perforated and there was abnormal red fluid and pale material found in the thoracic cavity. A thickened pericardium was also recorded during the macroscopic examination for this animal.
In view of these unexpected deaths, a modification was made to the dosing procedure to include dipping the dosing equipment in corn oil prior to dosing the animal.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall body weight gain was slightly higher (1.13X control) for F0 males given 1000 mg/kg/day when compared to the controls during the treatment period. Overall mean body weight gains for F0 males receiving 100 or 300 mg/kg/day were similar to control.
Prior to mating, mean body weight gain was higher for all F0 females receiving the test item. During Days 0-20 of gestation, group mean body weight gain for females given 300 or 1000 mg/kg/day was slightly lower compared to controls (0.92X and 0.94X control, respectively). These slightly lower gains during gestation for females given 300 or 1000 mg/kg/day had no impact on the mean body weight for Day 1 of lactation in both groups, as all groups receiving the test item had a similar mean body weight to controls.
Following parturition, females given 1000 mg/kg/day showed a slight mean body weight loss of 6 g during Days 1-4 of lactation compared to a mean 3 g gain in controls; thereafter, from Days 4-21 of lactation there was no effect of treatment on body weight performance. The body weight performance of females given 100 or 300 mg/kg/day was unaffected during the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment at any dose level investigated on the mean food consumption for F0 males and females during the 2-week pre-pairing period and for F0 females during the gestation and lactation periods.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

One female in the control and 1000 mg/kg/day groups exhibited irregular estrous cycles and one female at 100 mg/kg/day and 1000 mg/kg/day were acyclic, all remaining females had regular estrous cycles. This incidence of females not showing regular oestrous cycles had no effect on the pre-coital interval, with all females at all dose levels showing positive evidence of mating within the four days of pairing, however the acyclic female at 1000 mg/kg/day failed to achieve pregnancy.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Pre-coital interval and mating performance (percentage mating) were unaffected by treatment at all dose levels investigated.
At 1000 mg/kg/day, two of the six mated females failed to achieve pregnancy resulting in conception rate and fertility index being only 67%. One of eight mated females at 100 mg/kg/day also failed to achieve pregnancy with both the rate of conception rate and fertility index being 88%. All control females and females receiving 300 mg/kg/day achieved pregnancy therefore the rate of conception and fertility index were 100%.
There was no effect of treatment on gestation length or gestation index up to and including 1000 mg/kg/day, with all pregnant females successfully giving birth to live young.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs observed in the F1 offspring between birth and weaning that were considered to be related to parental treatment with the test item.

One F1 male that received 100 mg/kg/day (2M No. 411) was dispatched for animal welfare reasons after receiving its fifth dose on Day 25 of age. Signs observed 1-2 hours after dosing this animal included wet rales and piloerection, additional clinical signs of hunched posture, head tilt, salivation and a superficial subcutaneous mass on the left forelimb were recorded for the animal. Macroscopic examination identified abnormal contents in the skin/subcutis region of the left dorsocranial and trauma was observed in the left ribs.
One F1 male receiving 100 mg/kg/day (2M No. 414) was observed as having piloerection 1-2 hours after receiving its third dose on Day 23 of age and this sign was still observed at the end of the working day. Clinical signs observed for this animal included decreased activity, piloerection, hunched posture, uncoordinated gait and a superficial subcutaneous mass located on the left forelimb and therefore this animal was euthanised due to poor clinical condition. Macroscopic examination confirmed the presence of abnormal contents in the left forelimb of this animal and no other findings were recorded. Both deaths were suspected to be related to dosing traumas but as there was no damage to the oesophagus or trachea observed in either animal it cannot be definitively confirmed.
With the exception of the signs observed during the aforementioned mortality incidents; there were no clinical signs or signs associated with dosing related to test-item administration for the selected F1 generation.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Mean number of uterine implantations and Day 1 total litter size were essentially similar to the control group for females that received 1000 mg/kg/day; however, mean live birth index (%) was lower due to two females (No. 226 and 230) which each lost two offspring. Subsequent mean Day 4 viability index (%) was lower, as two females (No. 230 and 232) lost four and three offspring, respectively, during the first four days of lactation, and resulted in the lower litter size on Day 4 of lactation (prior to culls) attaining statistically significance when compared with the controls. Sex ratio for the offspring did not indicate any selective effect on survival for either sex. Offspring survival after culling of litters on Day 4 to weaning was similar to control.
There was no effect of parental treatment with the test item at doses up to and including 300 mg/kg/day on the mean number of uterine implantations, Day 1 total and live litter size, post-birth offspring survival, or sex ratio.

One F1 male that received 100 mg/kg/day (2M No. 411) was dispatched for animal welfare reasons after receiving its fifth dose on Day 25 of age. Signs observed 1-2 hours after dosing this animal included wet rales and piloerection, additional clinical signs of hunched posture, head tilt, salivation and a superficial subcutaneous mass on the left forelimb were recorded for the animal. Macroscopic examination identified abnormal contents in the skin/subcutis region of the left dorsocranial and trauma was observed in the left ribs.
One F1 male receiving 100 mg/kg/day (2M No. 414) was observed as having piloerection 1-2 hours after receiving its third dose on Day 23 of age and this sign was still observed at the end of the working day. Clinical signs observed for this animal included decreased activity, piloerection, hunched posture, uncoordinated gait and a superficial subcutaneous mass located on the left forelimb and therefore this animal was euthanised due to poor clinical condition. Macroscopic examination confirmed the presence of abnormal contents in the left forelimb of this animal and no other findings were recorded. Both deaths were suspected to be related to dosing traumas but as there was no damage to the oesophagus or trachea observed in either animal it cannot be definitively confirmed.
With the exception of the signs observed during the aforementioned mortality incidents; there were no clinical signs or signs associated with dosing related to test-item administration for the selected F1 generation.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean birth weights of male and female offspring were essentially similar in all groups and unaffected by parental treatment. There was no effect of parental treatment at doses up to
and including 1000 mg/kg/day on offspring body weight gain from birth to weaning on Day 21 of age.

Group mean body weight gain for selected F1 males and females given 1000 mg/kg/day for the treatment period was statistically significantly lower than controls (0.87X and 0.88X control for males and females, respectively). Body weight performance for selected F1 males and females given 100 or 300 mg/kg/day was unaffected by Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate] administration.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Group mean food consumption for selected F1 males and females was unaffected by treatment at all dose levels investigated.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related macroscopic abnormalities detected in the F1 offspring which died prematurely, or among the unselected F1 offspring killed at scheduled termination on Day 21 of age. There were no test item-related macroscopic abnormalities detected among the F1 males and females at scheduled termination.

One female receiving 100 mg/kg/day (2F No. 214) and two females receiving 1000 mg/kg/day (4F No.’s 231 and 234) were non-pregnant, additionally two females that received 1000 mg/kg/day were killed prematurely as result of mal-dosing. The assessment is therefore based on 8 litters in each of the Control and 300 mg/kg/day groups, 7 litters in the 100 mg/kg/day group and 4 litters in the 1000 mg/kg/day group.
Direct oral gavage administration of B-TEGME to the selected F1 animals commenced on Day 21 of age.

Conclusions:

Oral administration of B-TEGME to F0 animals at dose levels of 100, 300 or 1000 mg/kg/day was generally well tolerated with no adverse effects on clinical condition, body weight performance, food consumption or macropathology. At 1000 mg/kg/day, two out of the six mated females failed to achieve pregnancy and live birth index and Day 4 viability index were lower, although a clear relationship to treatment was not established. While these effects have the potential to compromise reproductive assessment at this dose level, there was considered to be insufficient evidence from this preliminary study, that they would be repeated in the subsequent OECD 443 study for this test item. The clinical condition and body weight gain of the offspring was unaffected by maternal treatment at 1000 mg/kg/day.
Oral administration of B-TEGME to F1 animals at dose levels of 100, 300 or 1000 mg/kg/day was also well tolerated, with no adverse effects on clinical condition, body weight performance or macropathology. Body weight gain of directly dose selected F1 offspring was slightly lower than control at 1000 mg/kg/day but not at a level precluding further investigation of toxicity at this dose level.
Based on the results obtained in this preliminary study of reproductive performance; it was concluded that the high dose level for the main extended one-generation (OECD 443) study should be set at 1000 mg/kg/day.

Executive summary:

Administration of B-TEGME at dose levels of 100, 300 and 1000 mg/kg/day to parental F0 animals for two weeks before pairing throughout gestation and lactation and to F1 selected animals from weaning up Day 28 of age was generally well tolerated. General condition (F0/F1), food consumption (F0/F1), mating performance (F0) and macropathology (F0/F1) were unaffected by treatment.

There were three premature deaths in the F0 generation, all animals were receiving 1000 mg/kg/day; one male and one female was found dead after dosing on Day 8 and 14 of treatment, respectively and an additional female was killed for welfare reasons on Day 7 of dosing. Macroscopic findings for these animals suggested mal dosing and therefore these deaths were considered not to be related to test-item toxicity. There were two premature decedents during dosing the F1 generation, both males were receiving 100 mg/kg/day and were killed for welfare reasons on Day 23 or Day 25 of age. Macroscopic examination revealed abnormal contents under the skin in the left forelimb of both animals, and one male had trauma to the left ribs; these findings are suggestive of mis-dosing but without trauma to the esophagus or trachea this relationship cannot be definitively confirmed.

One mated female receiving 100 mg/kg/day and two mated females receiving 1000 mg/kg/day were not pregnant. At 1000 mg/kg/day, this resulted in a lower rate of conception and fertility index and, in combination with the mortalities, a lower than optimum number of pregnant females/litters for assessment at 1000 mg/kg/day. One of these non-pregnant females at 1000 mg/kg/day was noted to have been acyclic, however, a similarly acyclic females receiving 100 mg/kg/day did achieve pregnancy. Epididymal and ovaries weights were also slightly lower for males or females receiving 1000 mg/kg/day, but there was no clear correlation between the low organ weights observed and the two females that failed to achieve pregnancy or the males that had suspect fertility. Whilst the highest incidence of animals failing to conceive was at 1000 mg/kg/day, the aetiology of the findings was unclear, there was no accompanying effect on implantation count for females that did attain pregnancy and no association with treatment was considered proven. A similar pregnancy rate in the subsequent main OECD 443 study would have the potential to lead to an inadequate number of litters/offspring for assessment but there is insufficient evidence from this preliminary study to indicate that such a low pregnancy rate could reasonably be expected to occur in a study of larger dimensions.

At 1000 mg/kg/day, mean live birth index was lower, due to two females which each lost two offspring, and subsequent mean Day 4 viability index was also low, as two females lost four and three offspring, respectively, during the first four days of lactation. This resulted in a statistically significantly lower litter size on Day 4 of lactation (prior to culls) at this dose level compared with the controls. However, sex ratio for the offspring did not indicate any selective effect on survival for either sex and the size of all the individual litters at 1000 mg/kg/day were within the concurrent control range. In view of the small group size available for assessment, this slight effect on offspring mortality may well represent normal biological variation and, at the magnitude observed, was insufficient to preclude this dose level from investigation in the subsequent OECD 443 study.

Differences in body weight gain from control for animals receiving 1000 mg/kg/day in the F0 generation were considered to be minor and insufficient to preclude this dose level from further investigation of toxicity. Overall body weight gain was slightly higher for males at 1000 mg/kg/day over the duration of the study and also for all treated females during the pre-pairing phase of the study. Mean overall body weight gain during gestation was slightly lower that the control but the similarity of the mean body weight on the Day 1 of lactation to that of the control suggest there was no underlying effect of treatment on the parental female. At the start of lactation, females given 1000 mg/kg/day showed a slight mean body weight loss (Days 1-4) but for the remainder of the lactation period showed improvement and overall gain during lactation was similar to the control group. There was no effect on body weight gain of the pre-weaning offspring at 1000 mg/kg/day but the selected F1 generation given 1000 mg/kg/day showed slightly low body weight gain in both sexes for the direct treatment period. However, all individual offspring weights showed satisfactory body weight gain during the treatment period and the extent of any body weight differences observed was considered not to be adverse.

Overall, there were no clear treatment related effects observed at 1000 mg/kg/day that precluded this dose level from being selected as a suitable high dose level for the subsequent OECD 443 study. Although possible effects were apparent for fertility and offspring mortality were observed, any relationship to treatment was equivocal. While these effects have the potential to compromise the reproductive assessment at this high dose level, there is insufficient evidence from this preliminary study that they would occur in a study of larger dimension, therefore, it is considered that dosage of 1000 mg/kg/day should be included in the next investigation of toxicity.

Reference 2

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: Two weeks. Animals of Cohort 1B are mated to produce the F2 generation and, thus, the premating exposure duration is 10 weeks for these Cohort 1B animals and the fertility parameters are covered allowing an evaluation of the full spectrum of effects on fertility in these animals.
- Basis for dose level selection: Based on the results obtained in a range-finding study of reproductive performance it was concluded that the high dose level for the main extended one-generation reproductive toxicity study (OECD TG 443) should be set at 1000 mg/kg/day (limit test concept).
- Inclusion/exclusion of extension of Cohort 1B: Inclusion of extension of Cohort 1B according to ECHA final decision TPE-D-2114460726-43-01/F.
- Termination time for F2: At weaning, according to OECD TG 443.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Inclusion of developmental neurotoxicity Cohorts 2A and 2B according to ECHA final decision TPE-D-2114460726-43-01/F.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: Inclusion of developmental immunotoxicity Cohort 3 according to ECHA final decision TPE-D-2114460726-43-01/F.
- Route of administration: Oral route according to ECHA final decision TPE-D-2114460726-43-01/F. The oral route is the most appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction, and B-TEGME is a liquid.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: The rat was selected as animal species because of the extent of background data and the comparability to general toxicity tests. The rat is the preferred species according to OECD TG 443 and ECHA final decision TPE-D-2114460726-43-01/F. Sprague Dawley rats were selected as this strain is accepted by regulatory agencies and historical control data are available.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as test species because of the extent of background data and the comparability to general toxicity tests. The rat is the preferred species according to OECD TG 443 and ECHA final decision TPE-D-2114460726-43-01/F. Sprague Dawley rats were selected as this strain is accepted by regulatory agencies and historical control data are available in the test facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age of the F0 animals at the start of the treatment: Males 74 to 81 days old; Females 68 to 74 days old.
- Weight range of the F0 animals at the start of the treatment: Males 347 to 444 g; Females 209 to 278 g.
- Housing: The test facility has a policy of improvement of animal welfare which includes permitting social interaction through multiple housing of rats whenever possible. This policy was followed for this study, except after mating when females were housed singly to permit collection of food consumption data individually for pregnant females and during lactation, when adult females were singly housed with their litter for good husbandry practice. Animals were also housed singly (overnight duration) to permit in cage observation which were performed as part of the functional observation battery (Cohort 2A).
For details on animal accomodation see table. Grid bottomed cages were suspended above absorbent paper which were changed daily during pairing. Solid bottomed cages had bedding which was changed at appropriate intervals. Cages and cage-trays were changed at appropriate intervals.
- Diet (e.g. ad libitum): SDS VRF1 Certified, pelleted diet ad libitum (except overnight for blood sampling for hematology, blood chemistry, urinalysis and thyroid hormone investigations for F0 and F1 Cohort 1A animals). Food hoppers were changed at appropriate intervals. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum (except overnight for blood sampling for hematology, blood chemistry, urinalysis and thyroid hormone investigations for F0 and F1 Cohort 1A animals). Water bottles were changed at appropriate intervals.
- Acclimation period: Five days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Correction factor: A correction factor of 1.14 was used when calculating quantities of test item used during dose preparation. This calculation was used to correct for the test item purity.
- Frequency of preparation: Weekly, may have been prepared in advance of the first day of dosing.
- Storage of formulation: Refrigerated (2 to 8ºC).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): The test item is instable in water (hydrolyzes to 3x methyltriglycol + boric acid).
- Amount of vehicle (if gavage): 4 mL/kg

ADMINISTRATION:
- Route: Oral gavage.
- Treated at: Constant doses in mg/kg/day.
- Volume dose: 4 mL/kg body weight.
- Individual dose volume: Calculated from the most recently recorded scheduled body weight.
- Control (Group 1): Vehicle at the same volume dose as treated groups.
- Frequency: Once daily at approximately the same time each day.
- Formulation: A daily record of the usage of formulation was maintained based on weights before and after dosing. Thie difference between actual and expected usage was monitored as a check of correct administration. After filling the dose equipment for each animal, the semi-ridged cannula was wiped dry and dipped in corn oil before administration. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Details on mating procedure:

- Time schedule: F0 pairing: After 2 weeks of treatment. F1 Cohort 1B pairing: After 10 weeks of formal treatment (nominal Week 14 of age).
- M/F ratio per cage: 1:1 (sibling pairing was not permitted).
- Length of cohabitation: Up to 2 weeks.
- Proof of pregnancy: Ejected copulation plugs and sperm within vaginal smear; referred to as Day 0 of gestation.
- M/F separation: Day when mating evidence detected.
- Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of formulations during storage were determined as part of another study, Covance Study Number MQ60WK. In that study formulations in the range 2 to 250 mg/mL were determined to be stable for 24 hours at ambient temperature (15 to 25°C) or 15 days when stored refrigerated (2 to 8°C). Samples of each formulation prepared for administration in Week 1 of the F0 and F1 generation and the last week of treatment were analyzed for achieved concentration of the test item. The analytical method involved dilution with acetone followed by further dilution with water:methanol (80:20 v:v) prior to quantitation performed using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). Sample concentrations were determined with reference to external calibration standards freshly prepared in the concentration range 0.5 ng/mL to 50 ng/mL.

