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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

 


1a- An extended one generation study is available (OECD 443, Covance 2O21): 


The purpose of this study was to assess the influence of Hexylene glycol on reproductive performance when administered continuously 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 and estrous cycles and one cohort was assessed for reproductive performance (as requested in ECHA Decision number: TPE-D-2114453322-58-01/F).


In the F0 generation, three groups of 25 male and 25 female rats received Hexylene glycol at dose levels of 100, 250 or 800 mg/kg/day at a volume dose of 5 mL/kg/day. Males were treated for two weeks before pairing, up to necropsy after the 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, 20 males and 20 females were treated from weaning to scheduled termination in adulthood (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation. A similarly constituted Control group in each generation received the vehicle, purified water, at the same volume dose as the treated groups.


For the F0 generation data were recorded on clinical observations, detailed physical examination and arena 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.


The F1 generation comprised of two cohorts:


For F1 Cohort 1A, data were recorded on clinical condition, detailed physical examination and arena observations, 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, detailed physical examination and arena observations, body weight, food consumption, sexual maturation, estrous cycles, mating performance, fertility, gestation length and gestation index, organ weight and macroscopic 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.


Results


There were four unscheduled deaths (one male at 100 mg/kg/day, one male at 250 mg/kg/day and two males at 800 mg/kg/day) in the F0 generation and one unscheduled death (one Control male) in the F1 generation, none were treatment related.


There were no signs attributed to oral gavage administration and no clinical signs (detailed physical examination and arena observations) attributable to treatment in the F0 or F1 adults.


Body weight gain and food consumption were unaffected by treatment during the two (F0) and 10 (F1) week pre-pairing periods and during gestation or lactation and in the F1 and F2 offspring body weights were unaffected by parental treatment.


Pre-pairing estrous cycles in the F0 and F1 females, estrous cycles at 10 weeks of age (F1) and estrous cycles following weaning of litters and before termination (F0 and F1) were unaffected by treatment.


Mating performance and fertility, gestation length and gestation index in the F0 and F1adults were unaffected by the treatment. The numbers of implantations and total offspring born were unaffected by parental treatment at 100, 250 or 800 mg/kg/day.


In the F1 litters, the live birth index was marginally low, reflecting a higher number of litters with offspring deaths up to Day 1 of age at 800 mg/kg/day and this was considered treatment related. Subsequent survival of the offspring was unaffected. In the F2 litters, a higher number of litters had offspring deaths between Days 1 and 4 of age, resulting in a decreased viability index and a marginally low litter size and this was considered treatment related.


The ratio of male to female offspring and the ano-genital distance of the F1 and F2 offspring were considered unaffected by treatment and no F1 male offspring had nipples. One F2 male at 100 mg/kg/day had one nipple; however this was considered not to be toxicologically important.


Absolute body weights of selected F1 males and females at 800 mg/kg/day were slightly but statistically significantly low on Day 1 of the formal F1 generation, when compared with Control.


Sexual maturation of F1 males and females and the period of time to first estrus following maturation was unaffected by treatment.


Hematology, blood chemistry and urinary parameters were not adversely affected by treatment at any dose in the F0 or F1. Adult serum TSH and T4 concentrations were unaffected by treatment in the F0 (adults) or F1 (offspring on Day 22 of age or adults).


Sperm count, motility and motion and morphology; splenic immunophenotyping parameters, ovarian follicle and corpora lutea counts were unaffected by treatment in the F1.


Administration of Hexylene glycol at 800 mg/kg/day resulted in centrilobular hepatocyte hypertrophy, an adaptive change and therefore considered not to be an adverse effect, in the liver of males and females in both F0 and F1 generations and correlated with an increase in organ weight.


Administration of Hexylene glycol resulted in hypertrophy, an adaptive change and therefore considered not to be an adverse effect, of the cortex of the adrenal glands of males and females given 800 mg/kg/day. This correlated with an increase in organ weight and hypertrophy.


Administration of Hexylene glycol to males at 100, 250 or 800 mg/kg in the F0 generation and 250 or 800 mg/kg/day in the F1 generation resulted in cortical vacuolation of the adrenal glands, an adaptive change and therefore considered not to be an adverse effect.


Administration of Hexylene glycol at all dose levels caused an increase in hyaline droplets in the kidney of males. This was considered non-adverse in the F0 generation and in the F1 males given 100 mg/kg/day. Adverse changes were apparent in F1 males given 250 or 800 mg/kg/day (basophilic tubules and granular casts), however these changes are not thought to be significant to man.


Other organ weight changes were considered not to be pathologically significant as there was no associated histopathological change.


Organ weights of the unselected F1 and the F2 offspring on Day 22 of age were unaffected by parental treatment and there were no macroscopic findings related to treatment in the F0 (adults), F1 (offspring and adults), F2 offspring.


Conclusion


Based on the results of this study it is concluded that the No Observed Adverse Effect Level (NOAEL) for systemic toxicity which has the potential to be harmful to human health in the F0 parent animals and the F1 Cohort 1A and 1B adult animals is 800 mg/kg/day. The NOAEL for kidney changes in F0 and F1 males consistent with the accumulation of alpha-2µ-globulin which was deemed adverse for the animals (but this finding is not generally considered relevant to human health) is 100 mg/kg/day. The NOAEL for reproductive performance of the F0 and F1B parent animals and survival and growth of the F1 and F2 offspring was concluded to be 250 mg/kg/day, based on transient reduced survival of the F1 and F2 offspring during the early post-natal period.


1b- A preliminary study was performed in order to select the dose-levels of the OECD 443 (Covance 2021).


In the F0 generation, two groups of six female rats received Hexylene glycol at doses of 500 or 800 mg/kg/day by oral administration from Day 6 after mating to Day 20 of lactation. A similarly constituted Control group received the vehicle, reverse osmosis water, at the same volume dose as the treated groups. Animals were allowed to litter and rear their offspring to weaning and were terminated on Day 21 of lactation. In the F1 generation, six males and six females were treated at the same doses and volume-dose as the F0 generation, from Day 21 to 27 of age, before termination on Day 28 of age.


Clinical condition, body weight, food consumption, gestation length and parturition observations, and macroscopic pathology investigations were undertaken on the F0 generation. The clinical condition, litter size and survival, sex ratio and body weight for all F1 offspring were assessed. After weaning, the F1 generation offspring were assessed for clinical signs, body weight, food consumption and macroscopic pathology investigations.


Results


F0 females and F1 offspring to weaning


There were no signs attributed to dosing or effect on clinical condition, body weight gain or food intake and there were no treatment related macroscopic findings at 500 or 800 mg/kg/day.


Gestation length, gestation index and parturition were unaffected at 500 or 800 mg/kg/day.


Mean implantation count (13.8) and the resultant total number of offspring born (13.0) at 800 mg/kg/day were both lower than the Historical Control Data (HCD) range (14.7-17.1 and 13.7-16.3, respectively).


There was no conclusive effect on litter size or sex ratio or on the subsequent survival and growth of the offspring to weaning at 500 or 800 mg/kg/day. One litter at 800 mg/kg/day showed poor growth; a relationship to treatment is uncertain.


F1 selected offspring


There were no signs attributed to dosing or effect on clinical condition, body weight gain or food intake and there were no treatment related macroscopic findings at 500 or 800 mg/kg/day.


One male receiving 800 mg/kg/day was found with a perforated esophagus, at the scheduled necropsy on Day 28 of age. It was considered that the animal had been mis-dosed.


Conclusion


It is considered that 800 mg/kg/day is suitable for investigation in the follow-on main extended one-generation reproductive performance study (OECD 443).


 


2-The potential toxic effects of Hexylene glycol (HG) was evaluated following daily oral administration (gavage) to male and female rats from before mating, through mating and through the gestation and lactation periods until day 4 post-partum (p. p.) (Allen, 2010). This study provides initial information on possible toxicological effects likely to arise from repeated exposure on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The study was conducted as a reproduction/developmental screening test according to the OECD 421 guideline and GLP.Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, HG (purity 99.95%), by daily oral (gavage) administration for 4 weeks before mating, through mating, gestation and the beginning of the lactation period (until day 4p. p.). The dose-levels were 200, 500 and 1000 mg/kg/day. Another group of 10 males and 10 females received the vehicle, purified water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg/day. The concentration of the dosage forms were checked during each study period (weeks 1, 5, 8 and 10). Clinical signs and mortality were checked daily. Body weight and food consumption were recorded at designated intervals throughout the study. Males were sacrificed after at least 10 weeks of treatment, females on day 5 p. p.. The adults were submitted for a macroscopic post-mortem examination, with seminology examination for the males, and designated organs were weighed. In addition, the numbers of corpora lutea and implantation sites were recorded for each female. A microscopic examination was performed on macroscopic lesions, livers, kidneys and forestomachs of males and females from all groups, and on reproductive organs (epididymides, testes and ovaries with oviducts) from the control and high-dose animals. The pups were observed daily for clinical signs, sexed and weighed on post-natal days (PND) 1 and 5. After their sacrifice on PND 5, they were examined to detect gross external and macroscopic abnormalities.