Duration of treatment / exposure:

F0 animals: Males: 2 weeks pre-pairing to necropsy after selection of the F1 generation receiving at least 10 weeks of treatment; Females: 2 weeks pre-pairing to necropsy on or soon after Day 28 of lactation following selection of the F1 generation.
F1 animals: Direct treatment of F1 offspring per gavage commenced at weaning (Day 21 of age) until termination of respective cohort (all offspring had potential indirect exposure in-utero and via milk during lactation):
- Unselected F1 offspring: No direct treatment; killed on Day 22 of age, organs retained as specified.
- Cohort 1A: Primary assessment of effects upon reproductive systems and of general toxicity; treated from weaning to approximately 13 weeks of age (Day 90).
- Cohort 1B: Follow-up assessment of reproductive performance; treated from weaning to 21/22 weeks of age following breeding at ca. 14 weeks of age.
- Cohort 2A: Developmental neurotoxicity testing (neurobehavioral testing followed by neurohistopathology assessment as adults); treated from weaning up to approximately Day 75 of age.
- Cohort 2B: Developmental neurotoxicity testing; no direct treatment, assigned to neurohistopathology assessment at weaning (Day 21 or Day 22 of age).
- Cohort 3: Developmental immunotoxicity testing; treated from weaning up to Day 60 of age.
F2 animals: No direct treatment; F2 animals had potential indirect exposure in-utero and via milk during lactation.

Frequency of treatment:

Once daily, at approximately the same time each day.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

F0: 24 animals per sex per dose;
F1, Cohort 1A: 20 animals per sex per dose;
F1, Cohort 1B: 24 animals per sex per dose;
F1, Cohort 2A: 10 animals per sex per dose;
F1, Cohort 2B: 10 animals per sex per dose;
F1, Cohort 3: 10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for this study have been selected based on the findings of the Preliminary Reproductive/Development Toxicity Screening Study in Sprague Dawley Rat by Oral Gavage Administration (Covance Study Number: 8436521).
In that study, F0 males and females were dosed with 100, 300 or 1000 mg/kg/day of the test item by oral gavage for 15 days before pairing until termination either after all litters had been born for the males or at weaning of F1 generation for the females; selected F1 generation animals were dosed at the same dose levels from Day 21 to Day 28 of age. A dosage of 1000 mg/kg/day did not result in any toxicologically significant effects for the pregnant or lactating F0 female, or for the pre-weaning and post-weaning F1 offspring that precluded this dose level from further assessment of reproductive toxicity. Effects on post-weaning body weight gain was observed for directly dosed F1 offspring but there were no associated effects on survival or clinical condition.
The high dose level for this main extended one generation reproductive toxicity study (EOGRTS; OECD 443) was therefore set at 1000 mg/kg/day. The low and intermediate dose levels were set at 100 and 300 mg/kg/day respectively, to achieve a dose response and/or aid in the determination of a No Observed Adverse Effect Level (NOAEL).

- Rationale for animal assignment (if not random):
F0: Allocation after acclimatization period; By sex; After exclusion of animals showing signs of ill-health; Animals at the extremes of the body weight range were not selected if alternatives were available. At commencement of the study the weight variation did not exceed 20% of the mean weight of each sex.
Selection of offspring to form F1 generation: Selection on Day 18-20 of age. Allocation at weaning on Day 21 of age. Formal start of F1 generation nominally on Day 28 of age (± 2 days of age) (direct dose administration commenced on Day 21 of age).
Where possible, two male and two female offspring were selected from each selected litter for F1 Cohorts 1A and 1B (up to the required number of offspring). For F1 Cohorts 2A, 2B and 3 at least 1 male or 1 female offspring was selected from each selected litter (up to the number required). Selected animals were microchipped on Day 18-21 of age and separated from littermates on Day 21 of age. Formal commencement of the F1 generation was on a nominal Day 28 of age (where possible 28±2 days of age for selected F1 animals). Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortalities. These spares had body weights and clinical signs monitored weekly and were terminated after commencement of the F1 generation; they did not receive direct treatment unless used as a replacement animal.

- Fasting period before blood sampling for clinical biochemistry (F0 and F1 Cohort 1A generations, 10 animals per sex per group): Overnight deprivation of food. Samples collected under light general anesthesia (isoflurane).
Blood sampling from sublingual vein of F0 and F1 Cohort 1A animals coincided with urine collection, and animals were therefore deprived of water overnight but had access to water for a minimum period of one hour prior to blood sampling.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS (F0 generation and F1 generation):
- Time schedule: At least twice daily for evidence of reaction to treatment or ill-health.
- Deviations from normal recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition.

PHYSICAL EXAMINATIONS:
- Time schedule: Once each week for all F0 and selected F1 generation animals. For F0 and F1 Cohort 1B females on Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
- A detailed physical examination was performed at nominally the same time of day on each occasion by an observer. After removal from the home cage, animals were assessed for physical condition and behavior during handling. Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviations from normal were recorded with respect to nature, and, where appropriate, degree of severity.

DETAILED CLINICAL OBSERVATIONS (F0 and formal F1 generation animals):
- Time schedule: Week 1 – Daily; Weeks 2 to 4 – twice weekly (middle and end of the week); Week 5 onward – once each week (Days 0, 7, 14 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 and F1 Cohort 1B females).

BODY WEIGHT (F0 and F1 generation):
- Time schedule for examinations:
F0 Males: Day that treatment commenced; Each week; Before necropsy (at least 10 weeks dosing).
F0 Females: Day that treatment commenced; Each week until mating detected; Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating; Days 1, 4, 7, 14, 21 and 28 of lactation; Before necropsy.
F1 selected animals: From nominal Week 4 of age, for Cohorts 1A, 1B (males only), 2A and 3: Twice during Week 1 of the F1 generation and weekly thereafter; On the day of attainment of sexual maturation; Before necropsy. For F1 Cohort 1B females: Twice during Week 1 of the F1 generation and weekly thereafter until mating detected; Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating; Days 1, 4, 7, 14, 21 and Day 28 of lactation; Before necropsy.

FOOD CONSUMPTION (F0 and F1 generation):
- Time schedule:
F0 animals: Weekly. Food consumption was not recorded for males and females during the period when paired for mating, but for males it was resumed after maiting of all animals was completed. For females after mating, the food consumption schedule coincided with the body weight schedule: Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating; Days 1-4, 4-7, 7-14 and 14-21 of lactation.
F1 selected animals: For Cohorts 1A, 1B (males only), 2A and 3: From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.
Cohort 1B females: Twice during Week 1 of the F1 generation and weekly thereafter until mating detected; Days 0-2, 2-4, 4-6, 6-8, 8-10, 10-12, 12-14, 14-16, 16-18, 18-20 after mating; Days 1-4, 4-7, 7-14 and 14-21 of lactation.

Food consumption was not recorded for Cohorts 1B males and females during the period when paired for mating but was continued for Cohorts 1B males once pairing of all the animals was completed.

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
Duration of gestation: Time that elapsed between mating and commencement of parturition.
Parturition observations: From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded and any difficulties observed were noted.

HEMATOLOGY, PERIPHERAL BLOOD:
F0 and F1 Cohort 1A animals, 10 animals per sex per group. Blood sampling at termination.
Parameters examined: Hematocrit; Hemoglobin concentration; Erythrocyte count; Total leucocyte count; Differential leucocyte count; Platelet count; Mean cell hemoglobin; Mean cell volume; Mean cell hemoglobin concentration; Red cell distribution width; Reticulocytes (absolute and relative counts); Prothrombin time; Activated partial thromboplastin time.
A blood film was prepared for examination at the discretion of the analyst, for samples showing abnormalities in the automated analysis.

BLOOD CHEMISTRY:
F0 and F1 Cohort 1A animals, 10 animals per sex per group. Blood sampling at termination.
Parameters examined: Alkaline phosphatase; Alanine amino-transferase; Aspartate amino-transferase; Gamma glutamyl transpeptidase; Glucose; Bilirubin – total; Cholesterol – total; Creatinine; Urea; Total protein; Albumin - by chemical assay; Albumin/globulin ratio; Sodium; Potassium; Chloride; Calcium; Phosphorus; Bile Acids.

BIOMARKERS - TSH AND T4:
F0: 10 animals per sex per group at termination.
F1 offspring: 10 litters per group – pooled litter sample of pups culled on Day 4 of age (if analysis required, restricted to T4 only). 10 males and 10 females per group on Day 22 of age from as many litters as possible (1 male or 1 female per litter where possible).
F1 Cohort 1A: 10 animals per sex per group at termination.
Conditions: F1 Offspring on Day 4 and Day 22 of age: No overnight deprivation of food. F0 and F1 Cohort 1A animals: Following overnight deprivation of food. Blood sampling of F0 and F1 Cohort 1A animals coincided with urine collection and and animals were therefore deprived of water overnight but had access to water for a minimum period of one hour prior to blood sampling.
Following preparation, serum aliquots were placed on dry ice and stored frozen (-60˚C to -80˚C).

URINALYSIS:
F0 and F1 Cohort 1A animals, 10 animals per sex per group. Overnight urine collection at termination in individual metabolism cages with deprivation of food and water.
Parameters examined: Clarity/color (appearance); Volume; pH; Specific gravity; Glucose; Ketone; Bilirubin (bile pigments); Blood pigments; Protein (total and concentration); Sodium (total and concentration); Potassium (total and concentration); Chloride (total and concentration)
After centrifugation, the pellet was examined microscopically for: Epithelial cells; Leucocytes; Erythrocytes; Crystals; Casts; Spermatozoa and precursors; Other abnormal components

Oestrous cyclicity (parental animals):

F0:
Dry smears: Daily for 15 days before pairing.
Wet smears: Daily after pairing until proof of mating; For four days before scheduled termination (nominally Days 25 to 28 post partum).
Females showing no evidence of mating were separated from the males after completion of the pairing period and vaginal smearing continued for up to 5 days or until the first estrus smear was seen. If a female showed an estrus smear during this period, it was killed as soon as practically possible and subjected to macroscopic examination. If necropsy was not possible on the day of estrus, smears were continued until the morning of necropsy. If a female did not show an estrus smear, wet smears began again on Day 22 after separation from mating (with day of separation = Day 0) for a period of 4 days with the last smear on the morning of necropsy (Day 25 after mating).

F1 Cohort 1A:
Wet smears: Following onset of vaginal patency until the first cornified (estrus) smear was recorded. For at least three days prior to the start of the necropsy phase and on the day of termination.
Dry smears: For 14 days from nominal Day 75 of age (± 2 days of age).

F1 Cohort 1B:
Wet smears: After pairing until evidence of mating confirmed; For at least three days prior to the start of the necropsy phase and on the day of termination.
Females showing no evidence of mating were separated from the males after completion of the pairing period and vaginal smearing continued for up to 5 days or until the first estrus smear was seen. If a female showed an estrus smear during this period, it was killed as soon as practically possible and subject to macroscopic examination. If necropsy was not possible on the day of estrus, smears were continued until the morning of necropsy. If a female did not show an estrus smear, wet smears were continued on Day 22 after separation from pairing (with day of separation = Day 0) for a period of 4 days with the last smear on the morning of necropsy (Day 25 after mating).

Sperm parameters (parental animals):

Parameters examined in F0 males and F1 Cohort 1A males:
- Vas deferens (from left side) – each animal in each group: For all males in all groups, sperm sample (at least 200 spermatozoa, where possible) assessed for motility using a computer assisted sperm analyzer (CASA). A manual assessment of sperm morphology (at least 200 spermatozoa, where possible) was performed. For the F0 males and F1 Cohort 1A males in Groups 2 and 3 a manual assessment of sperm morphology was performed.
- Cauda epididymis (from left side): For all males in all groups, the cauda epididymis was weighed and homogenized and the number of sperm was counted using a computer assisted sperm analyzer (CASA).
- Testis (from left side): For all males in all groups, the testis was homogenized and the number of homogenization-resistant spermatids was counted using a computer assisted sperm analyzer (CASA).

Litter observations:

RECORDS MADE DURING LITTERING PHASE (F0 and F1 Cohort 1B generations):
- Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill-health or reaction to treatment. On Day 1 of age, all offspring received a qualitative assessment of body temperature, state of activity and reaction to handling.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
- Sex ratio of each litter: Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
- Individual offspring body weights: All offspring: Days 1, 4, 7, 14 and 21 of age. Unselected F1: Day 22 of age. Selected F1 (not including Cohort 2B): 23, 25, 27* and 29* of age. (*Only applicable before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ± 2 days).
- Weaning of offspring: Day 21 of age.
- Ano-genital distance: Offspring on Day 1 of age, corrected for the body weight, using analysis of covariance.
- Nipple count: Male offspring Day 13 of age.

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Litters culled to 10 (where possible 5 males and 5 females).
- All culled offspring were macroscopically examined (with thyroid hormone samples collected from 10 F1 litters per group).

SEXUAL MATURATION (All Selected F1 generation animals not including F1 Cohort 2B animals):
Males: Examined daily from Day 38 of age for the completion of balano preputial separation. Body weight recorded on day of completion of separation.
Females: Examined daily from Day 28 of age until vaginal opening occurs. Body weight recorded on day of vaginal opening.
Females in F1 Cohort 1A: A wet smear was taken daily from the day of vaginal opening until first estrus, and for at least three days prior to the start of necropsy and on the day of termination. A dry smear was taken for 14 days from approximately Day 75 of age.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
F1 Cohort 2A:
Sensory Function – Quantitative:
At approximately Day 24 of age, the animals were tested in an automated system for auditory startle habituation. Startle amplitudes measured over five consecutive blocks of 10 trials (total 50 trials).

Neurobehavioral Screening:
The functional observation battery recordings were performed at approx. the same time of day at approximately Day 63-75 of age (nominally Day 70±1). Not all animals were tested in one day, but the time of testing was balanced across the groups.
- In cage observations: Abnormal motor movements (e.g. fasciculation, convulsions, stereotypy, excessive sniffing, licking, grooming and twitches); Palpebral closure; Posture; Tremor
- In hand observations: Ease of removal from cage; Exophthalmos; Fur condition; Piloerection; Reactivity to handling; Salivation/lacrimation; Vocalization on handling
- Observation in the arena (2-min recording period): Activity count; Arousal; Abnormal motor movements; Fecal count; Gait; Palpebral closure; Posture; Rearing count; Urination; Tremor
- Reactivity investigations: Approach response; Body temperature; Body weight; Grip strength (fore and hindlimb); Landing footsplay; Pupil closure reflex; Righting reflex; Auditory startle reflex; Tail pinch response; Touch response (pinna reflex); Proprioception

Motor Activity:
Between Days 63-75 of age (nominally Day 65±1) motor activity was measured by automated infra-red equipment. High and low beams recorded rearing and cage floor activity, respectively. For testing, designated animals were placed singly into observation cages. The test session for each animal was one hour. Data were automatically collected and reported at regular intervals throughout the session.

F1 Cohort 2B:
No specific investigations in life, animals despatched to necropsy at weaning (Day 21 or Day 22 of age) for histopathological examinations.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
F1 Cohort 3:
T cell dependent antibody response (TDAR) using keyhole limpet hemocyanin (KLH):
- Administration of KLH: Intravenous (bolus) at 300 µg KLH/animal/occassion (0.1 mLvolume per animal) on Day 47 and Day 54 of age.
- Blood sampling for evaluation of IgM response to KLH administration: On Day 46 of age (pre 1st KLH administration), Day 53 and 60 of age (six days after each KLH immunization) from sublingual vein under isoflurane anaesthesis. Blood volume 0.3 mL. Number of blood samples per occasion: Day 46 of age - 80; Day 53 of age - 80; Day 60 of age - 80. Blood samples allowed to clot for 0.5 to 2 hours after collection at room temperature before preparation of single serum aliquots. Serum was placed on dry ice following collection and stored frozen (-60˚C to -80˚C).
- Analysis: Specific anti-KLH IgM antibodies were measured in the Department of Immunology and Immunotoxicology (I&I) using suitable validated methods (Method No. I&I/0090: Method for the Measurement of Anti-KLH IgM in Rat serum). Details are documented in a separate Methodology Validation report.

Postmortem examinations (parental animals):

NECROPSY:
- Time of necroppsy:
F0 adult animals: Females on Day 28 post-partum. Males after at least 10 weeks of treatment and after weaning of the F1 animals, after confirmation that no further mating required.
F0 females failing to mate: If an estrus smear was seen following completion of the pairing period animals were terminated as soon as logistically possible. If no estrus smear was seen, animals were terminated on Day 25 after the last day of pairing.
F0 females failing to produce a viable litter and those with total litter loss: Terminated with first cohort of females with live litters.
F1 Cohort 1B animals: Males at approx. 21-22 weeks of age. Females on Day 28 post-partum.
F1 Cohort 1B females failing to mate: If an estrus smear was seen following completion of the pairing period animals were terminated as soon as logistically possible. If no estrus smear was seen, animals were terminated on Day 25 after the last day of pairing.
F1 Cohort 1B females failing to produce a viable litter and those with total litter loss: Terminated with first cohort of females with live litters.
- Macroscopic Pathology:
Complete: All animals, including surplus F1 and F2 offspring culled on Day 4 of age, and unselected F1 and F2 offspring. Where possible, decedent offspring ≤21 days of age (found dead or welfare kill) were examined and carcass retained.
Implantation site count: F0 females and F1 Cohort 1B females.

HISTOPATHOLOGY / ORGAN WEIGHTS:
- Organ weights: For bilateral organs, left and right organs were weighed together unless otherwise specified. Organ weights were not routinely recorded for animals killed or that died prematurely; organ weights were recorded for groups terminated prematurely.
- Fixation (F0 animals, F1 Cohort 1B animals): Standard 10% neutral buffered formalin. Where possible, carcass retained for decedent offspring ≤21 days of age. Testes in modified Davidson’s fluid. Eyes in Davidson’s fluid.
- Histology: See tables.

Postmortem examinations (offspring):

NECROPSY:
- Time of necroppsy:
F1 unselected offspring: On Day 4 and Day 22 of age.
F1 Cohort 1A animals: At approx. 13 weeks of age.
F1 Cohort 2A animals: At approx. Day 75 of age.
F1 Cohort 2B animals: At Day 21/22 of age.
F1 Cohort 3 animals: At approx. Day 60 of age.
F2 offspring: On Day 4 and Day 22 of age.
- Macroscopic Pathology:
Complete: All animals, including surplus F1 and F2 offspring culled on Day 4 of age, and unselected F1 and F2 offspring. Where possible, decedent offspring ≤21 days of age (found dead or welfare kill) were examined and carcass retained.

HISTOPATHOLOGY / ORGAN WEIGHTS:
- Organ weights: For bilateral organs, left and right organs were weighed together unless otherwise specified. Organ weights were not routinely recorded for animals killed or that died prematurely; organ weights were recorded for groups terminated prematurely.
- Fixation:
Unselected F1 and F2 animals (including decedent offspring), F1 animals Cohort 1A and Cohort 3: Standard 10% neutral buffered formalin. Where possible, carcass retained for decedent offspring ≤21 days of age. Testes (F1 animals only) in modified Davidson’s fluid. Eyes in Davidson’s fluid.
F1 generation Cohort 2A and 2B: Standard glutaraldehyde:paraformaldehyde fixation by in situ perfusion followed by immersion. Peripheral nerves: Left nerve specimens were retained in situ in the carcass and could be teased and evaluated by light microscopy if any evidence of neurological change was detected in the resin sections or if neurobehavioral studies indicated evidence of peripheral neuropathy.
- Histology: See tables.

IMMUNOPHENOTYPING OF SPLEEN LEUCOCYTES:
F1 Cohort 1A animals: Ten males and ten females per group from F1 Cohort 1A animals, from as many litters as possible, were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter (i.e. all surviving litters should be represented by at least one offspring). The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological examination. The remaining portions of the spleen were weighed and then placed into a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held on wet ice until processing for analysis. Cell suspensions from each tissue section were prepared by mechanical dissociation, following method IAI/0304 and the samples were stained with a cocktail of directly conjugated monoclonal antibodies. Any contaminating red cells were removed using lysis buffer and the samples were fixed by re-suspension in phosphate buffered saline containing 1% formaldehyde. Samples were analyzed immediately or stored at 2-8°C until analysis. Each sample was analyzed for cell markers using a combination of antibody markers.

Statistics:

DATA TYPES:
The following data types will be analyzed at each timepoint separately, where required, in support of interpretation:
- body weight, using absolute weights and gains over appropriate study periods.
- food consumption, over appropriate study periods.
- estrous cycles, vaginal opening to first estrous and pre-coital interval.
- mating performance and fertility.
- gestation length.
- litter size and survival indices.
- pre-weaning examination (ano-genital distance, surface and righting reflexes)
- sexual maturation, age and body weight at completion.
- clinical pathology (hematology, blood chemistry, urinalysis).
- thyroid hormone analysis (TSH and T4).
- immunophenotyping.
- behavioral data (sensory function, arena rearing and activity counts, grip strength, landing footsplay, body weight, body temperature and motor activity).
- organ weights, both absolute and body weight relative.
- sperm analysis, motility, morphology and counts.

METHODS:
For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group.
For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary.
Under the advice of the Associate Director, Global Statistics, or other qualified Statistician, alternative or additional methods may be carried out if deemed appropriate following data review.

Reproductive indices:

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

MATING PERFORMANCE & FERTILITY:
Individual data was tabulated. Group values calculated for males and females separately for the following:
Percentage mating (%) = (# of animals mating / Animals paired) x 100
Conception rate (%) = (# of animals achieving pregnancy / Animals mated) x 100
Fertility index (%) = (# of animals achieving pregnancy / Animals paired) x 100

GESTATION LENGTH & GESTATION INDEX:
Gestation length: Individual values tabulated for the number of days from mating to the start of parturition (inclusive), with half a day subtracted where parturition started overnight. Percentage of animals in appropriate categories tabulated for each group.
Gestation index was calculated for each group as:
Gestation index (%) = (# of live litters born / # pregnant) x 100

Offspring viability indices:

The following were calculated for each litter:
- Post implantation survival index (%) = (Total # of offspring born / Total # of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
- Live birth index (%) = (# of offspring on Day 1 after littering / Total # of offspring born) x 100
- Viability index (%) = (# of live offspring on Day 4 before culling / # of live offspring on Day 1 after littering) x 100
- Lactation index (%) = (# of live offspring on Day 21 after littering / # of live offspring on Day 4 (after culling)) x 100
Group mean values were calculated from individual litter values.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test-item related clinical signs or post-dose observations related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Eight females from the F0 generation were euthanized for welfare reasons or died prematurely during the study. None of the deaths were associated with the administration of the test item:
On Day 14 of treatment, one female (3F No. 249) that received 300 mg/kg/day was euthanized for welfare reasons due to abnormal gait with hind limbs splayed; this animal had also previously shown hunched body posture and whole-body pallor. Macroscopic examination revealed dark and thickened meninges, thickened pericardium, enlargement of the spleen, pancreatic, lumbar and mediastinal lymph nodes and thick pale fluid in the thoracic cavity. The cause of death was considered lymphoma.
On Day 16 of treatment, one female (4F No. 275) that received 1000 mg/kg/day was found dead, this female had no previous clinical observations or observations relating to dose administration. Macroscopic examination revealed a perforated esophagus, clotted blood and pale caseous material in the pericardium, and thickened pericardium. These findings indicated that this female was mal-dosed.
One female (4F No. 281) that received 1000 mg/kg/day was underactive, slightly hunched with irregular breathing and pale eyes prior to dosing on Day 18 of treatment. This animal was not dosed, their condition deteriorated during the day and was therefore euthanized for welfare reasons. Macroscopic examination revealed a perforated esophagus, thickened pericardium and abnormal contents of the pericardium, dark areas of the jejunum and many organs were noted as pale. These findings indicated that this female was mal-dosed.
On Gestation Day 6, one Control female (1F No. 218) was found dead and had macroscopic findings that suggested the death was due to a dosing accident. The findings included a distended esophagus with firm and pale contents and there was abnormal fluid and clotted blood in the heart.
On Day 1 of lactation, one female that received 100 mg/kg/day (2F No. 235) was observed gasping and the offspring were observed as having little or no milk in their stomachs and therefore this female and the litter were killed for welfare reasons. Macroscopic findings included a pale and inactive mammary gland, and the jejunum was pale in colour; no specific cause of death was established at histopathology.
One female receiving 1000 mg/kg/day (4F No. 284) was found dead in the cage, partly cannibalised on Day 24 of lactation. The only macroscopic finding for this female was that all lobes of the lungs were described as firm all other findings were related to the damage caused by the cannibalisation. This female was in good clinical condition and has no signs observed related to the dosing procedure in days prior to the death, a cause of death was not established at histopathology.
One female receiving 100 mg/kg/day (2F No 236) was euthanized on Day 49 and one female receiving 1000 mg/kg/day (4F No. 276) was euthanized on Day 44 due to failure to litter, both females were found to be not pregnant.
All other F0 animals survived to their scheduled sacrifice.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall mean body weight gain for males at all dose levels was unaffected by treatment.
Body weight gains for females prior to pairing and during the gestation period were unaffected by test item administration.
Group mean body weight gain for females receiving 300 or 1000 mg/kg/day were statistically significantly higher during Days 7 to 14 of lactation, when compared to controls. As a result, overall weight gain during Days 1 to 21 of lactation was statistically significantly higher at 1000 mg/kg/day. Conversely, a statistically significantly greater mean body weight loss was seen in females receiving 1000 mg/kg/day during Days 21 to 28 of gestation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption for males receiving 1000 mg/kg/day prior to pairing was generally slightly higher compared to controls with differences attaining statistical significance during Weeks 5 and 7 to 9 of treatment, resulting in the overall mean being statistically significantly higher compared to controls (1.06X control). Mean food consumption for males receiving 100 or 300 mg/kg/day was similar to controls. Mean food intake for females at all dose levels before pairing and during gestation and lactation was unaffected by treatment.

Food efficiency:

no effects observed

Description (incidence and severity):