 


The test item concentrations in the administered dosage forms remained within an acceptable range of variation in all analyzed weeks. No unscheduled deaths occurred in any male groups during the study. Two F0 females from the high-dose group were prematurely sacrificed on lactation day 2 or 3 following the death of their litter. Soft feces was noted in a few test item-treated parent males from all dose-levels for 1 or several days during the first half of the treatment period but was considered as non adverse. No relevant clinical signs were recorded for the parent females. Body weight and food consumption were considered not to have been affected by the test item treatment. There were no effects of treatment with the test item on mating performance or on the fertility index. All pregnant females gave birth to live pups, the length of gestation was similar in all groups and no difficulties at delivery were observed. Pre-implantation loss was similar in all groups. At 1000 mg/kg/day, there was a marked increase in pup mortality during the five days after birth and body weight gain of the surviving pups was reduced. At 500 mg/kg/day, there was a slight increase in post-implantation loss, together with a slight increase in pup mortality up to PND 5. Pup sex ratio on PND all groups and pup body weight gain up to PND 5 at 200 and 500 mg/kg/day were unaffected by the treatment of the parents with the test item. There were no relevant external or macroscopic abnormalities in the pups from any group. Test item treatment had no effects on spermatozoa motility, morphology, or epididymal and testicular sperm count. A dose-related increase in kidney and liver weights was noted in the males at 200 or 500 mg/kg/day and in the males and females at 1000 mg/kg/day. No treatment-related macroscopicpost-mortemfindings were noted. At microscopic examination, adverse altered liver cell foci (clear cell- and basophilic cell-types) were recorded in some males at 500 or 1000 mg/kg/day and hepatocellular hypertrophy was noted in the males at 200 mg/kg/day and in males and females at 500 or 1000 mg/kg/day. Hyaline droplets along with basophilic tubules were found in the tubular epithelium of the kidneys from the males at 200, 500 or 1000 mg/kg/day. Minimal hyperplasia of squamous cells together with hyperkeratosis of the forestomach was seen in some males at 500 or 1000 mg/kg/day.


 


Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) for HG was considered to be 1000 mg/kg/day for the parent females and 200 mg/kg/day for the parent males, because of the adverse microscopic findings in the liver at 500 and 1000 mg/kg/day in males only. The No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day. The conservative NOEL for the F1 offspring up to PND 5 was considered to be 500 mg/kg/day, based on the increased pup mortality and reduced body weight gain at 1000 mg/kg/day.


3-Because of the adverse findings, increased pup mortality and reduced pup body weight gain observed at 1000 mg/kg/day in the OECD 421 study (Allen, 2010), a study was conducted to provide general information concerning the effects of the test item, Hexylene glycol, on rat gestation, parturition and early lactation, and on the growth and development of the offspring up to day 5post-partum (p.p.) (Spezia, 2011). Two groups (group 3 and group 4) of 10 mated female Sprague-Dawley rats received the test item, Hexylene glycol, by daily oral (gavage) administration during gestation and lactation (day 6 post-coitum to day 4 post-partum). The dose-level was 1000 mg/kg/day. Two other groups (group 1 and group 2) of 10 mated females received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions and acted as control groups. The dosing volume was 5 mL/kg/day. At birth three litters from control group 2 were exchanged with three litters from test item-treated group 4 (cross-fostering procedure). This permitted the evaluation of any adverse effects after pre-natal (group 4), post-natal (group 2) or prenatal and post-natal (group 3) exposure of pups on growth and developmental parameters.


The test item concentration in the administered dosage form remained within the acceptable range of variation. In dams and when compared with controls, the test item (Hexylene glycol, purity 99.84%) administered at 1000 mg/kg/day by gavage from day 6post-coitum(p.c.) to day 4post-partum(p.p.), could be associated with increased duration of littering, increased mean progesterone and decreased mean prolactine serum levels. However, given the uncertainty in littering time differences and in biological variations of hormones, the toxicological significance of such findings remained doubtful. On histopathological examination, all females had mammary glands in lactation but the limited number of litters analyzed precludes the drawing of a definitive conclusion. In pups, there were no effects on mean litter size or sex ratio and there were no external abnormalities. In non-cross fostered litter, there was a statistically significant increase in the number of pups found dead in the treated group (15.1% vs. 4.0% in the control group) on days 1-4 p.p., resulting in a statistically significant decrease in the viability index on day 4 p.p. (84.9% vs. 96.0% in control). Pups had no clinical signs or abnormal behavior. There were no effects on body weight on days 1 and 5 p.p. In cross-fostered litters, there were no significant differences in the number of pups found dead in the treated group when compared to respective controls. Pups had no clinical signs or abnormal behavior. There was a slightly higher pup mean body weight on days 1 p.p. (vs. in controls). At pups necropsy on day 5 p.p., there were no treatment-related findings. Overall, there were no biologically significant findings in cross-fostered pups but the limited numbers of litters exchanged precludes the drawing of a definitive conclusion.


When compared with controls, no or no clear treatment-related effect of Hexylene glycol administered at 1000 mg/kg/day by gavage from day 6 post-coitum (p.c.) to day 4 post-partum (p.p.), was observed on littering duration, mean progesterone and prolactine serum levels. The significant decrease of the pup viability index observed on day 4 p.p. in non cross-fostered litters was not observed in cross-fostered litters. However, the limited number of cross-fostered litters analyzed precludes the drawing of a definitive conclusion.


 


4- In a study performed to evaluate the exposure of pups to Hexylene glycol and/or its metabolites on day 1 post-partum (p.p.) via dams, one group of 20 time-mated female Sprague-Dawley rats received the test item, Hexylene glycol, from day 6 post-coitum (p.c.) until 20 or 21 p.c. with non-radiolabelled Hexylene glycol, and on day 1 post-partum (p.p.) with [14C]-Hexylene glycol, by oral route (gavage) at the dose-level of 1000 mg/kg/day (Chevalier, 2014). One additional group of five rats received the vehicle, drinking water, under the same experimental conditions, from day 6 until day 1 post-partum (p.p.). A constant dose-volume of 5 mL/kg was used. The test item concentrations in the formulations prepared for use on day 6 p.c. and on day 20 p.c., the total radioactivity of the radiolabelled test item solution and the radiochemical purity of radiolabelled solution were measured. Clinical signs and mortality were checked daily. Body weight of dams was recorded at designated intervals throughout the study. On the day of parturition which was closely monitored, the pups were identified, examined, weighed, sexed and carefully examined for presence of milk in the stomach. Once the parturition was completed, the radiolabelled test item was given orally to the dams, presence of milk was checked 1, 3 and 5 hours post-dosing, and blood was collected from the dams before sacrifice for determination of radioactivity in plasma (see section 7.1.1). Dams were sacrificed, examined macroscopically, the number of corpora lutea and implantation sites were recorded and the mammary glands were preserved. After their sacrifice on PND 1, all pups were examined to detect gross external and macroscopic abnormalities. Then, two male and two female pups per litter were selected for determination of total radioactivity by liquid scintillation counting (see section 7.1.1).


The test item concentrations in the administered dose formulations prepared for use on day 6 p.c. and on day 20 p.c. remained within an acceptable range of approximately -6% when compared to the nominal values, the total radioactivity of the radiolabelled test item solution was -3% when compared to the nominal value and the radiochemical purity of the radiolabelled dose formulation was close to 97%. A total of 4/5 and 19/20 females were pregnant in the control and 1000 mg/kg/day group, respectively. At 1000 mg/kg/day, two females were sacrificed during pregnancy on day 22 p.c. due to difficulties to deliver and one dam was prematurely sacrificed during lactation (on day 1 p.p.) since the litter was entirely dead. During gestation, staggering gait was observed in all females, and during lactation day 1 p.p., almost all the females treated at 1000 mg/kg/day had piloerection and loss of balance. Signs of hypoactivity, ptyalism and half-closed eyes were noted in one or two females. Clinical signs and incidence of premature sacrifices were test item-related. Seventeen pregnant females gave birth to live pups. Repeated administrations of hexylene glycol at this dose provoked implantation losses. The percentage of dead pups in the hexylene glycol treated group was 8.3 vs. none in the control group, it was mainly due to the dead litter on day 1 p.p. (13 pups). The length of gestation in test item-treated females was similar to the control animals. There was a tendency towards increase of the length of parturition when compared to the control values. Specifically, females treated at 1000 mg/kg/day required 3.5 hours for complete parturition versus 2.3 hours for the control group. A total of three litters treated at 1000 mg/kg/day had few pups with hematoma on head, abdomen and/or on the back including one pup with increased size of head. These observations were considered to be test item-related.