The efficiency of food utilization for both males and females during the two week treatment period before pairing was unaffected by administration of the test item.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological investigations prior to the termination of the F0 generation revealed statistically significantly lower lymphocyte, eosinophil and basophil counts compared with controls (0.75X, 0.58X and 0.57X control, respectively) in males receiving 1000 mg/kg/day.
Males receiving 1000 mg/kg/day had statistically significantly low mean haematocrit (0.95X control), reticulocyte counts (0.72X control) and mean cell volume (0.95X control) compared with controls; mean cell volume was also significantly low among males at 300 mg/kg/day and females at 1000 mg/kg/day (0.96X and 0.97X control, respectively).
All other hematological differences from control observed during the treatment period were generally minor, confined to one sex or lacked dose relationship and were therefore attributed to normal biological variation. These included slight reductions in mean corpuscular haemoglobin concentration and red cell distribution width in females receiving 1000 mg/kg/day.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Biochemical analysis of the plasma at scheduled termination of the F0 animals revealed, when compared to controls, statistically significantly slightly higher mean urea concentrations in males receiving 1000 mg/kg/day (1.15X control).
All other biochemical differences from controls observed at scheduled termination were minor, limited to one sex and lacked a clear dose relationship and were therefore attributed to normal biological variation; this included a slight increase in sodium concentration in males at 300 mg/kg/day.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Mean T4 serum concentrations in F0 males given 1000 mg/kg/day was statistically significantly lower compared to controls although in absence of dose response. No other statistically significant difference in T4 concentrations was observed between the control and treatment groups.
Mean serum TSH concentrations were statistically significantly higher compared to controls in all groups of F0 males (2.19X, 2.18X and 2.65X control, at 100, 300 or 1000 mg/kg/day, respectively) although there was no dose response apparent; a similar difference was not apparent in F0 females.
Since these differences were slight and not accompanied by histological alterations in the thyroid, they were not considered adverse. Additionally, the majority of values were within the historical control data (HCD) range of the rat strain used, with only one F0 male TSH concentration (4M No. 82) outside the HCD range.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females given 1000 mg/kg/day had a statistically significantly higher total protein concentration compared with controls (approximately 1.45X control) and urinary pH was significantly lower; total chloride concentration was also significantly higher in females given 1000 mg/kg/day (1.42X control).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings related to the administration of the test item were present in the kidneys of F0 females and the thymus and epididymis of F0 males:
There was a higher incidence and severity of basophilic tubules in the kidneys of females given 1000 mg/kg/day, with more of multifocal distribution. This correlated with the higher organ weight at necropsy. The females given 100 or 300 mg/kg/day showed a slightly higher incidence of basophilic tubules when compared to contemporaneous controls. Whilst no increase in severity of the finding was present, in those given 300 mg/kg/day the distribution was multifocal similar to those given 1000 mg/kg/day. There was no true dose relationship established and the severity was minimal thus the significance of this change in females given 300 mg/kg/day is considered equivocal and in those given 100 mg/kg the severity and distribution were considered similar to controls and not histopathologically significant.
In males there was cellular debris present in the epididymis in those given 1000 mg/kg/day. Whilst there was no correlating change within the testes making interpretation of this change unclear, there were alterations in the sperm analysis indicating adverse changes in the male reproductive system. No change was present in males given 100 or 300 mg/kg/day.
There was a slightly higher incidence of decreased lymphocytes in the cortex of the thymus of males given 1000 mg/kg/day. This correlated with a slightly lower weight at necropsy but was minor, of low incidence and therefore of equivocal significance. No change was present in males given 100 or 300 mg/kg/day.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Mineralisation in the aorta was present only in females given 1000 mg/kg/day but this is seen as a background finding of variable incidence in Sprague Dawley rats in this laboratory and was considered to be incidental.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Five females receiving 1000 mg/kg/day exhibited irregular estrous cycles compared to one female exhibiting an irregular cycle in the controls. This incidence of females not showing regular estrous cycles had no effect on the pre-coital interval, with the majority of females at all dose levels showing positive evidence of mating within the four days of pairing. Estrous cycles of the F0 females at termination were unaffected by treatment.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, the percentage of motile and progressively motile sperm was lower (motile sperm: 81% versus 94% in controls), total testicular spermatid number was higher (221 millions/g versus 187 millions/g in controls), and all motion parameters assessed were statistically significantly lower with the exception of medium and static sperm, percentage of slow sperm which were higher when compared to controls. Sperm morphology was also affected for males given 1000 mg/kg/day with a higher percentage of abnormal sperm observed 20.1% compared to 2.2% in controls, included abnormalities such as decapitated sperm (13.3%), abnormal head (3.5%), abnormal tail (3.6%), abnormal midpiece (2.2%) and flat head (3.4%).
Sperm motility, counts and morphology were unaffected for males that received 300 mg/kg/day although numerous motion parameters (VAP, VCL, ALH, STR and LIN) were statistically significantly lower compared to controls.
At 100 mg/kg/day, as with 300 mg/kg/day but to a lesser extent, some motion parameters (VAP, VCL and ALH) were statistically significant lower compared to controls. Sperm morphology and counts were unaffected by test item administration.

Reproductive performance:

no effects observed

Description (incidence and severity):

Pre-coital interval was unaffected by test item administration. All mating pairs showed positive evidence of mating within six days of pairing.
Mating performance and fertility were unaffected by the test item. The percentage mating was 100% in all groups, and conception rate and fertility index were 95-100 % in all groups; one F0 female given 100 mg/kg/day (2F No. 236) and one F0 female given 1000 mg/kg/day (4F No. 276) were not pregnant. In addition, for one control female found dead on Day 6 of gestation pregnancy status could not be determined.
A slight, but statistically significant, shift to gestation lengths was apparent for females given 1000 mg/kg/day compared to controls. The expected gestation length in CD rats ranges from 22 to 23 days. In the 1000 mg/kg/day group, more females had a 23-day gestation length, one female showed a 23.5-day gestation length and another female showed a 24-day gestation length. There was no effect of treatment on gestation index up to and including 1000 mg/kg/day, with all pregnant females successfully giving birth to live pups.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

reproductive function (sperm measures)

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the F1 generation males lower T4 serum concentrations were observed in males that received 300 or 1000 mg/kg/day, with the difference from control attaining statistical significance. No other statistically significant difference in T4 concentrations was observed between the control and treatment groups.
There was no effect of test item administration on mean serum TSH concentration in F1 offspring on Day 22 of age or in Cohort 1A animals when compared to controls.
Since these differences were slight and not accompanied by histological alterations in the thyroid, they were not considered adverse. Additionally, the majority of values were within the historical control data (HCD) range of the rat strain used, with only one F1 male T4 concentration (4M No. 476) outside the HCD range.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

F1 generation (Cohort 1B):
Limited tissues were examined from all Cohort 1B males and females from Group 1, 3 and 4 due to a significant reduction in the number of females producing and maintaining litters in animals given 1000 mg/kg/day. In those females given 1000 mg/kg/day eleven animals failed to litter and a further animal did not mate. Of those females given 1000 mg/kg/day littering as expected, four suffered total litter losses resulting in only eight with litters surviving to completion of the study. Microscopically there were no consistent changes present to account for the differences. Of those animals failing to litter one had involuting implantation site(s) (Animal 956), another had a large necrotic fetus present in the uterus (Animal 961), one had inflammation in the uterus and cervix which may have affected its ability to conceive or maintain pregnancy (Animal 960) and another had no corpora lutea present in the ovaries (Animal 971) suggesting abnormal cyclical activity. No histological changes were present in the remaining seven. Of the eight females with viable litters at study completion there was a higher weight of the uterus/cervix/oviducts which correlated with a higher proportion with estrus morphology in the vagina. It is not clear, due to the comparatively small pool of animals given 1000 mg/kg/day remaining, if there is any disruption of resumption of normal cyclical activity.
In males, findings related to the administration of the test item were present in the testes and epididymis of males. Cellular debris in the epididymis was present in most males given 1000 mg/kg/day and occasional males given 300 mg/kg/day. There was minimal degeneration of occasional cells in the seminiferous tubules of males given 1000 mg/kg/day indicating progression of the findings seen in the F0 males and F1A males. The F1A males received the test item for a shorter duration of time which may account for the difference.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous Cycles - Cohort 1A:
A slightly higher number of females showing irregular cycles was observed in F1 Cohort 1A females compared to controls, with two females showing irregular cycles in females given 300 or 1000 mg/kg/day and three females exhibited irregular cycles at 100 mg/kg/day compared to no control females showing irregular cycles. Furthermore, one female in both the 300 and 1000 mg/kg/day groups were acyclic. As the majority of females receiving the test item showed 4-, 4/5- or 5-day cycles, and as no dose relationship was apparent, it is considered that estrous cycle regularity of the Cohort 1A females during the last two weeks of treatment was unaffected by test item administration. In addition, estrous cycle at termination for these females were unaffected at all dose levels investigated.

Stage of Estrous Cycle at Termination - Cohort 1B:
The majority of females that received 100, 300 or 1000 mg/kg/day were showing estrous morphology at termination.

Ovarian Follicle Counts and Corpora Lutea - Cohort 1A:
At scheduled termination of the Cohort 1A females, ovarian follicle counts were slightly lower in females given 1000 mg/kg/day when compared to Controls. There was no similar decrease in the number of corpora lutea present.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Sperm Assessment - Cohort 1A:
At 1000 mg/kg/day, the percentage of motile and progressively motile sperm was lower (90% versus 95% in controls for motile sperm), total epididymal sperm count was lower (103 millions/g versus 140 millions/g in controls) and VAP, VSL, VCL, ALH and Rapid motion parameters assessed were statistically significantly lower and medium, slow and static sperm were significantly higher than in controls. Sperm morphology was also affected for males given 1000 mg/kg/day with a higher percentage of abnormal sperm observed 15.8 % compared to 2.8% in controls, included abnormalities such as decapitated sperm (6.4%), abnormal head (7.1%), abnormal tail (3.3%), and flat head (6.7%).
Sperm motility and morphology were unaffected for males that received 300 or 100 mg/kg/day although epididymal sperm counts were slightly lower for both groups but were within the historical control data (HCD) range.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Pre-Coital Interval - Cohort 1B:
The pre-coital interval for all F1 Cohort 1B females receiving 100 mg/kg/day including controls was within 1 to 4 days. Among females receiving 300 mg/kg/day, two females had pre-coital interval of 5 to 8 days and one female had a pre-coital interval of 9 to 12 days, the remaining 21 females had pre-coital interval of 1 to 4 days. A similar trend was observed in F1 Cohort 1B females that received 1000 mg/kg/day with the majority of females (21) having a pre-coital interval of 1 to 4 days, two females had longer pre-coital intervals with one female having a 5 to 8 days pre-coital interval and one female having a 13 to 14 days pre-coital interval. As the majority of females receiving the test item showed a positive evidence of mating within a 4-day period it is considered that these low incidences of extended pre-coital intervals are due to normal biological variation and therefore pre-coital interval is unaffected by test item administration at all dose levels investigated.

Mating Performance and Fertility - Cohort 1B:
The conception rate and fertility index were statistically significantly lower compared to controls, at 88% or 57% for conception rate and 88% or 54% for fertility index, for F1 Cohort 1B females that received 300 or 1000 mg/kg/day, respectively. At 300 and 1000 mg/kg/day, 24 and 23 females were confirmed to have mated with 21 and 13 females achieving pregnancy, respectively. Mating performance and fertility of the Cohort 1B animals given 100 mg/kg/day were unaffected by test item administration.

Gestation Length and Gestation Index - Cohort 1B:
A slight, but statistically significant, shift in gestation length was apparent for females given 300 and 1000 mg/kg/day compared to controls. The expected gestation length in CD rats ranges from 22 to 23 days. In the 300 mg/kg/day group, three females showed a 23.5-day gestation length compared to two females given 100 mg/kg/day and one control female. Since this difference was very slight, it was considered non-adverse. In the 1000 mg/kg/day group, three females showed a 23.5-day gestation length, four females showed a 24-day gestation length and two females showed a 24.5-day gestation length. Gestation index was unaffected by test item administration.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

reproductive performance

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the F1 offspring that were considered to be related to parental treatment with the test item.
There were no signs associated to dose administration and there were no test item-related changes in general clinical condition among F1 males and females of all cohorts throughout the study, at any dose level investigated.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F1 Litter Responses:
Mean number of uterine implantations and Day 1 total or live litter sizes were essentially similar to the control group for females that received 1000 mg/kg/day; however, Day 4 viability index (%) was lower, as offspring deaths during the first four days of lactation occurred in 9/21 litters in females given 1000 mg/kg/day compared to offspring deaths in 2/23 litters in controls during the first four days of lactation and the difference attained statistical significance. Sex ratio for the offspring did not indicate any selective effect on survival for either sex. Offspring survival after culling of litters on Day 4 to weaning was similar to control.
There was no effect of parental treatment with the test item at 100 or 300 mg/kg/day on the mean number of uterine implantations, Day 1 total and live litter size, post-birth offspring survival, or sex ratio.

F1 Generation:
Increased premature euthanasia related to the test item administration was seen in females, due to reduced fertility in the F1 generation in Cohort 1B. At 1000 mg/kg/day, twelve animals were euthanized early, eleven due to failure to litter and one which failed to mate. A further four were euthanized due to total litter loss. One control female (1F No. 887) was euthanized for welfare reasons on Day 42 after the formal commencement of F1 treatment and this was due to a liver lobe torsion. One female given 300 mg/kg/day (3F No. 947) was euthanized on Day 13 of gestation due to a mammary tumor. In addition, three females given 300 mg/kg/day failed to litter where one female (3F No. 938) was paired with a male (3M No. 542) with notable tubular atrophy in the testes which would have resulted in infertility. The remaining two females had no obvious cause for the lack of litter but at this low incidence it was considered incidental and not related to treatment. One female that received 100 mg/kg/day (1F No. 921) was euthanized for welfare reasons on Day 14 of lactation. From Day 10 of lactation onwards the animal had decreased activity, thin build, rapid breathing, piloerection, hunched posture and its perigenital area was dark with a firm superficial mass present that ruptured on Day 14 of lactation. Macroscopic examination of the animal found that the adrenal glands had dark areas, the lungs and bronchi had pale areas, there were depressions on the tongue and ruptured masses present on the mammary tissue in the genital region.
Two further premature deaths occurred in the F1 generation; however these deaths were not attributable to test item administration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring Body Weight:
The group mean body weight of male and female F1 offspring on Day 1 of age, and subsequent body weight gain of the offspring to weaning on Day 21 of age, was unaffected by parental treatment with the test item at all dose levels investigated.

F1 Generation Body Weight:
A slight reduction in body weight and body weight gain was recorded from weaning until Day 25 of age for males and females given 1000 mg/kg/day. From the formal start of the F1 generation, body weight gains in males only receiving 1000 mg/kg/day continued to be reduced during the first 4 weeks of the F1 generation (0.89X control from Day 1 to 29 of the formal start of F1 generation); thereafter there was no conclusive or consistent effect of treatment. Body weight gains of males and of females before pairing were unaffected by treatment at 100 or 300 mg/kg/day.
Mean body weight gain for the F1 Cohort 1B females that received 1000 mg/kg/day was statistically significantly lower compared to control during the gestation period (0.57X control). Group mean body weight gain for the F1 Cohort 1B females given 100 or 300 mg/kg/day during gestation and all dose levels investigated during lactation was essentially similar to controls.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The overall mean food consumption value for males that received 1000 mg/kg/day was slightly lower (0.95X control) compared to controls, this difference attained statistical significance. Mean food intake values for individual periods for males given 1000 mg/kg/day were low at the start of the treatment and towards the end of the treatment period showed slightly higher mean intake values compared to controls. As these differences were minor, lacked consistency and showed no dose response relationship, they were attributed to normal biological variation. Food consumption for males at 100 or 300 mg/kg/day was unaffected by treatment.
Food consumption was statistically significantly lower (0.92X control during Days 1 to 4) than control for females at 1000 mg/kg/day for the first week of treatment (Days 1-8), thereafter food consumption was comparable with control. Overall food consumption was unaffected at any dose level for F1 females or for F1 Cohort 1B females before pairing and during gestation.
Mean food consumption for females that received 1000 mg/kg/day was statistically significantly lower compared to control for the lactation period (0.75X control). Mean food consumption for females at 100 or 300 mg/kg/day was unaffected by treatment during lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion efficiency for F1 Cohort 1B animals prior to pairing was unaffected by test item administration.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology - Cohort 1A:
Haematological investigations prior to the termination of the F1 Cohort 1A animals revealed slightly lower mean cell hemoglobin, mean cell volume and red cell distribution width in males and females that received 1000 mg/kg/day when compared with controls, with all differences attaining statistical significance. Mean cell hemoglobin was lower in males and females receiving 300 mg/kg/day, and mean cell volume was lower in females given 300 mg/kg/day, with all differences from control attaining statistical significance. Red blood cell concentration was slightly higher (1.05X control) for females that received 1000 mg/kg/day when compared to controls.
All other differences from control were minor, limited to one sex or lacked a dose response relationship and were therefore attributed to normal biological variation, such as the low eosinophil counts among males at 1000 mg/kg/day and high leukocytes and lymphocytes in females that received 300 mg/kg/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood Chemistry - Cohort 1A:
Biochemical analysis of the plasma at scheduled termination of the F1 Cohort 1A animals revealed, when compared to controls, slightly higher mean urea concentrations in males and females receiving 1000 mg/kg/day (1.22X and 1.14X control, for males and females, respectively). These differences attained statistical significance, but no dose relationship was apparent in both sexes.
Mean plasma bile acids were moderately higher and achieved statistical significance in all groups of treated females although in the absence of a dose response relationship.
All other biochemical differences from controls observed at scheduled termination were minor or lacked clear dose relationship or were confined to one sex and therefore attributed to normal biological variation. These differences included slightly high gamma-glutamyl transferase activity in males at 100 mg/kg/day and statistically significant differences in glucose concentration were conflicting for males and females at 1000 mg/kg/day, mean glucose concentration were also low for males receiving 100 or 300 mg/kg/day, but a dose relationship was not apparent.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis - Cohort 1A:
Analysis of the clarity/color and composition of the urine of F1 Cohort 1A animals prior to scheduled termination revealed, when compared to controls, statistically significantly lower pH in males and females in the 1000 mg/kg/day group. Males in this dose group also showed slightly lower urinary protein, sodium, potassium, and chloride concentrations. Microscopy of the urine sediment did not reveal any test item-related changes.

Sexual maturation:

no effects observed

Description (incidence and severity):

The age and body weight at which F1 females attained vaginal opening in all treatment groups, and at which F1 males at 100 or 300 mg/kg/day attained balano preputial separation was unaffected by test item administration.
The body weight at completion of balano preputial separation was statistically significantly lower for F1 males that received 1000 mg/kg/day when compared to controls, however the age of completion was similar to controls.

Vaginal Opening - Cohort 1A: There was no effect of test item administration on the duration between vaginal opening and the first estrus occurring in the Cohort 1A females.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distance of F1 offspring at dose levels up to 1000 mg/kg/day on Day 1 of age was unaffected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

All F1 male offspring were assessed on Day 13 of age for the presence of nipples; no nipples were observed.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Unselected Offspring:
There was no clear effect of parental test item administration on the weights of the brain, liver, spleen or thymus of the unselected F1 offspring killed on Day 22 of age.
It was noted that at 1000 mg/kg/day there was a statistically significantly lower absolute brain, spleen and thymus weight in males and lower absolute brain weight in females. At 300 mg/kg/day there was a statistically significantly lower absolute brain weight in females. After adjustment for body weight the thymus weight of males was lower at 1000 mg/kg/day but the variation was not statistically significant. After adjustment for body weight the spleen weight was lower for males but higher for females, neither change reaching statistical significance. The brain weight showed no variation from contemporaneous control animals after adjustment for body weight.
All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

F1 generation (Cohorts 1A and 1B):
In Cohort 1A there were several statistically significant lower absolute organ weights, particularly in males, which were considered likely to be related to the lower body weight in animals given 1000 mg/kg/day. In males, lower weights were present in the brain, epididymis, pituitary, spleen, thymus, thyroids and parathyroids and the prostate. After adjustment for body weight there was a statistically significant higher weight in the heart, kidneys and seminal vesicles with coagulating gland. There was a statistically significant lower weight in the pituitary and thymus. In Cohort 1B there was a statistically significant lower absolute pituitary and epididymides weight in males given 1000 mg/kg/day. After adjustment for body weight there was still a marginal, although not statistically significant, lower pituitary weight. After adjustment for body weight there was a statistically significant higher seminal vesicles with coagulating gland weight. No changes were noted at pathology in the seminal vesicles and the variation occurred only in this cohort, thus this is considered to be incidental.
In F1A females given 1000 mg/kg/day lower absolute weights were present in the adrenal glands, brain, ovaries and pituitary. After adjustment for body weight there was a statistically significant higher weight in the kidneys and lower weight in the pituitary. In the F1B females given 300 and 1000 mg/kg/day there was a statistically significant lower pituitary weight and higher uterus, cervix and oviduct weight, both absolute and body weight adjusted. The higher weight of the uterus/cervix/ovaries is likely to be related to cyclical activity with an increased proportion of females in estrus compared to control animals, however the low number of surviving females given 1000 mg/kg/day makes interpretation complex thus relationship to treatment is equivocal.
Whilst there were no histopathology correlates for any of the organ weight changes in the F1 Cohort 1A animals, the lower weight of the thymus and pituitary and higher weight of the kidneys were consistent with the F0 animals and thus were considered to be related to the administration of the test item.

F1 generation (Cohorts 2A and 2B):
There was a marginally lower brain weight, both absolute and relative to body weight in animals given 1000 mg/kg/day in both Cohorts 2A and 2B.

F1 generation (Cohort 3):
No changes were apparent in the weight of the spleen from Cohort 3 animals which could be related to the administration of the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The predominant macroscopic observation detected in the F1 offspring that died prior to scheduled termination was the absence of milk in the stomach.
There were no macroscopic abnormalities detected among the unselected F1 offspring killed at scheduled termination on Day 22 of age.

The macroscopic examination performed at scheduled termination of F1 Cohort 1A, 1B, 2A, 2B and 3 animals revealed no test item-related lesions. The incidence and distribution of all findings were considered to be unrelated to treatment.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 generation (Cohort 1A, 2A and 2B):
In the F1 Cohort 1A findings related to the administration of the test item were present in the epididymis of males. There was minimal cellular debris present in the epididymis in those given 1000 mg/kg/day. Whilst there was no correlating change within the testes making interpretation of this change unclear, there were alterations in the sperm analysis indicating adverse changes in the male reproductive system. No change was present in males given 100 or 300 mg/kg/day. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
No changes related to the administration of the test item were seen in the brain and nerves, including both paraffin and resin sections from Cohort 2A and the brain sections from Cohort 2B including brain morphometry from both cohorts.
All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for this strain. Therefore, they were considered not test article related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Auditory Startle Response Habituation - Cohort 2A:
Auditory startle response habituation for F1 Cohort 2A animals at Day 24±1 of age, both in terms of latency to peak amplitude values and peak amplitude values, was unaffected by treatment with the test item at all dose levels investigated. The group mean percent habituation values in both sexes at all dose levels was considered to be unaffected by test item administration. There was no effect on the latency to the peak response or on the amplitude of the peak response during the initial 10 trials, only one individual block of 10 trials for mean latency to peak attained statistical significance in males given 1000 mg/kg/day during trials 21-30 which was marginally higher compared to controls.

Motor Activity - Cohort 2A:
The locomotor activity of Cohort 2A F1 animals on nominal Day 65±1 of age was unaffected by test item administration. One difference from the control group attained statistical significance in treated females, with a lower number of low beam breaks recorded during the 36-minute test for females given 300 mg/kg/day. The overall total number of high and low beam breaks during the 1-hour test was unaffected, and therefore this difference was considered incidental and unrelated to test item administration.

In-Cage Observations - Cohort 2A:
A detailed functional observational battery was performed for all Cohort 2A animals on nominal Day 70±1 of age. There were no test item-related changes evident in the behavior of the Cohort 2A animals during the in-cage observations.

In-Hand Observations - Cohort 2A:
During the in-hand observations there were no findings observed which represented neurological changes related to administration of the test item.

Arena Observations - Cohort 2A:
The 2-minute arena observation of the Cohort 2A animals did not reveal any neurological changes in the Cohort 2A animals at any dose level investigated.

Reactivity Investigations - Cohort 2A:
Reactivity investigations of the Cohort 2A animals revealed, when compared to controls, statistically significantly lower mean body weight in males given 1000 mg/kg/day and a statistically significantly lower hind limb grip strength in males in the 1000 mg/kg/day, although in the absence of a dose relationship.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

T-Cell Dependent Antibody Response (F1 Cohort 3):
No treatment-related differences in IgM responses were observed in F1 Cohort 3 male and female rats when the test item was administered at 100, 300 and 1000 mg/kg/day when compared to the control. Apparent differences, observed when comparing mean IgM levels in some of the treated groups to the control group, were considered to represent normal variation based on the high variability in the individual results and the lack of a clear dose-related response.

Spleen Cell Immunophenotyping (F1 Cohort 1A):
Statistically significantly lower male NK cell cells/spleen were observed at doses 300 mg/kg/day and 1000 mg/kg/day when compared to the control group. Statistically significantly lower female WBC, lymphocyte, T cell (including CD4+ and CD8+ T cells), NK cell and monocyte cells/spleen were observed at dose 1000 mg/kg/day when compared to control group, as well as a statistically significantly lower female NK cell and monocyte cells/spleen at dose 300 mg/kg/day when compared to the control group. A statistically significantly lower female monocyte percentage was observed at dose 1000 mg/kg/day when compared to the control group.
Whilst the aforementioned statistically significant differences were observed in Sprague-Dawley rat spleen leukocytes, these differences were not related to the oral gavage administration of the test item as the differences were slight and the variation observed in the immunophenotyping cell percentage and cells/spleen parameters at doses 300 mg/kg/day and 1000 mg/kg/day were within the ranges observed in the control animals.
There were no further observable or statistically significant effects on the immunophenotyping parameters measured in Sprague-Dawley rat spleen leukocytes as a result of the oral gavage administration of the test item.

Spleen Weight (F1 Cohort 3):
No changes were apparent in the weight of the spleen from Cohort 3 animals which could be related to the administration of the test item.

As two F0 females (F0 female No. 236 given 100 mg/kg/day and F0 female No. 276 given 1000 mg/kg/day) were not pregnant, three F0 females died prematurely and all deaths were related to mis-dosing (1F No. 218, 4F No’s. 275 and 281) and two additional F0 females were killed for welfare reasons (2F No. 235 and 3F 249), the assessment of F1 litter responses is based on 23, 22, 23 and 21 litters at 0, 100, 300 and 1000 mg/kg/day, respectively.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed among the F2 offspring that were considered to be related to parental treatment with the test item.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Litter Size, Survival Indices and Sex Ratio: The mean number of implantation sites, litter size, post-implantation survival and live birth index were all statistically significantly lower than controls in the 1000 mg/kg/day group (implantation sites: 7.9 versus 15.1 in controls; litter size: 7.2 versus 14.3 in controls; post-implantation survival: 84.8% versus 94.1% in controls). Mean litter size on Day 1 to Day 4 of lactation before the cull was lower compared to controls, litter size remained stable following the Day 4 cull to the Day 21 of age. The lower Viability index reflected one total litter loss and pup mortality in two other litters.
There was no effect on post-implantation survival, litter size or offspring survival from Day 1 of lactation at 100 or 300 mg/kg/day. Offspring sex ratio was unaffected by test item administration at all dose levels investigated.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The group mean body weight of male and female F2 offspring on Day 1 of age, and subsequent body weight gain of the offspring to weaning on Day 21 of age, was unaffected by parental treatment with the test item for males at all dose levels investigated and for females at 100 or 300 mg/kg/day. For female offspring at 1000 mg/kg/day, overall weight gain was slightly lower at 90% of controls; in the absence of any effect on male sibling weight gain an effect of treatment is considered highly unlikely.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, mean ano-genital distance was slightly longer in male and female F2 offspring, both differences attained statistical significance. There was no effect of treatment on the ano-genital distance of F2 offspring in the 100 or 300 mg/kg/day groups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

All F2 male offspring were assessed on Day 13 of age for the presence of nipples; no nipples were observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The predominant macroscopic observation detected in the F2 offspring that died prior to scheduled termination was the absence of milk in the stomach. There were no macroscopic abnormalities detected among the F2 offspring killed at scheduled termination on Day 21 of age.

Eleven Cohort 1B females in the 1000 mg/kg/day group (No’s. 953, 955, 956, 960, 961, 965, 969, 971, 973, 975 and 976) and three Cohort 1B females in the 300 mg/kg/day group (No’s. 931, 938 and 943) failed to litter, one female given 1000 mg/kg/day (No. 964) failed to mate, one Control female (No. 887) was killed for welfare reasons prior to pairing and one female given 300 mg/kg/day (No. 947) was killed for welfare reasons on Day 13 of gestation. In addition, three females given 1000 mg/kg/day (No’s. 958, 959, 966 and 970) had a total litter loss on Days 4, 1, 1 and 6 of lactation, respectively. Therefore, the assessment of F2 litter responses is based on 23, 24, 20 and 8 litters at 0, 100, 300 and 1000 mg/kg/day, respectively.

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Summary of Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F0)

	Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
Sex	Males				Females
Dose Level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Kidneys
Absolute Weight (g)	3.76	102	97	103	2.15	97	102	109**
Body Weight Relative (%)	0.67	100	100	106*	0.74	96	101	106**
Testes
Absolute Weight (g)	3.70	107	103	107**
Body Weight Relative (%)	0.66	104	105	110*
Thymus
Absolute Weight (g)	0.25	109	95	80*	0.26	98	109	97
Body Weight Relative (%)	0.044	107	97	83*	0.090	97	109	95
Pituitary
Absolute Weight (g)	0.014	100	100	100	0.017	94	100	94*
Body Weight Relative (%)	0.0026	92	96	100	0.0059	93	97	90*
* = Statistically significant difference (absolute or relative) compared with respective control mean value *p 0.05; **p 0.001. Note: Values for absolute weight and body weight adjusted weight for dosed groups expressed as percentage control mean value.

 

Summary of Incidence and Severity of Test Article-Related Microscopic Findings – Terminal Sacrifice (F0)

	Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
Sex	Males				Females
Dose Level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Kidneys
Number Examined	24	0	2	24	23	22	23	20
Basophilia, Tubular, Focal
Minimal	7	0	1	5	2	5	1	3
Slight	0	0	0	0	0	0	0	1
Basophilia, Tubular, Multifocal
Minimal	1	0	1	2	1	0	5	1
Slight	0	0	0	0	0	1	0	3
Moderate	0	0	0	0	0	0	0	1
Total Incidence of Basophilic  Tubules	8	0	2	7	3	6	6	9
Epididymides
Number Examined	24	24	24	24	NA	NA	NA	NA
Debris, Cellular
Minimal	0	0	0	10
Total	0	0	0	10
Thymus
Number Examined	24	24	24	24	23	0	0	20
Lymphocytes Decreased, Cortical
Minimal	1	2	1	0	0	0	0	2
Slight	1	0	0	7	0	0	0	0
NA = Not applicable

 

Summary Table: Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F1 Cohort 1A)

	Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
Sex	Males				Females
Dose Level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Kidneys
Absolute Weight (g)	3.23	104	107	104	1.86	100	102	103
Body Weight Relative (%)	0.67	103	104	113**	0.70	101	99	105*
Thymus
Absolute Weight (g)	0.40	96	99	78**	0.32	112	112	94
Body Weight Relative (%)	0.083	96	96	85**	0.12	113	110	96
Pituitary
Absolute Weight (g)	0.013	100	100	85**	0.015	107	100	80**
Body Weight Relative (%)	0.0028	96	96	86**	0.0058	103	93	81**
NA = Not applicable. * = Statistically significant difference (absolute or relative) compared with respective control mean value *p 0.05; **p0.001. Note: Values for absolute weight and body weight adjusted weight for dosed groups expressed as percentage control mean value.

 

Summary Table: Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F1 Cohort 1B)

	Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
Sex	Males				Females
Dose Level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pituitary
Absolute Weight (g)	0.015	100	93	80**	0.018	100	89*	72**
Body Weight Relative (%)	0.0025	100	100	92	0.0058	100	90*	76**
Seminal Vesicles with Coagulating Gland
Absolute Weight (g)	2.31	105	95	107	NA	NA	NA	NA
Body Weight Relative (%)	0.40	105	96	117**	NA	NA	NA	NA
Epididymis
Absolute Weight (g)	1.49	103	99	90**	NA	NA	NA	NA
Body Weight Relative (%)	0.25	103	101	98	NA	NA	NA	NA
Uterus, Cervix and Oviducts
Absolute Weight (g)	NA	NA	NA	NA	0.64	102	126*	135**
Body Weight Relative (%)	NA	NA	NA	NA	0.21	102	127*	140*
NA = Not applicable. * = Statistically significant difference (absolute or relative) compared with respective control mean value *p 0.05; **p0.001. Note: Values for absolute weight and body weight adjusted weight for dosed groups expressed as percentage control mean value.