The administration of Hexylene glycol during the gestation and on day 1 p.p. caused clinical signs in dams, difficulties to deliver, and pup mortality. Compared to the control group, the increase of the length of littering and the high value of post-implantation losses recorded in the Hexylene group given 1000 mg/kg/day could not be attributed to the test item treatment with certainty.


 


In a reproduction/developmental toxicity screening test following oral administration of HG to male and female rats, the NOAEL for was considered to be 1000 mg/kg/day for the parent females and 200 mg/kg/day for the parent males, because of the adverse microscopic findings in the liver at 500 and 1000 mg/kg/day in males only. The NOEL for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day. The conservative NOEL for the F1 offspring up to PND 5 was considered to be 500 mg/kg/day, based on the increased pup mortality and reduced body weight gain at 1000 mg/kg/day. Further studies have shown that HG, administered at 1000 mg/kg/day by gavage from day 6 post-coitum (p. c.) to day 4 post-partum (p. p.), have no or no clear treatment-related effect on littering duration, mean progesterone and prolactine serum levels. The significant decrease of the pup viability index observed on day 4 p. p. in non cross-fostered litters was not observed in cross-fostered litters. However, the limited number of cross-fostered litters analyzed precludes the drawing of a definitive conclusion. In another study, 1000 mg/kg/day of cold HG administered during the gestation and radiolabelled HG on day 1 p. p. caused clinical signs in dams, difficulties to deliver, and pup mortality. Compared to the control group, the increase of the length of littering and the high value of post-implantation losses recorded in the Hexylene group given 1000 mg/kg/day could not be attributed to the test item treatment with certainty. Radioactivity was detected in all pups sacrificed on day 1 p. p., demonstrating the transfer of test item from the plasma through the milk, although the amounts detected were very low (ca. 0.09% of the dose). All together these data suggest that the reduction of the pups viability is most probably secondary to maternal toxic effects than a direct effects on pups.


 

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (Animal arrival) 26 August 2020
Experimental completion date (pathology): JUNE 15
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:
Purpose
The purpose of this study was to assess the influence of Hexylene glycol on reproductive performance when administered continuously 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 and estrous cycles and one cohort was assessed for reproductive performance (as requested in ECHA Decision number: TPE-D-2114453322-58-01/F).



Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Animal Model
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:
Supplier Charles River (UK) Ltd.

Number of animals ordered 108 males and 108 females; unrelated (males not related to females).

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Six days before commencement of treatment.

Age of the F0 animals at the start of the treatment
Males: 77 to 83 days old.
Females: 70 to 76 days old.

Weight range of the F0 animals at the start of the treatment
Males: 359 to 445 g.
Females: 216 to 298 g.

Allocation to Treatment Groups (F0 Generation)
Allocation By sex, after a period of acclimatization.

Animals showing signs of ill health were excluded. Animals at the extreme of the weight range were not selected if alternatives were available.

At commencement of the study the body weight of animals did not exceed ¿20% of the mean for each sex.

Selection of Offspring to Form F1 Generation

Selection On Day 18 to 21 of age.

Allocation - formal start of F1 generation Day 21 of age (direct dose administration commenced on Day 21 of age).

Method 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, two males and two females were selected from each selected litter (if more were required, up to three males and three females were selected from each selected litter) and allocated to each of the two cohorts.

Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age.

Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

Identification
Identification of animals Unique for each F0 animal and selected F1 offspring within study. All pre-weaning offspring were numbered individually within each litter on Day 1 of age.

Method Microchip (F0 generation and selected F1 generation).

Toe tattoo (pre-weaning offspring).

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24°C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages 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.

Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization and after selection up to four animals of one sex
Pairing one male and one female
Males to termination* up to four animals
Females after mating (from Day 0 after mating) one animal
Females during littering (from Day 20 after mating) one animal + litter
Females to termination (after weaning) up to four animals
Offspring maturation (from weaning until selection) litter
F1A animals* up to four animals of one sex
F1B animals until pairing* up to four animals of one sex

* Except when animals were separated into single housing overnight prior to urine collection )

Environmental Enrichment
Aspen wood based products A soft white untreated wood products; provided to each cage throughout the study (except for F0/F1 Cohort 1B females during lactation and when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced when necessary.

Plastic shelter Provided to each cage throughout the study (except for F0/F1 Cohort 1B animals when paired for mating and lactation and when F1 Cohort 1A animals were separated into single housing overnight during urine collection) and replaced at the same time as the cages .

Paper shavings From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Diet Supply
Diet SDS VRF1 Certified, pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted (diet was removed overnight before blood sampling for hematology, blood chemistry and thyroid hormones and during the period of urine collection).

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted (except during urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen wood based products.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Purified
Details on exposure:
Method of preparation
The required amount of test item was weighed out and 50% of the final volume of purified water was added. The mixture was stirred using a magnetic stirrer. The formulation was made up to the required volume. The formulation was returned to the container and mixed using a magnetic stirrer until homogenous. It was then transferred to 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.

Storage of formulation Refrigerated (2 to 8°C).

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.



Details on mating procedure:
Mating Procedure - F0 and F1 Cohort 1B Generation
F0 pairing commenced After two weeks of treatment.

F1 Cohort 1B pairing commenced After ten weeks from selection.

Male/female ratio 1:1 from within the same treatment groups (sibling pairing was not permitted).

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:
Formulation Analysis
Stability and homogeneity Homogeneity and stability of the dose formulation at 5 and 200 mg/mL was established for one day at ambient temperature (15-25°C) and seven days refrigerated (2-8ºC) as part of Covance Study no. PY70VS.

Achieved concentration Samples of each formulation prepared for administration in Week 1 (F0 generation) and Week 1 and the last week of treatment (F1 generation) were analyzed for achieved concentration of the test item.

ADDITIONAL DETAILS TO BE ADDED WHEN FORMULATION ANALYSIS REPORT AVAILABLE
Duration of treatment / exposure:
Duration of Treatment
F0 animals For two weeks before pairing until termination after litters were Day 28 of age.

F1 animals From weaning until termination of respective cohort; although direct treatment started at weaning, all offspring had potential for exposure in utero and via the milk during lactation

Cohort 1A : General toxicity and pathology of the tissues of the male and female reproductive systems - treated from weaning to 13 weeks of age.

Cohort 1B : Treated from weaning throughout pairing, gestation and lactation up to weaning of F2 litters.
Frequency of treatment:
Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Details on study schedule:
Selection On Day 18 to 21 of age.

Allocation - formal start of F1 generation Day 21 of age (direct dose administration commenced on Day 21 of age).

Method 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, two males and two females were selected from each selected litter (if more were required, up to three males and three females were selected from each selected litter) and allocated to each of the two cohorts.

Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age.

Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
F0 generation, Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
F0 generation, Group 2
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
F0 generation, Group 3
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
F0 generation, Group 4
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1A
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1A
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1A
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1A
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1B - control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1B
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1B
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Remarks:
F1 generation, Cohort 1B
No. of animals per sex per dose:
20 males, 20 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection
The doses used in this study (0, 100, 250 and 800 mg/kg/day) were selected in conjunction with the Sponsor and based on the results of a preliminary Reproduction/Developmental Toxicity Screening Test (CIT Study number 36609 RSR) and on the findings from a preliminary Pre and Post-natal Development study conducted at these laboratories (Covance Study no. WL45XP).

In the preliminary Reproduction/Developmental Toxicity Screening Test, treatment at 1000 mg/kg/day was tolerated by the dams, but there was a marked increase in pup mortality and two litters out of ten at that dose died. In the preliminary Pre and Post-natal Development study (females only), doses of 250 or 800 mg/kg/day were well-tolerated in both the F0 and the F1 generation (treated from Day 21 to Day 27 of age). There were no signs attributed to dosing or effect on clinical condition, body weight gain or food intake and there were no treatment related macroscopic findings in the F0. Gestation length, gestation index and parturition were unaffected and there was no conclusive effect on litter size or sex ratio or on the subsequent survival and growth of the offspring to weaning.

The offspring in one litter at 800 mg/kg/day showed poor growth and a relationship to treatment was uncertain. There were no signs attributed to dosing or effect on clinical condition of the treated F1 and the animals continued to grow after weaning. Food intake was unaffected, and there were no treatment related macroscopic findings.

It was therefore considered that 800 mg/kg/day was suitable for investigation as the high dose in this extended one-generation main reproductive performance study (OECD 443). The dose of 100 mg/kg/day was selected as the low dose and 250 mg/kg/day, the low dose in the preliminary study, was selected as the intermediate dose.
Parental animals: Observations and examinations:
Serial Observations
Clinical Observations - F0 and F1 Generation
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 generation 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 20 of lactation for females).

F1 generation From Day 21 of age - daily.
Formal 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 20 of lactation for females).

Detailed observations were recorded at the following times in relation to dose administration:
• Prior to dosing.
• One to two hours after completion of dosing
• As late as possible in the working day.

Detailed Physical Examination and Arena Observations
Before treatment commenced, during each week of treatment, after F1 formal selection, Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Body Weight - F0 and F1 Generation
The weight of animals was recorded as follows:

F0 males Day that treatment commenced.
Each week.
Before necropsy.

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 Days 21, 23, 25, 27* and 29* of age.
Each week.

F1 Cohort 1B females on Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating and Days 1, 4, 7, 14, 21 and 28 post-partum.

Before necropsy.
* Only applicable before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ±2 days; not reported).

Food Consumption - F0 and F1 Generation
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 males and females Weekly, from the day that treatment commenced, until paired for mating.

Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.

For females after mating food consumption was performed to match the body weight recording:

Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18 19 after mating

Days 1-3, 4-6, 7-13, and 14-20 of lactation.(fed ad-libitum from Day 21 of lactation).

F1 selected animals Cohort 1A: Twice weekly during nominal Week 4, then weekly until termination.

Cohort 1B: Twice weekly during nominal Week 4, then weekly until paired for mating.
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18 19 after mating
Days 1-3, 4-6, 7-13, and 14-20 of lactation (fed ad-libitum from Day 21 of lactation).

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

Urinalysis - F0 and F1 Cohort 1A Generation
Urine samples were collected after overnight withdrawal of food and water at the following occasions:

Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group
F1 Cohort 1A Ten male and ten female animals per group

The individual samples were examined for the following characteristics:

• Using manual methods:
• Clarity and Color (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter

• Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
• Glucose (Gluc)
• Ketones (Keto)
• Bile pigments (Bili)
• Blood pigments (UBld)

• Using a Cobas 6000 Analyzer:
• Protein - total (T-Prot) and concentration (Prot)
• Sodium - total (T-Na) and concentration (U-Na)
• Potassium - total (T-K) and concentration (U-K)
• Chloride - total (T-Cl) and concentration (U-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.

• Epithelial cells (Epi)
• Leucocytes (WBC)
• Erythrocytes (RBC)
• Casts
• Spermatozoa
• Other abnormal components (A)

Thyroid Hormone Analysis - TSH and T4
Blood samples were collected at the following occasions:
Occasion Generation Animals
Termination F0 Adults Ten male and ten female animals per group

F1 Offspring Ten litters per group - pooled litter sample Day 4 of age#
Ten male and ten female animals per group on Day 22 of age (from as many litters as possible)

F1 Adults - Cohort 1A Ten male and ten female animals per group (approx. 13 weeks of age)
# T4 only.

Conditions Adults: Following overnight deprivation of food.

Offspring Day 4 and Day 22: No overnight deprivation of food.
Blood sample site Adults: Sublingual vein.
Offspring Day 4 of age: Decapitation
Offspring Day 22 of age: Orbital sinus
Anaesthetic Adults and offspring on Day 22 of age: Isoflurane.
Offspring Day 4 of age: not required
Anticoagulant None.
Blood tube Greiner Minicollect - with clot activator.
Blood volume Adults and offspring Day 22 of age: 1.0 mL.
Offspring Day 4 of age: maximum possible.
Treatment of samples Samples were kept at ambient temperature (15 to 25¿C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Serum tubes Serum was transferred to appropriately labelled polypropylene “cryo” tubes using plastic disposable pipettes.
Number of aliquots Adults and offspring Day 22 of age: Two per animal.

• Aliquot 1: 0.2 mL serum for T4
• Aliquot 2: residual serum for TSH

Offspring Day 4 of age: single aliquot.
• Maximum possible
Final storage conditions Deep frozen (approximately -60°C to -90¿C).
Fate of samples Aliquot 1 (T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2 (TSH): dispatched to the Department of Immunology and Immunotoxicity, Covance.
T4 Performed by the Department of Bioanalysis, Covance.

Oestrous cyclicity (parental animals):
Estrous Cycle Monitoring - F0 and F1 Cohort 1B Generation
Dry and wet smears were taken as follows:

Dry smears - F0 females only For 15 days before pairing, using cotton swabs.

Wet smears - F0 and F1 Cohort 1B females After pairing until evidence of mating confirmed.

For four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females.
Sperm parameters (parental animals):
Sperm Analysis - F0 and F1 Cohort 1A
Immediately after scheduled sacrifice of each F0 and F1 Cohort 1A male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.

The following tests were performed:
Sperm motility: all groups A sample of sperm was expressed from the left vas deferens (with the exception of F0 males Group 1; No. 19 and Group 3; No. 51 where tissues were taken from the right side) into prewarmed (target 37°C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 µm depth cannula by capillary action and, at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyzer (CASA).

Sperm morphology: Groups 1 and 4 A 200 ¿L aliquot of the sperm/medium mixture (described above) was diluted with 800 ¿L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, with the exception of F0 male Group 1; No. 7, F1A males Group 1; No. 418 and Group 4; No. 471 where assessment of 200 sperm was unable.

Sperm morphology: Groups 2 and 3 Fixed samples retained for possible future assessment.
Sperm count: Groups 1 and 4 The left cauda epididymis (with the exception of F0 males Group 1; No. 19 and Group 3; No. 51 where tissues were taken from the right side) of each male was weighed and then the tunica was removed. The portion obtained was weighed then frozen. Prior to analysis the cauda epididymis portions were allowed to thaw then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3 Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4 After removal of the tunica, the left testis (with the exception of F0 males Group 1; No. 19 and Group 3; No. 51 where tissues were taken from the right side) of each male was frozen. Prior to analysis, the testes were allowed to thaw then homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed.

Homogenization-resistant spermatid counts: Groups 2 and 3 Samples frozen for possible future assessment.
Litter observations:
Records Made During Littering Phase - F0 and F1 Cohort 1B Generation
The records maintained were as follows:

Clinical observations Observed approximately 24 hours after birth (Day 1 of age) and then daily 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 during 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 Recorded on Days 1, 4 (before culling), 7, 14, 21 and 22 (day of necropsy) of age.

Weaning of offspring The dam was removed from the litter cage and offspring were weaned on Day 21 of age.

Ano-genital distance Day 1 of age - all offspring.
Nipple/areolae count Day 13 of age - male offspring.
Postmortem examinations (parental animals):
Terminal Investigations
Time of Necropsy
F0 males After weaning of the F1 animals, after confirmation that no further mating required.

F0 females Day 28 post partum.

F0 females failing to produce a viable litter Terminated with first cohort of females with live litters.

Macroscopic examination
All animals, including surplus offspring culled on Day 4 of age and Day 22 of age unselected offspring were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents (offspring
For F0 and F1 Cohort 1B females the implantation site count was recorded.

For females of F1 Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.

Pathology procedures - F0 (Parental) Animals
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides ¿
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Optic nerves
Ovaries ¿
Pancreas
Pituitary ¿
Prostate - dorsolateral and ventral combined ¿
Rectum
Sciatic nerves
Seminal vesicles (with coagulating gland) ¿
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes ¿
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix and oviducts¿
Vagina ¿
Vas Deferens ¿

Histology
F0 animals
Wet tissues Wet tissues were dispatched to the Test Site (Covance Harrogate, UK) for processing.

Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

All procedures were performed in compliance with local Standard Operating Procedures.

Full List All adult animals killed or dying prematurely.

All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.

Processing - Males and females; liver, adrenal glands and abnormalities only

Males only kidneys All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Processing - reproductive organs only The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant.

Routine staining Sections were stained with hematoxylin and eosin.

On completion of the study phase the Histology slides were transferred back to the Test Facility with any necessary supporting documentation. The wet tissues, blocks and raw paper data were returned to the Test Facility.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures table. Requisite organs were weighed for animals killed at scheduled intervals.

Light Microscopy - F0 Animals
Premature deaths F0, F1A, F1B All animals from all groups.

Scheduled kill
F0 animals All animals of Groups 1 and 4.
All animals of Groups 2 and 3. Liver, adrenal glands and abnormalities only
Males only from Groups 2 and 3 Kidney
All animals of Groups 2 and 3 with suspect fertility Reproductive organs.