 

Summary Table: Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F1 Cohort 2A)

	Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
Sex	Males				Females
Dose Level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Brain
Absolute Weight (g)	1.99	98	99	95*	1.85	100	99	93*
Body Weight Relative (%)	0.456	99	100	105	0.744	104	99	93
* = Statistically significant difference (absolute or relative) compared with respective control mean value *p 0.05; **p0.001. Note: Values for absolute weight and ratio of organ weights (relative to body) for dosed groups expressed as percentage control mean value.

 

Summary Table: Test Article-Related Effects in Organ Weight Parameters – Terminal Sacrifice (F1 Cohort 2B)

	Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
Sex	Males				Females
Dose Level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Brain
Absolute Weight (g)	1.47	103	98	94	1.43	101	105	96
Body Weight Relative (%)	2.86	98	97	93	2.75	96	103	99
Note: Values for absolute weight and ratio of organ weights (relative to body) for dosed groups expressed as percentage control mean value.

 

Summary Table: Incidence and Severity of Test Article-Related Microscopic Findings – Terminal Sacrifice (F1 Cohort 1A)

	Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate
Sex	Males
Dose Level (mg/kg/day)	0	100	300	1000
Epididymides
Number Examined	19	20	19	20
Debris, Cellular
Minimal Moderate	0 1	0 0	0 0	6 0
Total	1	0	0	6

 

Summary Table: Incidence and Severity of Test Article-Related Microscopic Findings – Terminal Sacrifice (F1 Cohort 1B)

	Tris[2-[2-(2-methoxyethoxy) ethoxy]ethyl] orthoborate
Sex	Males
Dose Level (mg/kg/day)	0	100	300	1000
Epididymides
Number Examined	24	0	24	24
Debris, Cellular
Minimal	0	0	2	9
Slight	0	0	0	4
Total	1	0	2	13
Testes
Number Examined	24	0	24	24
Degeneration, Tubular
Minimal	0	-	0	8
Total	0	-	0	8

 

 

Conclusions:

Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 300 mg/kg/day due to the high incidences of reduced fertility in females of F1 Cohort 1B receiving 1000 mg/kg/day and the increased incidences of minimal epididymal cellular debris coupled with sperm motility and morphology changes in both F0 and F1 Cohort 1B males given 1000 mg/kg/day, accompanied with degeneration in the testes for F1 Cohort 1B males at 1000 mg/kg/day only.
Aside from the above-mentioned instances of reduced fertility and male reproductive system changes at 1000 mg/kg/day, increased incidences of basophilic tubules in the kidneys of F0 females and increased incidence of decreased lymphocytes in the cortex of the thymus in the F0 generation males were observed at 1000 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 300 mg/kg/day.
The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 300 mg/kg/day due to reduced early post-partum survival at 1000 mg/kg/day in both generations and low litter size in F2 litters.
There was no evidence of developmental neurotoxicity or developmental immunotoxicity in this study, therefore the NOAEL for these endpoints was concluded to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to assess the influence of Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate on reproductive performance when administered by oral gavage to Sprague-Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation, estrous cycles, and reproductive performance. In addition, cohorts of F1 animals were used to assess developmental neurotoxicity and developmental immunotoxicity.

In the F0 generation, three groups of 24 male and 24 female rats received Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate at dose levels of 100, 300 or 1000 mg/kg/day at a volume dose of 4 mL/kg/day. Males were treated for two weeks before pairing, up to necropsy after litters were weaned. Females were treated for two weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. In the F1 generation, 74 males and 74 females were treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation. Similarly constituted control groups received the vehicle, corn oil, at the same volume dose and throughout the same relevant period.

For the F0 generation data were recorded on clinical observations, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid related hormones, sperm assessment, organ weight, macroscopic pathology and microscopic pathology investigations were performed.

For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age. Serum samples that were collected from selected offspring on Day 22 of age were analyzed for thyroid-related hormones.

At weaning the F1 generation was split into five cohorts:

For F1 Cohort 1A, data were recorded on clinical signs and condition, body weight, food consumption, sexual maturation and estrous cycles. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid‑related hormones, sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and immunophenotyping investigations were performed.

For F1 Cohort 1B, data was recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles, mating performance, fertility, gestation length and gestation index. Organ weight, macroscopic pathology and microscopic pathology investigations were performed. For F2 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano‑genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age.

For F1 Cohort 2A, data was recorded on clinical condition, body weight, food consumption and sexual maturation. Sensory function, neurobehavioural screening, brain weight, macroscopic pathology and microscopic pathology investigations were performed.

For F1 Cohort 2B, animals received no direct treatment and no specific in-life investigations were performed. Animals were dispatched to necropsy at weaning for microscopic pathology investigations of the brain.

For F1 Cohort 3, data was recorded on clinical signs, body weight, food consumption and sexual maturation. Spleen weight, macroscopic pathology and T-cell dependent antibody response investigations were performed.

Results:

The mean concentrations of Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate in test formulations analyzed during the study were within +10/-15% of the nominal concentration, confirming the accuracy of formulation.

There were slightly lower circulating levels of thyroxine (T4) in F0 adult males at 1000 mg/kg/day and the F1 generation males at 300 or 1000 mg/kg/day. 

F0 responses:

Administration of Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption or food conversion efficiency.

Estrous cycles, mating performance, pre-coital interval, fertility and gestation index were unaffected by treatment. Females in the 1000 mg/kg/day group showed a trend towards slightly extended gestation lengths.

The hematological examinations for F0 animals revealed, when compared with controls, lower values in some white blood cell parameters (lymphocyte, eosinophil and basophil counts), haematocrit, reticulocyte counts and mean cell volume in males given 1000 mg/kg/day. Mean cell volume were slightly lower than that of controls for males receiving 300 mg/kg/day and females receiving 1000 mg/kg/day. Blood chemistry investigations revealed higher mean urea concentrations in males at 1000 mg/kg/day. The analysis of urine prior to the termination of F0 animals, revealed higher total urinary protein and lower urinary pH in both sexes at 1000 mg/kg/day, total chloride concentration was also higher for females given 1000 mg/kg/day.

Sperm motility was reduced, and abnormal sperm morphology was increased, and total testicular spermatid counts were higher in males given 1000 mg/kg/day. Only slight changes in sperm motility were observed in males at 100 or 300 mg/kg/day.

Changes in organ weights consisted of slightly higher body weight relative kidney weights in males and females at 1000 mg/kg/day. In males at 1000 mg/kg/day, slightly higher absolute and body weight relative testes weights and lower absolute and body weight relative thymus weights were seen. In addition, absolute and body weight relative pituitary weight were slightly lower in females given 1000 mg/kg/day when compared to controls.

There were no test item related macroscopic findings observed at the scheduled sacrifice.

Histopathological evaluation of a full list of retained tissues revealed treatment related changes in the kidneys of the high dose females and the thymus and epididymis of the high dose males. In the kidneys, a higher incidence and severity of basophilic tubules with more multifocal distribution was observed in females at 1000 mg/kg/day. Minimal cellular debris was observed in the epididymis and a slight reduction in lymphocytes in the cortex of the thymus was observed in males that received 1000 mg/kg/day.

F1 litter responses:

The general condition of offspring, litter size on Day 1 of age, sex ratio, offspring body weight or development and macropathology were unaffected by treatment. Offspring survival was lower during the first four days of lactation in the litters of females given 1000 mg/kg/day.

Organ weights for unselected F1 offspring on Day 22 of age were unaffected by parental treatment and there were no macroscopic findings for offspring that either died prematurely or at scheduled termination that could be attributed to Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate.

F1 selected animals:

Administration of Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate was generally well tolerated with no treatment-related mortality in F1 generation cohorts 1A or 1B, 2A, 2B and 3. Early euthanasia related to the test item was required in females, related to reduced fertility in the F1 generation in Cohort 1B. At 1000 mg/kg/day, there were twelve animals euthanized early, eleven due to failure to litter and one which failed to mate. A further four females that received 1000 mg/kg/day were euthanized due to total litter loss.

There were no adverse effects on general condition or food conversion efficiency. Body weight gain was generally unaffected during all phases of the study with the exception of Cohort 1B females during the gestation period, where body weight gain for females given 1000 mg/kg/day was statistically lower compared to controls. The food intake for animals during all phases in the study was generally similar to controls, the only difference was observed for F1 Cohort 1B females at 1000 mg/kg/day during the lactation period. There was also no effect of treatment on the attainment of sexual maturation of the F1 animals, or on the duration between vaginal opening and first estrus in the F1 Cohort 1A females, or the stage of estrus at termination of the F0 females or the F1 Cohort 1A and 1B females

Estrous cycle regularity and pre-coital interval were unaffected by test item administration. A slight shift towards longer gestation lengths was apparent for females receiving 300 mg/kg/day and a stronger shift to longer gestation length was seen in females at 1000 mg/kg/day in comparison with the concurrent control. The number of females with live litters born were lower due to treatment at 1000 mg/kg/day. The rate of conception and fertility index for females receiving 1000 mg/kg/day was low compared to controls. The majority of females that received 100, 300 or 1000 mg/kg/day were showing estrous morphology at termination.

The hematological examinations at schedule termination revealed, when compared with controls, a lower mean cell hemoglobin, mean cell volume and red cell distribution width amongst males and females receiving 1000 mg/kg/day. Mean cell haemoglobin were slightly lower than that of controls in both sexes receiving 300 mg/kg/day and mean cell volume was low in females at 300 mg/kg/day. 

Blood chemistry investigations at scheduled termination for F1 animals revealed statistically significant higher urea concentration for males and females at 1000 mg/kg/day and higher bile acid concentrations at all dose levels in females. Urinary assessment revealed lower urinary pH in males and females given 1000 mg/kg/day and slightly lower urinary protein, sodium, potassium, and chloride concentrations in males at 1000 mg/kg/day.

Assessment of the F1 Cohort 2A and 2B animals did not reveal any evidence of developmental neurobehavioural or neuropathology changes.  Auditory startle response habituation, locomotor activity and the performance of the F1 Cohort 2A animals in the Functional Observational Battery (FOB) tests were unaffected by treatment.  During the reactivity investigations in the FOB, F1 Cohort 2A males at 1000 mg/kg/day showed a slightly lower hind limb grip strength; this difference was considered related to the lower mean body weight for this group. Brain weights were slightly lower in F1 Cohort 2A and 2B animals given 1000 mg/kg/day although the brain morphometry of the F1 Cohort 2A and 2B animals and detailed histopathological evaluation of the neurological tissues of the F1
Cohort 2A animals were unaffected by test item administration. 

The qualitative evaluation of the ovaries of F1 Cohort 1A females given 1000 mg/kg/day revealed a slightly lower number of follicles. In the absence of an effect on the number of corpora lutea or any histopathological changes in the ovary of the F1 Cohort 1B females, this finding was considered to be of no toxicological significance.

Sperm motility was reduced, epididymal sperm counts were lower compared to controls and normal sperm morphology was reduced in males receiving 1000 mg/kg/day. Sperm motility, counts and morphology of the F1 Cohort 1A males receiving 100 or 300 mg/kg/day were unaffected by test item administration.

Several changes in organ weights for the Cohorts 1A and 1B animals were observed, only the low thymus weights in males given 1000 mg/kg/day, the low pituitary weights and high kidney weight in both sexes given 1000 mg/kg/day were considered test item-related as these changes were consistent with the F0 generation.

There were no test item related macroscopic findings observed at the scheduled termination of F1 Cohort 1A, 1B, 2A, 2B and 3 animals.

Histopathological evaluation of full list retained tissues revealed treatment related changes in the epididymis of the high dose males in F1 Cohort 1A and 1B. Minimal cellular debris was observed in the epididymis of the F1 Cohort 1A males, minimal to slight cellular debris in the epididymis as well as minimal tubular degeneration of the testes was observed in F1 Cohort 1B males given 1000 mg/kg/day.

Assessment of the F1 Cohort 3 animals did not reveal any evidence of developmental immunotoxicity. Spleen weights at scheduled termination were unaffected, and there was no impact on the T-Cell Dependent Antibody Response (TDAR) following immunization with 300 µg of Keyhole Limpet Hemocyanin (KLH) on Days 47 and 54 of age.

F2 litter responses:

The general condition of offspring, sex ratio, offspring body weight or development and macropathology were unaffected by treatment. Ano-genital distances were similar to controls and there were no nipples observed in male offspring assessed on Day 13 of age.

Mean number of implantation sites, litter size, post-implantation survival and live birth index were all statistically significantly lower than controls in the 1000 mg/kg/day group. Mean litter size during the first four days of lactation prior to scheduled cull on Day 4 was lower compared to controls and Day 4 Viability index was low, litter size remained stable following the Day 4 cull to Day 21 of age. There was no effect on post-implantation survival, litter size or offspring survival from Day 1 of lactation at 100 or 300 mg/kg/day.

Conclusion:

Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance of the F0 and F1 Cohort 1B animals was 300 mg/kg/day due to the high incidences of reduced fertility in females of F1 Cohort 1B receiving 1000 mg/kg/day and the increased incidences of minimal epididymal cellular debris coupled with sperm motility and morphology changes in both F0 and F1 Cohort 1B males given 1000 mg/kg/day, accompanied with degeneration in the testes for F1 Cohort 1B males at 1000 mg/kg/day only.

Aside from the above-mentioned instances of reduced fertility and male reproductive system changes at 1000 mg/kg/day, increased incidences of basophilic tubules in the kidneys of F0 females and increased incidence of decreased lymphocytes in the cortex of the thymus in the F0 generation males were observed at 1000 mg/kg/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 300 mg/kg/day.

The NOAEL for the F1 and F2 offspring up to weaning was concluded to be 300 mg/kg/day due to reduced early post-partum survival at 1000 mg/kg/day in both generations and low litter size in F2 litters.

There was no evidence of developmental neurotoxicity or developmental immunotoxicity in this study, therefore the NOAEL for these endpoints was concluded to be 1000 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

The maternal/developmental NOAEL in a Charnoff & Kavlock rat study was determined to be at least 170 mg B-TEGME/kg bw/day based on a toxicity study with oral application of up to 1000 mg brake fluid/kg bw/day.

 

Prenatal developmental toxicity was further investigated in a prenatal development study in rats dosed at 30, 300 and 1000 mg/kg body weigtht B-TEGME. A further group of twenty-four time-mated females was exposed to the vehicle only (Methyltriglycol or TEGME) to serve as a control. The oral administration of B-TEGME and TEGME by oral gavage in pregnant rats resulted in some delayed ossification effects in foetuses; however, these effects were not considered of toxicological relevance and are most probably secondary to maternal toxicity effects of TEGME that has been used as vehicle in concentrations from ca. 3200 mg/kg bw/d (high dose group) to ca. 4200 mg/kg bw/d (control/vehicle group). Developmental toxicity experiments conducted with TEGME indicate developmental effects at doses > 1,000 mg/kg/day. Effects observed in offspring from rats treated with 1250 mg/kg/day TEGME or rabbits treated with 1500 mg/kg/day TEGME during gestation included skeletal variants and decreased body weight gain (see attached OECD SIDS document for TEGME, CAS 112-35-6, August 2005).