Right testis A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Ovaries F0- Qualitative evaluation of 1 section from each ovary.

Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).
Postmortem examinations (offspring):
Terminal Investigations
Time of Necropsy
F1 Cohort 1B females Day 28 post partum.

F1 Cohort 1B females failing to produce a viable litter Terminated with first cohort of females with live litters.

Unselected offspring Culled on Day 4 and Day 22 of age.

F1 Cohort 1A animals At approximately 13 weeks of age.

F1 Cohort 1B males At approximately 17 weeks of age (after weaning of F2 offspring).


Macroscopic examination
All animals, including surplus offspring culled on Day 4 of age and Day 22 of age unselected offspring were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents (offspring ¿21 days of age that were found dead or welfare kill), where possible, were examined and carcass retained.

F1 Cohort 1B females the implantation site count was recorded.

For females of F1 Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea.


Pathology procedures - F1 Adult animals (Cohort 1A)
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
left axillary
Optic nerves
Ovaries
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulating gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix and oviducts
Vagina
Vas Deferens


Pathology procedures - F1 Adult animals (Cohort 1B)
Tissue and regions examined

Abnormalities
Adrenals
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femurs - (longitudinal section through joint)
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
left axillary
Optic nerves
Ovaries
Pancreas
Pituitary
Prostate - dorsolateral and ventral combined
Rectum
Sciatic nerves
Seminal vesicles (with coagulation gland)
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum - bone marrow
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix and oviducts
Vagina
Vas Deferens

Pathology procedures - Unselected F1 and F2 offspring (Day 22 of age)
Ten male and ten females per group; one male or one female from each litter to ensure that all litters are represented

Tissue and regions examined

Abnormalities
Brain (cerebellum, cerebrum, midbrain)
Epididymides
Ovaries
Pituitary
Prostate
Seminal vesicles
Skin with mammary glands (inguinal area)
Spleen
Testes
Thymus
Uterus with cervix and oviducts
Vagina

PLEASE REFER TO SECTION ABOVE "SPERM PARAMETERS - PARENTAL ANIMALS" RE SPERM PARAMETRS FOR OFFSPRING

Histology
F0 animals and F1 Cohort 1A
Wet tissues Wet tissues were dispatched to the Test Site (Covance Harrogate, UK) for processing.

Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

All procedures were performed in compliance with local Standard Operating Procedures.

Full List All adult animals killed or dying prematurely.

All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.

Processing - Males and females; liver, adrenal glands and abnormalities only

Males only kidneys All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Processing - reproductive organs only The reproductive organs were examined from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant.

Routine staining Sections were stained with hematoxylin and eosin.

On completion of the study phase the Histology slides were transferred back to the Test Facility with any necessary supporting documentation. The wet tissues, blocks and raw paper data were returned to the Test Facility.

F1 Cohort 1B
Processing Tissue samples were dehydrated and embedded in paraffin wax.

Limited list (to block) All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities (to block) All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.

Immunophenotyping of Spleen Leucocytes - F1 Cohort 1A
Ten males and ten females per group from F1 Cohort 1A were selected for immunophenotyping.

Where possible, one male or one female was assigned from each selected litter.

The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portions of the spleen was then weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis.

Samples were sent via courier to Department of Immunology and Immunotoxicology (I&I), Covance. A copy of the whole spleen and partial spleen weights were provided to I&I.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures table. Requisite organs were weighed for animals killed at scheduled intervals.

For unselected F1 offspring on Day 22 of age, organs were weighed from ten males and ten females per sex per group from as many litters as possible.

Immunophenotyping of Spleen Leucocytes - F1 Cohort 1A
Ten males and ten females per group from F1 Cohort 1A were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter.

The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portions of the spleen was then weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis.

Samples were sent via courier to Department of Immunology and Immunotoxicology (I&I), Covance. A copy of the whole spleen and partial spleen weights were provided to I&I.

F1 Cohort 1A
Tissues preserved for examination were examined as follows:
Premature deaths F0, F1A, F1B All animals from all groups.

F1A All animals of Groups 1 and 4. All specified in Table 3
All animals of Groups 2 and 3. Liver, adrenal glands and abnormalities only
Males only from Groups 2 and 3 Kidney
F1B All animals. Abnormalities only.

Right testis A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted.

Ovaries F0/F1A - Qualitative evaluation of 1 section from each ovary.
F1 Cohort 1A - Quantitative evaluation of 5 sections from the middle third from each ovary for the assessment of primordial follicle and small growing follicle populations as well as evaluation of corpora lutea numbers in one section from each ovary.

Vagina The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).
Statistics:
Data Evaluation
This report contains serial observations pertaining to all days or weeks of treatment completed, together with signs data collected during the necropsy period. No serial observations relating to the acclimatization period are included in this report.

Summary statistics (e.g. means and standard deviations) presented in this report 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 presentation purposes, however, they were usually rounded to fewer places. It is, therefore, not generally possible to reproduce the presented means and standard deviations exactly using the presented individual data.

Throughout the report the following abbreviations were used:
M Male
F Female
SD Standard deviation
N Number contributing to the mean (normally the number of animals/litters)

PLEASE REFER TO ANY OTHER INFORMATION ON MATERIALS AND METHODS"
Reproductive indices:
Pre-coital Interval - F0 and F1 Cohort 1B Generation
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 and Fertility - F0 and F1 Cohort 1B Generation
Individual data was tabulated. Group values were calculated for males and females separately for the following:

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

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

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

Gestation Length and Gestation Index - F0 and F1 Cohort 1B Generation
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day.

Gestation index was calculated for each group as:

Gestation index (%) = (Number of live litters born / Animals paired) x 100
Offspring viability indices:
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.

Survival Indices - F0 and F1 Cohort 1B Generation
The following were calculated for each litter:

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

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

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

Viability index (%) = (Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering / Number of live offspring on Day 4 (after culling)) x 100

Group mean values were calculated from individual litter values.
Sex Ratio - F0 and F1 Cohort 1B Generation

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

Group mean values were calculated from individual litter values.

Offspring Examinations - F0 and F1 Cohort 1B Generation
Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.

A check was performed to assess for the presence or absence of nipple/areolae for the male offspring on Day 13 of age. As no nipples were present for F1 males, no data are included.
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical Observations
There were no signs attributed to oral gavage administration in males, or in non-mated females during the two-week pre-pairing period, or females during gestation or lactation.

All clinical signs observed during the detailed physical examination and arena observations, showed no relationship to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were four decedents in the F0 generation, none were attributed to treatment with Hexylene glycol.

One male (No. 46) receiving 100 mg/kg/day that was previously clinically normal was found dead shortly after dosing on Study Day 54. Macroscopically, perforation and distension of the esophagus were present along with fluid, adhesions and abnormal contents in the thoracic cavity. Histopathology indicated inflammation and abscess formation in the thoracic cavity that were consistent with mis-dosing were apparent and this was the cause of death.

One male (No. 55) receiving 250 mg/kg/day was killed for welfare reasons on Study Day 36. Trauma wounds, including bruising, depressions and eschar formation were apparent. Macroscopically, discoloration and a depression with an open area were present on the skin. The major microscopic change was abscess formation in the skin/subcutaneous tissue and this was considered to be the cause of the poor clinical condition and therefore the cause of death.

One male (No. 78) receiving 800 mg/kg/day that was previously clinically normal was killed for welfare reasons on Study Day 54, due to signs of gasping respiration and decreased activity. Macroscopic perforation of the esophagus was consistent with mis-dosing and inflammatory change in the lungs correlated with this.

One male (No. 90) receiving 800 mg/kg/day that was previously clinically normal was found dead shortly after dosing on Study Day 36. Macroscopically, pale areas were present in the lungs and the glandular mucosa in the stomach was thickened. The major histopathology change was chronic, active inflammation in the heart and this was considered to be the cause of death. The aetiology of this was not clear but it was considered not to be related to treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight gain at 100, 250 or 800 mg/kg/day was unaffected by treatment during the two-week pre-pairing period for males and females or until scheduled necropsy of the males.

Overall body weight gain was unaffected by treatment during gestation at 100, 250 or 800 mg/kg/day.

Overall body weight gain was unaffected by treatment during lactation at 100, 250 or 800 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption at 100, 250 or 800 mg/kg/day was unaffected by treatment during the two week pre-pairing period for males and females or until scheduled necropsy of the males.

Overall food consumption was unaffected by treatment during gestation at 100, 250 or 800 mg/kg/day.

Overall food consumption was unaffected by treatment during lactation at 100, 250 or 800 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant differences in the cellular composition of the blood in Week 10 of treatment in males and on LD 28 in females. All inter-group differences were within the 95-percentile of the HCD range, unless indicated.
In males at 800 mg/kg/day and in females at 100, 250 or 800 mg/kg/day and without relationship to treatment, prothrombin time was short (88%* and 86%*, 73*%% or 83%** of Control, respectively).
In males at 800 mg/kg/day, neutrophil count was high (166%** of Control) that exceeded the HCD range; however, as this was seen in one sex and with no microscopic correlate, it was considered not to be toxicologically important. Monocyte count was high (155%* of Control) and platelet count was marginally high (116%* of Control) in males at 800 mg/kg/day. In females at 800 mg/kg/day, hematocrit and red cell count were marginally low (96%** and 95%*, respectively) and mean cell hemoglobin and mean cell hemoglobin concentrations were marginally high (104%* and 103%* respectively).


PLEASE REFER TO THE ATTACHED TABLE: "HAEMATOLOGY - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F0)"
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant differences in the constituents of the blood plasma in Week 10 of treatment in males and on LD 28 in females. All inter-group differences were within the 95-percentile of the HCD range, unless indicated.
In males and females at 800 mg/kg/day, glucose concentration was low (88%** and 86%* of Control, respectively) that exceeded the HCD range in males only and at 250 or 800 mg/kg/day, total protein concentrations were high (106%** or 114%** and 105% or 105%*, respectively) that exceeded the HCD range in males only at 800 mg/kg/day and correlated with marginally high albumin concentration (108%) that also exceeded the HCD range and low albumin globulin ratio at 100, 250 or 800 mg/kg/day (92%*, 91%** or 88%**, respectively). The changes in total protein and albumin concentrations in males only were attributed to the microscopic changes identified in the liver.
In males only at 250 or 800 mg/kg/day, alkaline phosphatase activities were increased (121%* or 138%** of Control, respectively) and at 800 mg/kg/day, alanine amino transferase activity was 200%** of Control. Aspartate amino transferase activity was 174%** of Control and exceeded the HCD range. Bilirubin, bile acid (no HCD available) and cholesterol concentrations were high (200%**, 264%* and 195%**, respectively) and creatinine concentration was low (90%*).


PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES AT SCHEDULED TERMNINATION (F0)"
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis - TSH and T4
Serum TSH and T4 concentrations at termination in F0 and F1 adults and F1 offspring on Day 22 of age were considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day. There was variation in the group mean results and mean serum T4 concentration was statistically significantly low in F1 males treated at 800 mg/kg/day; however, female concentrations were unaffected at any dose.

PLEASE REFER TO TABLES IN "ANY OTHER INFORMATION ON RESULTS": "GROUP MEAN SERUM TSH CONCENTRATION DATA IN MALE RATS"
and
"GROUP MEAN SERUM TSH CONCENTRATION DATA IN FEMALE RATS"
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant differences in the composition of the urine in Week 10 of treatment in males and on LD 28 in females.

Urinary pH was marginally low in males at 250 or 800 mg/kg/day and in females at 800 mg/kg/day (91%* or 79%** and 83%**, respectively). Specific gravity was marginally high in males and females at 800 mg/kg/day (both 101%**).

In males only, total sodium concentration was high at 800 mg/kg/day (171%**) and correlated with a high volume of urine in the same animals (140%*).

PLEASE REFER TO THE ATTACHED TABLE: "URINALYSIS - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F0)"
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to the administration of Hexylene glycol were seen in the adrenal glands and liver of males and females and the kidneys of males.
Diffuse cortical hypertrophy, minimal or slight, was present in the adrenal gland of males and females given 800 mg/kg/day. Vacuolation of the cortex was increased in males given all doses of Hexylene glycol. The hypertrophy, which correlated with the changes in organ weight, was considered to be treatment related.
Centrilobular hypertrophy was present in the liver of males and females given 800 mg/kg/day. Bile duct hyperplasia was present in males given 800 mg/kg/day.
In the kidneys of males given all doses of Hexylene glycol there was an increase in the incidence and severity of hyaline droplet accumulation in the tubular cells. Basophilic tubules were increased in those givem 100 or 250 mg/kg/day but this showed no relation to treatment and was not apparent in those given 800 mg/kg/day thus is considered unrelated to treatment. This is considered to be a specific effect to male rats and is not relevant to humans.


PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "INCIDENCE AND SEVERITY OF HEXYLENE-GLYCOL RELATED MICROSCOPIC FINDINGS - TERMINAL SACRIFICE (F0)
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycles at pairing were unaffected by treatment at 100, 250 or 800 mg/kg/day.

All females receiving 100, 250 or 800 mg/kg/day showed estrus following lactation, during the four day period immediately before termination (LD 25-28).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
When compared with Control, it was considered that there was no adverse effect on sperm motility, morphology or testicular or cauda epididymis sperm counts at 100, 250 or 800 mg/kg/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-Coital Interval
Pre-coital interval was unaffected by treatment at 100, 250 or 800 mg/kg/day.

Mating Performance and Fertility
Mating performance and fertility were unaffected by treatment at 100, 250 or 800 mg/kg/day. One pairing from the Control group (Female No. 216) failed to result in pregnancy, following evidence of a successful mating. Macroscopic examination of the female revealed no corpora lutea or implantation sites and there were no microscopic present to account for this.

Gestation Length and Gestation Index
Gestation length was considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day, as all animals littered within 22-23.5 days of mating

Gestation index was unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: All endpoints examined
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
There were no signs attributed to oral gavage administration in males, or in non-mated females or females during gestation or lactation.
All clinical signs observed during the detailed physical examination and arena observations, showed no relationship to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male (No. 500) from the Control group that was previously clinically normal was found dead shortly after dosing on Study Day 79; there were no macroscopic or microscopic findings and the cause of death was undetermined.
One male (No. 521) receiving 250 mg/kg/day showed decreased activity and impaired respiration (irregular and shallow respiration) and was killed for welfare reasons on Study Day 100. Macroscopically, there was perforation of the esophagus and abnormal contents/adhesions in the thoracic cavity. Microscopically inflammation of the pleura was present and death was caused by a dosing accident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 800 mg/kg bw/day, absolute body weight was transiently, but significantly low on Days 1, 8 and 15 in males and on Days 1 and 8 in females and this was considered to be treatment related.
Overall body weight gain at 100, 250 or 800 mg/kg/day was unaffected by treatment in males and in un-mated females from weaning to scheduled termination (Cohort 1A; 13 weeks of age; Cohort 1B after weaning of the F2 litters).
Mean absolute body weight of Cohort 1B females at 800 mg/kg/day was marginally low (93%**) on GD 0, when compared with Control. This was considered to be treatment related.
Investigation showed lower than Control body weight gains at 100, 250 or 800 mg/kg/day during the period of pairing. The reason for the low weight gain in treated animals during pairing was not known, but was considered not to be toxicologically significant.
Absolute body weight at 800 mg/kg/day remained low, when compared with Control, to Day 18 of gestation; however, overall body weight gain at 100, 250 or 800 mg/kg/day was unaffected by treatment.
Absolute body weight on LD 1 at 800 mg/kg/day was marginally, but statistically significantly low (95%* of Control); the difference was considered not to be toxicologically significant. Overall body weight gain (LD 1-21) at 100, 250 or 800 mg/kg/day was superior to that of the Control (2.3-3.3-fold**).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption at 100, 250 or 800 mg/kg/day was unaffected in males and females during the 10-week pre pairing period.
Overall food consumption was unaffected by treatment during gestation at 100, 250 or 800 mg/kg/day.
Overall food consumption was unaffected by treatment during lactation at 100, 250 or 800 mg/kg/day.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Description (incidence and severity):
see cohort 1 A below
Clinical biochemistry findings:
not examined
Description (incidence and severity):
see cohort 1 A below
Urinalysis findings:
not examined
Description (incidence and severity):
see cohort 1A below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Reproductive organs and thymus weights were unaffected by treatment in Cohort 1B.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for this animal age and/or strain and age. Consequently, they were considered not test article related.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
adaptative effects seen in the adrenals, the liver ,
non specific effects in the males kidneys
see dicussion below
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycles from Day 75 of age were considered unaffected by treatment at 100, 250 or 800 mg/kg/day.
One female at 100 mg/kg/day was acyclic (at least ten days without estrus), one female at 250 mg/kg/day had an irregular cycle (at least one cycle of two, three or six to ten days) and two females were acyclic and one female had an irregular cycle at 800 mg/kg/day that were within the HCD range and therefore considered not to be toxicologically significant (see Attachment 14.8).
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Clinical signs:
no effects observed
Description (incidence and severity):
Offspring remained in good clinical condition until scheduled termination on PND 22. Commonly seen signs of attached umbilical cord, little or no milk in stomach, bruising, cuts and patchy hair growth were seen at low incidence and showed no relationship to parental treatment at 100, 250 or 800 mg/kg/day.