 

In a prenatal developmental toxicity study in New Zealand White rabbits, B-TEGME was administered as pure substance without being formulated with a vehicle to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 0, 100, 250 and 500 mg/kg on gestation days (GD) 6 through 28. B-TEGME caused evidence of maternal toxicity at the high dose of 500 mg/kg bw/d, such as doe mortality, and abortion in the most sensitive individuals as well as slightly reduced food consumption. The NOAEL for maternal toxicity was 250 mg/kg bw/d. Reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, pre- and post-implantation loss as well as uterine weight, placental weight, fetal weight and sex distribution of the fetuses were unchanged. In the high-dose group (500 mg/kg bw/d) a number of fetuses from different litters were affected by visceral (multiple malformations of urogenital tract) or skeletal (malformations of axial skeleton) malformations, some of which exceeded their historical control ranges. In addition, significantly increased incidences of external, skeletal and cartilage variations, all above the historical control range, were noted. The total of these findings caused the significant increase of overall malformations and variations in the high-dose group, although these particular values were still within the historical control range of the test facility. Since there was evidence for treatment-related adverse effects of B-TEGME on fetal morphology at the high-dose of 500 mg/kg bw/d, the NOAEL for prenatal developmental toxicity was 250 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2017 - Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, 55116 Mainz

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF, B756
- Expiration date of the lot/batch: 01 Feb 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: guaranteed by sponsor
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle used

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no purification step before application to animals

FORM AS APPLIED IN THE TEST: pure, unformulated substance

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL(NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Breeder: Charles River Laboratories, France)
- Age at study initiation: 15-17 weeks
- Weight at study initiation: pregnant females between 3054 – 4070 g
- Fasting period before study: not applicable
- Housing: during acclimatization housed singly in Type 4X03B700CP cages (TECNIPLAST Deutschland GmbH, Hohenpeißen-berg, Germany; floor space 4264 mm², internal height 450 mm)
- Diet: ad libitum; pelleted Kliba maintenance diet (Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland)
- Water: ad libitum, potable tap water in water bottles
- Acclimation period: 5 days before artificial insemination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2
- Humidity (%): 45-65
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12/12 (6.00 h to 18.00 h / 18.00 h to 6.00 h)

IN-LIFE DATES: From: 2017-11-13 To: 2017-12-21

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: applied as pure substance without being formulated with a vehicle

DIET PREPARATION
- no diet applied

VEHICLE
- no vehicle used

Analytical verification of doses or concentrations:

no

Remarks:

no stability, homogeneity and concentration control analyses of test item formulations were conducted due to pure substance application

Details on mating procedure:

- Impregnation procedure: artificial insemination
- Day of insemination referred to as day 0 of pregnancy

Duration of treatment / exposure:

implantation to one day prior to the expected day of parturition (GD 6-28)

Frequency of treatment:

daily

Duration of test:

30d

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on dose-range finding studies with non-pregnant (300 and 1,000 mg/kg bw/d for 21 d) and pregnant (300 and 600 mg/kg bw/d for GD 6 through GD 28) female New Zealand White rabbits the doses were selected. In the first study at 1,000 mg/kg bw/d, one out of three female animals were found dead on study day 12 with similar necropsy findings in all three animals. At 300 mg/kg bw/d no adverse effects were observed. In the second study at 600 mg/kg bw/d, one out of five female animals were killed moribund on study day 21 after showing reduced nutritional condition. At 300 mg/kg bw/d no adverse effects were observed.
- Rationale for animal assignment: There are historical control data available from the test facility for New Zealand White rabbits and the specific strain has been proven to be sensitive to substances with a teratogenic potential.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity; animals examined several times daily (GD 0-29) if signs occured
- during administration period (GD 6-28): all animals checked daily for abnormal clinical signs before administration as well as within 5 hours after

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

FOOD CONSUMPTION: Yes
- Time schedule for examination: daily during GD 0-29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter (and the heads of any fetus which revealed severe findings during the external examina-tion, e.g. anophthalmia, microphthalmia or hydrocephalus)

Statistics:

DUNNETT-test (two-sided) for the hypothesis of equal means:
Food consumption ("mean of means" = values that allow a rough estimation of the total food consumption during different time intervals [pretreatment, treatment and entire study]), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions:
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

WILCOXON-test (one-sided) for the hypothe-sis of equal medians:
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

For all:
* for p < 0.05; ** for p < 0.01

Indices:

The conception rate (in %):
(number of pregnant animals / number of fertilized animals) * 100


The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice:
([number of corpora lutea – number of implantations] / number of corpora lutea) * 100


The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice:
([number of implantations – number of live fetuses] / number of implantations) * 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- control: one female had blood in bedding before and after treatment on GD 16, reduced defecation was observed in four females
- 100 mg/kg bw/d: reduced defecation in one animal observed
- 250 mg/kg bw/d: reduced defecation in one animal observed
- 500 mg/kg bw/d: reduced defecation in six females observed, no defecation observed in three animals

- adverse effect is indicated by higher incidence of both findings in the high-dose group

Mortality:

mortality observed, treatment-related

Description (incidence):

- 500 mg/kg bw/d: one female were found dead on the day of scheduled sacrifice (GD 29), two females were sacrificed after abortion ahead of schedule (GD 29 and GD 28)
- simultaneous abortion/death of 3 high-dose does immediately before term points to maternal toxicity

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no significant treatment related effect on body weights/body weight gain
- particularly no effect on carcass weights or corrected (net) body weight gain at all dose levels

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- the mean food consumption of the does in the 500 mg/kg bw/d group was reduced from GD 9 onwards
- this effect was statistical significant on GD 9-10 and GD 13-15
- afterwards (around GD 17) the effect recovered, being comparable to or exceeding the control values

- overall, the high-dose does consumed 7% less food than the control animals during the treatment period (GD 6-28)
- food consumption of the low- and mid-dose rabbits (100 and 250 mg/kg bw/d) was comparable to thecontrol

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

- gravid uterus weights were not significantly different from controls, differences between all groups were assessed to be without biological relevance and no dose-dependence was observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- spontaneous findings: noted in individual females of all groups, i.e. watery feces in one control and one high-dose doe, a blind ending uterine horn (left) in one high-dose doe, absence of uterine horn (left), absent kidney (left) and absent ureter (left) in one high-dose doe
- additional fiindings associated with unscheduled maternal death or sacrifice: findings after gavage error (thoracic cavity filled with blood) in one doe, watery feces in one doe (sacrificed after abortion on GD 29), an empty stomach in one doe No. 78 (died on GD 29), watery feces, stomach filled to distension with feed, very dry, hard feces in rectum in one doe

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

- no test substance-related and/or biologically relevant differences between the different test groups

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- no test substance-related and/or biologically relevant differences between the different test groups

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- no test substance-related and/or biologically relevant differences between the different test groups

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

- one dead fetus found at cesarean section of one 500 mg/kg bw/d doe
- it is considered a rare finding but may occur spontaneously in this rabbit strain

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

- conception rate: 88% in 100 mg/kg bw/d group , 96% in 250 and 500 mg/kg bw/d groups and 100% in the control group (0 mg/kg bw/d)

Details on maternal toxic effects:

All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

food consumption and compound intake

mortality

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

food consumption and compound intake

mortality

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

- mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance, no biologically relevant differences in comparison to the control group

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- distribution of the fetuses in test groups 100, 250 and 500 mg/kg bw/d was comparable to the control fetuses

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- occurred in test groups 100 and 250 mg/kg bw/d), as listed in Tab. 1
- male fetus No. 31-02 had multiple soft tissue and skeletal malformations additionally
- male fetus No. 70-04 had an associated soft tissue malformation

The distribution of external malformations about the dose groups does not indicate an association to the treatment, no statistically significant differences between the groups were noted (Tab. 2).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- detected in single fetuses of test groups 100 and 500 mg/kg bw/d, as shown in Tab. 7
- male 100 mg/kg bw/d fetus No. 31-02 had multiple skeletal malformations affecting the vertebral column, ribs, sternum and pelvic girdle, furthermore, additional external and multiple visceral malformations, considered to be spontaneous in origin and not treatment-related
- in the 500 mg/kg bw/d group the incidences for all individual malformations as well the significantly increased litter incidence and mean% affected fetuses per litter with skeletal malformations (Tab. 8) were covered by the historical control ranges
- exception: the litter and affected fetuses/litter incidences for severely fused sternebrae (bony plate) exceeded their historical control ranges at 500 mg/kg bw/d

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

- occurred in fetuses of the test groups 100, 250 or 500 mg/kg bw/d, as listed in Tab. 4
- male low-dose fetus No. 31-02 had multiple visceral malformations, i.e. absent kidney, absent ureter, absent adrenals and a right-sided aortic arch, furthermore, additional external and multiple skeletal malformations
- male mid-dose fetus No. 70-04 had an additional external malformation
- three fetuses (out of two litters) of the 500 mg/kg bw/d group multiple visceral malformations affecting the urogenital tract were recorded

- no statistically significant differences in the overall incidences between the groups were noted (Tab. 5)
- the 500 mg/kg bw/d group litter incidence (9.5%; historical range 0 – 4.8%) as well as affected fetuses/litter incidence (mean% 1.5; historical range 0 – 0.6) for fetuses with multiple visceral malformations were above the historical control

Details on embryotoxic / teratogenic effects:

Fetal external variations:
- one external variation, i.e. paw hyperflexion, was recorded in six fetuses of four 500 mg/kg bw/d litters - resulted in an increased litter incidence (19% vs. 0 in control) and mean% of affected fetuses per litter (4.6 vs. 0 in control)
- the increased overall incidences of external variations are given in Tab. 3

Fetal soft tissue variations:
- broad variety of soft tissue variations, i.e. cystic dilatation of the brain, malpositioned carotid branches, short innominate, absent lung lobe (Lobus inferior medialis) and dilated renal pelvis in individual fetuses of test groups 0, 100, 250 or 500 mg/kg bw/d
- incidences were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant (Tab. 6)

Fetal skeletal variations:
- skeletal variations of different bone structures were observed in all test groups, with or without effects on corresponding cartilages
- observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing
- overall affected fetuses/litter incidences of skeletal variations were statistically significantly increased in all substance-treated groups (Tab. 9)
- this effects were above the historical control range (HCD: mean% 93.9 [83.2 - 100.0])
- all skeletal variations with statistically significant differences between the control and the treated groups are given in table 10
- two of the findings – supernumerary thoracic vertebra and supernumerary rib (13th, cartilage present) had higher incidences than the controls in all treated groups
- this increase was not statistically significant and hovered around the upper limit of the historical range in the low-dose group and not dose-related for the supernumerary rib (13th, cartilage present).

Fetal skeletal unclassified cartilage observations:
- the results are given table 11
- incidence ‘knobby rib cartilage’ was statistically significantly increased in test group 250 mg/kg bw/d (affected fetuses/litter, mean%: 0.0/1.9/1.2*/1.1 [p≤0.05])
- the vaue was within the historical control range (mean% 0.4 [0.0 - 1.3]) and there is no dose-response - no association to the treatment was assumed
- incidence ‘branched rib cartilage’ was statistically significantly increased in test group 500 mg/kg bw/d (affected fetuses/litter, mean%: 0.0/0.7/0.8/3.1* [p≤0.05])
- litter and affected fetus-es/litter incidences were outside the historical control range (mean% 0.0 [0.0 - 0.5])

Fetal external unclassified observations (table 12):
- two unclassified external observations, i.e. placentae discolored and placentae necrobiotic, were recorded in one fetus of test groups 250 or 500 mg/kg bw/d
- the effects were not considered to be related to treatment

Fetal soft tissue unclassified observations: (table 13)
- in one control fetus, three low-dose, seven mid-dose and three high-dose fetuses: a blood coagulum around urinary bladder observed
- this finding is not considered to be treatment-related

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

other: significantly increased incidences of external, skeletal and cartilage variations above the historical control range

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

skeletal: rib

skeletal: vertebra

skeletal: pelvic girdle

visceral/soft tissue: urinary

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Table 1: Individual fetal external malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	31-02 M^a)b)	acaudate
	46-01 F	umbilical hernia
2 (250 mg/kg bw/d)	70-04 M^a)	umbilical hernia
3 (500 mg/kg bw/d)	none

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

^a)fetus with additional soft tissue malformation (see Tab. 4)

^b)fetus with additional skeletal malformation (see Tab. 7)

Table 2: Total external malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	2 (1.0)	1 (0.5)	0.0
Litter incidence	N (%)	0.0	2 (9.1)	1 (4.2)	0.0
Affected fetuses/litter	Mean%	0.0	1.2	0.6	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 3: Total external variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	0.0	0.0	6 (3.8)
Litter incidence	N (%)	0.0	0.0	0.0	4 (19)*^Fi
Affected fetuses/litter	Mean%	0.0	0.0	0.0	4.6*^Wi

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

*^Fi= p ≤ 0.05 (Fisher’s exact test [one-sided])   *^Wi= p ≤ 0.05 (Wilcoxon-test [one-sided])

Table 4: Individual fetal soft tissue malformations 

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	31-02 M^a)b)	multiple visceral malformations
2 (250 mg/kg bw/d)	70-04 M^a)	malpositioned kidney
3 (500 mg/kg bw/d)	82-02 F, 82-04 F	multiple visceral malformations
	83-04 F	multiple visceral malformations

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

^a)fetus with additional external malformation (see Tab. 2)

^b)fetus with additional skeletal malformation (see Tab. 7)

Table 5: Total soft tissue malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	1 (0.5)	1 (0.5)	3 (1.9)
Litter incidence	N (%)	0.0	1 (4.5)	1 (4.2)	2 (9.5)
Affected fetuses/litter	Mean%	0.0	0.6	0.6	1.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 6: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	10 (4.4)	3 (1.6)	5 (2.6)	8 (5.0)
Litter incidence	N (%)	7 (28)	3 (14)	4 (17)	3 (14)
Affected fetuses/litter	Mean%	4.9	1.8	3.2	4.5

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 7: Individual fetal skeletal malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	31-02 M^a)	multiple skeletal malformations
	37-06 M	misshapen thoracic vertebra
2 (250 mg/kg bw/d)	none
3 (500 mg/kg bw/d)	80-12 F	sternebrae severely fused (bony plate)
	87-09 F	severely malformed vertebral column and/or ribs
	88-03 M	misshapen lumbar vertebra
	92-09 F	thoracic hemivertebra, sternebrae severely fused (bony plate)

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

^a)fetus with additional external and soft tissue malformations (see Tabs. 1 and 4)

Table 8: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	2 (1.0)	0.0	4 (2.5)
Litter incidence	N (%)	0.0	2 (9.1)	0.0	4 (19)*^Fi
Affected fetuses/litter	Mean%	0.0	1.0	0.0	2.2*^Wi

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

*^Fi= p ≤ 0.05 (Fisher’s exact test [one-sided])   *^Wi= p ≤ 0.05 (Wilcoxon-test [one-sided])

 

Table 9: Total fetal skeletal variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	210 (93)	189 (98)	194 (99)	160 (100)
Litter incidence	N (%)	25 (100)	22 (100)	24 (100)	21 (100)
Affected fetuses/litter	Mean%	92.1	97.3*	99.1**	100.0**

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])           ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 10: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding	Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d	HCD Mean % (range)
Supernumerary thoracic vertebra	19.4	27.6	39.9**	46.9**	17.9 (7.8 - 25.6)
Supernumerary lumbar vertebra	0.7	0.6	1.5	7.2*	0.3 (0.0 - 2.4)
Unossified sternebra; unchanged cartilage	11.6	18.7*	21.9**	45.4**	15.4 (7.7 - 27.5)
Misshapen sternebra; unchanged cartilage	6.4	3.6	6.3	18.2**	9.8 (3.1 - 17.7)
Supernumerary rib (13^th); cartilage present	56.8	69.9	77.0**	70.7**	60.4 (50.0 - 70.9)
Incomplete ossification of talus; cartilage present	1.5	0.6	2.8	14.2**	2.1 (0.0 - 4.8)
Unossified talus; cartilage present	0.4	1.0	2.3	16.7**	0.9 (0.0 - 2.6)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p ≤ 0.05 (Wilcoxon-test [one-sided])            ** = p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 11: Total unclassified cartilage observations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	62 (27)	35 (18)	37 (19)	59 (37)
Litter incidence	N (%)	23 (92)	16 (73)	16 (67)	19 (90)
Affected fetuses/litter	Mean%	30.5	18.7	17.1	34.3

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Table 12: Total externalunclassified observations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	0.0	1 (0.5)	1 (0.6)
Litter incidence	N (%)	0.0	0.0	1 (4.2)	1 (4.8)
Affected fetuses/litter	Mean%	0.0	0.0	0.8	0.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 13: Total soft tissue unclassified observations

				Test group 0 0 mg/kg bw/d		Test group 1 100 mg/kg bw/d		Test group 2 250 mg/kg bw/d		Test group 3 500 mg/kg bw/d
Litter Fetuses		N N		25 226		22 192		24 196		21 160
Fetal incidence		N (%)		1 (0.4)		3 (1.6)		7 (3.6)		3 (1.9)
Litter incidence		N (%)		1 (4.0)		2 (9.1)		2 (8.3)		2 (9.5)
Affected fetuses/litter		Mean%		0.3		1.7		2.8		1.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

Conclusions:

The oral administration of B-TEGME to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at the high-dose level of 500 mg/kg bw/d, such as abortions/mortality and reduced food consumption. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg bw/d.

Since there was evidence for treatment-related adverse effects of the test substance on fetal morphology at the high-dose of 500 mg/kg bw/d, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.

Executive summary:

B-TEGME was tested for its prenatal developmental toxicity in New Zealand White rabbits according to OECD 414.

The test substance was administered as pure substance without being formulated with a vehicle to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 100, 250 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The control group, consisting of 25 females, was dosed with drinking water (same quantity as the high-dose animals) in parallel. At terminal sacrifice on GD 29, 21-25 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The following test substance-related adverse effects/findings were noted:

Test group 3 (500 mg/kg bw/d):

Dams

- Death or abortion in 3 females immediately before term (GD 28/29), corroborative necropsy findings indicating enteropathy

- Lower food consumption (7% below control)

Fetuses

- Increased incidence of fetuses showing visceral (urogenital tract) or skeletal (axial skeleton) malformations, some above historical range

- Increased incidences of external, skeletal and cartilage variations, all above historical control range

- Increased incidences of total malformations and variations

Test group 2 (250 mg/kg bw/d):

- No test substance-related adverse effects on does, gestational parameters or fetuses.

Test group 1 (100 mg/kg bw/d):       

- No test substance-related adverse effects on does, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of B-TEGME to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6 -28) caused evidence of maternal toxicity at the high-dose level of 500 mg/kg bw/d, such as abortions/mortality and reduced food consumption. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg bw/d.

Since there was evidence for treatment-related adverse effects of the test substance on fetal morphology at the high-dose of 500 mg/kg bw/d, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

BASF SE, 40R0618/11R275, 2018

B-TEGME was tested for its prenatal developmental toxicity in New Zealand White rabbits according to OECD 414.

The test substance was administered as pure substance without being formulated with a vehicle to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 100, 250 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The control group, consisting of 25 females, was dosed with drinking water (same quantity as the high-dose animals) in parallel. At terminal sacrifice on GD 29, 21-25 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The following test substance-related adverse effects/findings were noted:

Test group 3 (500 mg/kg bw/d):

Dams:

- Death or abortion in 3 females immediately before term (GD 28/29), corroborative necropsy findings indicating enteropathy

- Lower food consumption (7% below control)

Fetuses:

- Increased incidence of fetuses showing visceral (urogenital tract) or skeletal (axial skeleton) malformations, some above historical range

- Increased incidences of external, skeletal and cartilage variations, all above historical control range

- Increased incidences of total malformations and variations

Test group 2 (250 mg/kg bw/d):

- No test substance-related adverse effects on does, gestational parameters or fetuses.

Test group 1 (100 mg/kg bw/d):       

- No test substance-related adverse effects on does, gestational parameters or fetuses.

 

B-TEGME caused death and abortions in 3 high-dose females (500 mg/kg bw/d), in addition reduced or no defecation was observed at a higher frequency at this dose level. Necropsy observations in the decedents comprised watery feces, an empty stomach or a stomach filled to distension with feed combined with very dry, hard feces in rectum. These clinical observations indicate that 500 mg/kg bw/d of the test item were maternally toxic to sensitive individuals in the present study.

The mean food consumption of the high-dose dams (500 mg/kg bw/d) was significantly below the concurrent control shortly after the beginning of the treatment and recovered afterwards, however, overall these animals consumed about 7% less food than the control group. There was no significant treatment related effect on body weights/body weight gain, and particularly on carcass weights or corrected (net) body weight gain at all dose levels.

No differences of toxicological relevance between the control and the treated groups (100, 250 or 500 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

One dead fetus was found at cesarean section of high-dose doe No. 84 (500 mg/kg bw/d) which is a rare finding but may occur spontaneously in this rabbit strain.

Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose.

Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the mid-dose tested (250 mg/kg bw/d).

In the high-dose group (500 mg/kg bw/d) a number of fetuses from different litters were affected by visceral (multiple malformations of urogenital tract) or skeletal (malformations of axial skeleton) malformations, some of which exceeded their historical control ranges. In addition, significantly increased incidences of external, skeletal and cartilage variations, all above the historical control range, were noted. The total of these findings caused the significant increase of overall malformations and variations in the high-dose group, although these particular values were still within the historical control range of the test facility.

Thus, for the high-dose (500 mg/kg bw/d) the collective spectrum of observed anomalies may suggest beginning developmental toxicity.

 

Under the conditions of this prenatal developmental toxicity study, the oral administration of B-TEGME to pregnant New Zealnd White rabbits from implantation to one day prior to the expected day of parturition (GD 6 -28) caused evidence of maternal toxicity at the high-dose level of 500 mg/kg bw/d, such as abortions/mortality and reduced food consumption. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg bw/d.

Since there was evidence for treatment-related adverse effects of the test substance on fetal morphology at the high-dose of 500 mg/kg bw/d, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.

 

Shell, 41204726, 2015

In a supporting prenatal developmental toxicity study, the test item B-TEGME (89% pure) 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 at dose levels of 30, 300, and 1000 mg/kg bw/day (Shell, 2015). A further group of twenty-four time-mated females was exposed to the vehicle only (Methyltriglycol or TEGME) to serve as a control; B-TEGME was also used as vehicle in the dosed groups.

Clinical signs and necropsy findings did not indicate any effect of treatment in parental females at any treatment level. Body weight development and food consumption in treated groups did not show any significant intergroup differences when compared to the concurrent control females. However, statistical analysis of the control (TEGME) group against a background control group using a standard vehicle did reveal a reduction in body weight development and food consumption. Therefore, a true effect of B-TEGME on maternal toxicity may have been masked by the concurrent control using TEGME.

The predominant external finding detected throughout the 1000 mg/kg bw/day dose group was an increase in the percent of observed small foetuses. At this treatment level a number of litters had a high incidence of foetuses observed to be small. Lower mean foetal weight was evident at 1000 mg/kg bw/day with the majority of the small foetuses also having the lower body weights. Visceral foetal examinations did not show an overall increase in foetuses with findings and therefore no treatment effects. An observed, statistically significant increase in foetuses with small/no renal papilla development was not dosage related and was therefore considered insufficient evidence of a treatment related effect. Skeletal examinations showed an increase in the overall number of foetuses with skeletal findings in litters from the 1000 and 300 mg/kg bw/day treatment groups. At 1000 mg/kg bw/day delayed ossification of the sternebra, post lumbar vertebral centra and arches were evident together with a reduced number of metacarpals and an increased incidence of a short 13th rib. These findings all achieved statistical significance. There were other observations of delayed ossification that did not achieve statistical significance but are of biological relevance. An increase incidence of incomplete ossification was evident in one, or more than one cranial bone plus increased numbers of foetuses with no ossification of one thoracic vertebral centra and incomplete ossification in more than one lumbar arch. The overall incidence of the skeletal findings detected were not restricted to the identified small foetuses or to litters containing small foetuses but were equally distributed throughout the 1000 mg/kg bw/day treatment group. Although in the skeletal developmental parameters which showed a statistical significance, the mean control values were also outside of the background control values, therefore suggesting that the control vehicle (TEGME) that is also present in the test item may also contribute to the effects detected in the treated litters. The findings were enhanced in the treated groups and these findings are indicative of an overall delay in offspring development. The pattern of foetal development differences does indicate that this is a non-specific treatment effect on foetal growth and development rather than a direct effect upon the development of a body system. Although there was no clear correlation of lower ossification to smaller/lighter fetuses, the higher number of fetuses per litter observed in the high dose group is considered to have contributed to these findings.

At 300 mg/kg bw/day, lower mean foetal weight was evident and skeletal examinations showed that development had been slightly delayed as dosage related trends could be observed for the same skeletal parameters as has been seen at 1000 mg/kg bw/day albeit to a lesser extent.

No effect on post implantation losses were evident at 1000 or 300 mg/kg bw/day therefore the test item does not affect embryo lethality.

No effects were evident at 30 mg/kg bw/day.

The study is considered to be reliable with restrictions as TEGME (2-(2-(2-methoxyethoxy)ethoxy)ethanol, CAS 112-35-6) has been used as vehicle in concentrations from 3200 mg/kg bw/d (high dose group) to 4200 mg/kg bw/d (control/vehicle group). Developmental toxicity experiments conducted with TEGME indicate developmental effects at doses > 1,000 mg/kg/day. Effects observed in offspring from rats treated with 1,250 mg/kg/day TEGME or rabbits treated with 1,500 mg/kg/day TEGME during gestation included skeletal variations and decreased body weight gain (see attached OECD SIDS document for TEGME, CAS 112-35-6; August 2005). Therefore, the vehicle TEGME might have masked maternal toxicity in this study and/or might have contributed to the observed developmental effects (slight delay in development).

 

Shell, SBGR.92.233, 1993

A screening developmental toxicity was performed in rats with a brake fluid containing 17% B-TEGME. Doses administered were 4, 25 and 170 mg B-TEGME/kg bw/day, based upon doses of 25, 150 and 1000 mg brake fluid/kg bw/day. This study was applied according to a Charnoff & Kavlock (CKA) design and in compliance with GLP, which was considered to be adequate, reliable and relevant. This study can therefore support further developmental toxicity testing. Dams were allowed to litter and the pups observed and weighed on days 1 and 5 post-partum, at which point the study was terminated. As there were no skeletal and visceral examinations in this study, it was considered as a supporting study for a final prenatal developmental toxicity study in rats. In this screening study, no treatment related effects on birth, live birth, or viability indices were found, neither were there treatment-related clinical effects on dams or pups, or gross abnormalities seen in the pups. The NOAEL for this study was at least 170 mg B-TEGME /kg bw/day based on the NOAEL of 1000 mg brake fluid/kg bw/day. It must be taken into consideration that the brake fluid also contained other borated and non-borated glycol ethers and other components.

Justification for classification or non-classification

The developmental effects observed in a prenatal developmental toxicity study in rats with B-TEGME and TEGME (slight delay in development) are not considered to be sufficient to classify B-TEGME for developmental toxicity. Furthermore, the vehicle TEGME might have masked maternal toxicity and/or might have contributed to the observed developmental effects (slight delay in development). Therefore, the results of this study are considered inconclusive for classification.

The findings of a prenatal developmental toxicity study in rabbits demonstrate clear maternal toxicity at the high dose tested (i.e. 500 mg/kg bw/d): B-TEGME caused death or abortion in 3 females immediately before term (GD 28/29), and corroborative necropsy findings indicating enteropathy. Furthermore, lower food consumption (7% below control) was observed at this dose level. Fetal examinations revealed that there was no adverse effect of B-TEGME on the respective morphological structures up to the mid-dose tested (250 mg/kg bw/d). In the high-dose group (500 mg/kg bw/d), several fetuses from different litters were affected by visceral (multiple malformations of urogenital tract) or skeletal (malformations of axial skeleton) malformations, some of which exceeded their historical control ranges. In addition, significantly increased incidences of external, skeletal and cartilage variations, all above the historical control range, were noted. Although the total of these findings caused a significant increase of overall malformations and variations in the high-dose group (500 mg/kg bw/d), the particular values were still within the historical control range of the test facility. Thus, for the high-dose (500 mg/kg bw/d) the collective spectrum of observed anomalies may suggest beginning developmental toxicity.

Based on the results obtained in an extended one-generation reproducitve toxicity study (EOGRTS; OECD 443) it was concluded that the NOAEL for reproductive performance of the F0 and F1 Cohort 1B animals was 300 mg/kg bw/day due to the high incidences of reduced fertility in females of F1 Cohort 1B receiving the limit dose of 1000 mg/kg bw/day and the increased incidences of minimal epididymal cellular debris coupled with sperm motility and morphology changes in both F0 and F1 Cohort 1B males given 1000 mg/kg/day, accompanied with degeneration in the testes for F1 Cohort 1B males at the limit dose of 1000 mg/kg bw/day only. The effects of reduced fertility and male reproductive system changes at 1000 mg/kg bw/day were observed in the presence of other toxic effects, i.e. increased incidences of basophilic tubules in the kidneys of F0 females and increased incidence of decreased lymphocytes in the cortex of the thymus in the F0 generation males were observed at 1000 mg/kg bw/day, therefore the NOAEL for systemic toxicity in the F0 and F1 adult animals was concluded to be 300 mg/kg/day. The NOAEL for the F1 and F2 offspring up to weaning was concluded to be at the same dose level of 300 mg/kg/day due to reduced early post-partum survival at 1000 mg/kg/day in both generations and low litter size in F2 litters. No evidence of developmental neurotoxicity or developmental immunotoxicity was observed in this study.

In contrast, in a 90-day oral toxicity study in Wistar rats (OECD 408) no effects on male reproductive organs were observed up to 1000 mg/kg/d consistent with those observed in the EOGRTS (rat, oral). In this study, two males treated with 1000 mg/kg bw/day had small and flaccid testes. One of these males also had small epididymides. A further male treated with 1000 mg/kg bw/day had a small left testis. One male treated with 10 mg/kg bw/day had small testes and another male from this treatment group had a small right testis. No effect on testes or epididymides were observed at the mid dose of 100 mg/kg bw/day. Accordingly, these observations were incidental due to a missing dose-response relationship, and in the absence of any treatment-related histologic correlates the inter-group differences were considered not to be of toxicological importance.

Conclusion: Based on a total weight of evidence of all available information related to the determination of the intrinsic reproductive toxicity of B-TEGME (consistency of results, presence of maternal toxicity in experimental animal studies, irrelevance of oral route of administration to humans), the classification of B-TEGME in Repro category 2, H361fd (Suspected of damaging fertility. Suspected of damaging the unborn child.) is considered the appropriate classification.