There were no signs attributed to oral gavage administration in males, or in non-mated females or females during gestation or lactation.
All clinical signs observed during the detailed physical examination and arena observations, showed no relationship to treatment.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One male (No. 500) from the Control group that was previously clinically normal was found dead shortly after dosing on Study Day 79; there were no macroscopic or microscopic findings and the cause of death was undetermined.
One male (No. 521) receiving 250 mg/kg/day showed decreased activity and impaired respiration (irregular and shallow respiration) and was killed for welfare reasons on Study Day 100. Macroscopically, there was perforation of the oesophagus and abnormal contents/adhesions in the thoracic cavity. Microscopically inflammation of the pleura was present and death was caused by a dosing accident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall body weight gain PND 1 to PND 21 was unaffected by parental treatment at 100, 250 or 800 mg/kg/day.

The absolute body weight of male offspring on PND 1 was marginally, but statistically significantly high (107% of Control) at 800 mg/kg/day.

Overall body weight gain at 100, 250 or 800 mg/kg/day was unaffected by treatment in males and in un-mated females from weaning to scheduled termination (Cohort 1A; 13 weeks of age; Cohort 1B after weaning of the F2 litters).
Mean absolute body weight of Cohort 1B females at 800 mg/kg/day was marginally low (93%**) on GD 0, when compared with Control. Investigation showed lower than Control body weight
gains at 100, 250 or 800 mg/kg/day during the period of pairing. The reason for the low weight gain in treated animals during pairing was not known, but was considered not to be toxicologically significant.

Overall body weight gain during gestation at 100, 250 or 800 mg/kg/day was unaffected by treatment.
Absolute body weight on LD 1 at 800 mg/kg/day was marginally, but statistically significantly low (95%* of Control); the difference was considered not to be toxicologically significant. Overall body weight gain (LD 1-LD 21) at 100, 250 or 800 mg/kg/day was superior to that of the Control (2.3-3.3-fold**).


PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "BODY WEIGHT (G) - GROUP MEAN VALUES OF FEMALES BEFORE PAIRING AND ON GD 0"
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption at 100, 250 or 800 mg/kg/day was unaffected in males and females during the 10-week pre pairing period.
Overall food consumption was unaffected by treatment during gestation at 100, 250 or 800 mg/kg/day.
Overall food consumption was unaffected by treatment during lactation at 100, 250 or 800 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
There were no toxicologically significant differences in the cellular composition of the blood following 10 weeks of direct treatment (approximately Week 13 of age).

Platelet count was high in males treated at 800 mg/kg/day (115%** of Control).
In females at 250 or 800 mg/kg/day, reticulocyte count was slightly low, without relationship to dose (81%* or 83%** of Control, respectively). Neutrophil count was low in females treated at 800 mg/kg/day (63%*) and eosinophil count was low in males at 800 mg/kg/day (58%*) and females at 100, 250 or 800 mg/kg/day (62%*, 77%* or 46%** of Control, respectively); however, overall white cell counts were unaffected.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood Chemistry - Cohort 1A
There were no toxicologically significant differences in the constituents of the blood plasma following 10 weeks of direct treatment (approximately Week 13 of age).

Cholesterol concentration was high in males and females at 800 mg/kg/day (132%** or 125%* of Control, respectively). Total plasma protein concentrations were marginally high in males and females at 800 mg/kg/day (108%** or 106%*, respectively) and correlated in males only with marginally high albumin concentration at 250 or 800 mg/kg/day (both 106%*).
In males only, alanine amino-transferase activity was high without relationship to dose at 250 or 800 mg/kg/day (145% or 148% of Control, respectively). Bile acid concentration was markedly high in females treated at 800 mg/kg/day (7.5-fold**); however, only the values for two animals exceeded the Control group range; therefore no relationship to treatment is inferred. Glucose concentration was slightly low in males at 250 or 800 mg/kg/day (87%* or 80%*, respectively). Potassium concentration was marginally high in females at 250 or 800 mg/kg/day (107* or 111%**) and calcium concentration was marginally high in males at 800 mg/kg/day (105%**).

PLEASE REFER TO THE ATTACHED TABLE: "BLOOD CHEMISTRY - GROUP MEAN VALUES AT SCHEULED TERMINATION (F1 COHORT 1A)"
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A
There were no toxicologically significant differences in the composition of the urine following 10 weeks of direct treatment (approximately Week 13 of age).

Urinary pH was low in males and females at 250 or 800 mg/kg/day (88%** or 81%* and 94%* or 91%* of Control, respectively) and specific gravity was marginally high in males only at these doses (101%* or 102%*, respectively). In females only, total protein and potassium concentrations were high at 800 mg/kg/day (both 142%* of Control).

PLEASE REFER TO THE ATTACHED TABLE: "URINALAYSIS - GROUP MEAN VALUES AT SCHEDULED TERMINATION (F1 COHORT 1a)"
Sexual maturation:
no effects observed
Description (incidence and severity):
All animals at 100, 250 or 800 mg/kg/day showed sexual maturation within one day of Control and was therefore unaffected by treatment.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Ano-genital distance on Day 1 of age was unaffected by parental treatment at 100, 250 or 800 mg/kg/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
A check was performed to assess for the presence or absence of nipple/areolae for the male offspring on Day 13 of age. As no nipples were present for F1 males, no data are included.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no signs attributed to oral gavage administration in males, or in non-mated females or females during gestation or lactation.

All clinical signs observed during the detailed physical examination and arena observations, showed no relationship to treatment.

Body weight relative brain weight was marginally low in females following parental treatment at 800 mg/kg/day (91%*).

F1 generation Cohort 1A
There was an increase in absolute and body weight relative weight of the adrenal glands of males given 800 mg/kg and females given 250 and 800 mg/kg/day. There was an increase in absolute and body weight relative weight of the kidneys of males given 800 mg/kg/day and females given 250 and 800 mg/kg/day, in addition the body weight relative weight only was increased in males given 100 and 250 mg/kg/day. There was an increase in absolute and body weight relative weight of the liver of males and females given 800 mg/kg/day. There was an increase in body weight relative weight of the prostate of males given 800 mg/kg/day and seminal vesicle (including coagulating glands) of males given all doses and the absolute and body weight relative weight of the ovaries of females given all doses.

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.
Reproductive organs and thymus weights were unaffected by treatment in Cohort 1B.


PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "HEXYLENE GLYCOL-RELATED EFFECTS IN ORGAN WEIGHTS PARAMETERS - TERMINAL SACRIFICE (F1)"
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings in the F1 offspring on PND 22 were considered to be unrelated to parental treatment at 100, 250 or 800 mg/kg/day.

F1 generation Cohort 1A and 1B
No macroscopic changes were seen related to the administration of Hexylene Glycol.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or were as expected for this animal age and/or strain and age. Consequently, they were considered not test article related.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Histopathology - Cohort 1A
Changes related to the administration of Hexylene glycol were seen in the adrenal glands and liver of males and females and the kidneys of males.
Increased diffuse cortical hypertrophy, minimal, was present in the adrenal gland of males and females given 800 mg/kg/day. Vacuolation of the cortex was increased in males given 800 mg/kg/day and marginally in those given 250 mg/kg/day.
Centrilobular hypertrophy was present in the liver of males and females given 800 mg/kg/day. Bile duct hyperplasia was not increased in F1 males.
In the kidneys of males given all doses of Hexylene glycol there was an increase in the incidence and severity of hyaline droplet accumulation in the tubular cells. In addition there was an increase in incidence of basophilic tubules, with multifocal changes at minimal or slight severity in males given 250 or 800 mg/kg/day. Granular casts were also present in one male given 250 and three males given 800 mg/kg/day.


PLEASE REFER TO THE TABLE IN "ANY OTHER INFORMATION ON RESULTS": "INCIDENCE AND SEVERITY OF HEXYLENE-GLYCOL RELATED MICROSCOPIC FINDINGS - TERMINAL SACRIFICE (F1)"
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis - TSH and T4
Serum TSH and T4 concentrations at termination in F0 and F1 adults and F1 offspring on Day 22 of age were considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day. There was variation in the group mean results and mean serum T4 concentration was statistically significantly low in F1 males treated at 800 mg/kg/day; however, female concentrations were unaffected at any dose.

PLEASE REFER TO THE TABLES IN "ANY OTHER INFORMATION ON RESULTS": "MEAN SERUM T4 CONCENTRATIONS IN F1 OFFSPRING"
and
"MEAN SERUM T4 CONCENTRATIONS IN F0 AND F1 ANIMALS"
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Litter Size, Survival Indices and Sex Ratio
The numbers of implantations and total offspring born were unaffected by treatment at 100, 250 or 800 mg/kg/day.

A higher number of litters at 800 mg/kg/day showed offspring deaths following parturition to Day 1 of age, when compared with Control (14 v 5* litters) and was higher than the HCD range (See Attachment 14.8); live birth index was marginally low (91.2%* of Control) and also exceeded the Historical Control Data (HCD) range. Subsequently, litter size on Day 1 and on Day 4 (before culling) at 800 mg/kg/day was slightly, but statistically significantly low (90%* of Control), this was considered to be treatment related. Subsequent survival of the offspring to weaning was unaffected.
Offspring sex ratio was unaffected by treatment at 100, 250 or 800 mg/kg/day


Vaginal Opening - Cohort 1A
The interval between vaginal opening and first estrus was considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day.

Estrous Cycles - Cohort 1A
Estrous cycles from Day 75 of age were considered unaffected by treatment at 100, 250 or 800 mg/kg/day.

One female at 100 mg/kg/day was acyclic (at least ten days without estrus), one female at 250 mg/kg/day had an irregular cycle (at least one cycle of two, three or six to ten days) and two females were acyclic and one female had an irregular cycle at 800 mg/kg/day that were within the HCD range and therefore considered not to be toxicologically significant.

Pre-Coital Interval - Cohort 1B
Pre-coital interval was unaffected by treatment at 100, 250 or 800 mg/kg/day.

All pairs showed evidence of successful mating within the 14 day period, as seen in the Control.

Mating Performance and Fertility - Cohort 1B
Following successful evidence of mating, four pairings at 100 mg/kg/day (Female No’s 709, 717, 718 and 720) and two pairings at 800 mg/kg/day (Female No’s 747 and 754) failed to result in pregnancy. Macroscopic examination of the females revealed no corpora lutea or implantations; therefore the animals had not been pregnant. No changes were present microscopically to account for the infertility and no significant findings were present As there was no relationship to treatment and only a small number of animals affected, no relationship with treatment was inferred.

Gestation Length and Gestation Index - Cohort 1B
Gestation length was considered to be unaffected by treatment at 100, 250 or 800 mg/kg/day, as all animals littered within 22-23.5 days of mating (See HCD).

Gestation index was unaffected by treatment.

Stage of Estrous Cycle at Termination - Cohort 1B
Estrous cycles during the four day period before termination (LD 25-28) were unaffected by treatment at 100, 250 or 800 mg/kg/day.

All females receiving 100, 250 or 800 mg/kg/day showed estrus following lactation, during LD 25-28, with the exception of one female receiving 100 mg/kg/day. This was considered not to be toxicologically significant.

Ovarian Follicle Counts and Corpora Lutea - Cohort 1A
The numbers of ovarian follicles and corpora lutea was unaffected at 100, 250 or 800 mg/kg/day.

Sperm Assessment - Cohort 1A
When compared with Control, it was considered that there was no adverse effect on sperm motility, morphology or testicular or cauda epididymis sperm counts at 100, 250 or 800 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
General Condition of Offspring
Offspring remained in good clinical condition until scheduled termination on PND 22. Commonly seen signs of attached umbilical cord, bruising and patchy hair growth were seen at low incidence and showed no relationship to parental treatment at 100, 250 or 800 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Absolute body weight of male and female offspring on PND 1 was unaffected by treatment at 100, 250 or 800 mg/kg/day; subsequent body weight gain (Days 1-21 of age) was marginally, but statistically significantly low following parental treatment at 800 mg/kg/day (both 94%* of Control).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Ano-genital distance on Day 1 of age was unaffected by parental treatment at 100, 250 or 800 mg/kg/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
One male offspring, following parental treatment at 100 mg/kg/day, had 1 nipple. No other offspring at 100 mg/kg/day and no offspring at 250 or 800 mg/kg/day had nipples.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights in the F2 offspring on PND 22 were unaffected following parental treatment at 100, 250 or 800 mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings in the F2 offspring on PND 22 were considered to be unrelated to parental treatment at 100, 250 or 800 mg/kg/day.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Litter Size, Survival Indices and Sex Ratio
The numbers of implantations and total offspring born were unaffected by parental treatment at 100, 250 or 800 mg/kg/day.
A higher number of litters at 800 mg/kg/day showed offspring deaths during Days 1 to 4 of age (Viability Index (%), when compared with Control (7 v 1** litters) that was outside the HCD range, thus the viability index was decreased and as a consequence live litter size was marginally low at the same dose; these changes were considered to be a treatment related affect. Subsequent survival to weaning was unaffected.
The viability indices were unaffected at 100 or 250 mg/kg/day.
The ratio of male to female offspring was unaffected by parental treatment at 100, 250 or 800 mg/kg/day.


PLEASE REFER TO THE ATTACHED TABLE: "LITTER SIZE - GROUP MEAN VALUES (F1)"
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes

Formulation Analysis


The mean concentrations of Hexylene Glycol in dose formulations were within the nominal limits (-10/-15%), confirming the accuracy of formulations. The difference from mean remained within 4%, confirming precise analysis.


Analyzed Concentrations for Hexylene Glycol in Purified Water








































































































































Occasion



Group



Nominal concentration (mg/mL)



Analyzed concentration


(mg/mL)



RME (%)



Difference from mean (%)



Analysis 1



Analysis 2



Mean



Week 1 (F0 generation)



1



0



ND



ND



-



-



-



2



20



18.2



18.2



18.2



-9.0



±0.12



3



50



49.1



46.4



47.7



-4.6



±2.77



4



160



152



141



146



-8.8



±3.54



 



Week 1 (F1 generation)



1



0



ND



ND



-



-



-



2



20



19.9



19.9



19.9



-0.5



±0.14



3



50



50.4



50.5



50.5



+1.0



±0.10



4



160



162



162



162



+1.3



±0.09



 



Last week (F1 generation)



1



0



ND



ND



-



-



-



2



20



20.3



20.8



20.5



+2.5



±1.18



3



50



51.9



51.9



51.9



+3.8



±0.03



4



160



167



167



167



+4.4



±0.28



RME      Relative mean error, representing the deviation from nominal


ND          Not detected


Mean analyzed concentrations and difference from mean values were calculated using unrounded figures.


 


: Group Mean Serum TSH Concentration Data (pg/mL) in Male Rats















































































































































Group



Treatment



Dose (mg/kg/day)



 



F0 Adults
Termination



F1 Offspring Day 22
Termination



F1 Adults Cohort A
Week 13
Termination


 
 

1



Control



0



Mean



723



532



1410


 

SD



499



203



811


 

%CV



69.1



38.2



57.7


 

N



10



10



10


 

2



Hexylene
Glycol



100



Mean



1120



454



972


 

SD



499



263



379


 

%CV



44.6



57.9



39.0


 

N



10



10



10


 

3



Hexylene
Glycol



250



Mean



935



636



862


 

SD



540



333



295


 

%CV



57.7



52.4



34.2


 

N



10



10



10


 

4



Hexylene
Glycol



800



Mean



914



539



764*


 

SD



428



NA



687


 

%CV



46.8



NA



89.8


 

N



10



10



10


 

*              p<0.05


Text table 3: Group Mean Serum TSH Concentration Data (pg/mL) in Female Rats















































































































































Group



Treatment



Dose (mg/kg/day)



 



F0 Adults


Day 28 of Lactation
Termination



F1 Offspring Day 22
Termination



F1 Adults Cohort A
Week 13
Termination


 
 

1



Control



0



Mean



489



611



596


 

SD



239



378



320


 

%CV



48.8



61.9



53.6


 

N



10



10



10


 

2



Hexylene
Glycol



100



Mean



526



315



646


 

SD



265



108



245


 

%CV



50.4



34.3



37.8


 

N



10



10



10


 

3



Hexylene
Glycol



250



Mean



697



486



602


 

SD



326



231



372


 

%CV



46.7



47.5



61.7


 

N



10



10



10


 

4



Hexylene
Glycol



800



Mean



803*



561



658


 

SD



336



294



206


 

%CV



41.8



52.3



31.4


 

N



10



10



10


 

*              p<0.05


 


 


: Mean serum T4 concentrations (pg/mL) in F1 offspring










































































































Group



Treatment



Dose


 


(mg/kg/day)



Parameters



F1 offspring on Day 22 of age


(Male)



F1 offspring on Day 22 of age


(Female)



1



Control



0



Mean



36900



35800



SD



10300



9720



CV%



27.9



27.2



N



10



9



2


 



Hexylene glycol



100



Mean



40800



35300



SD



4950



6380



CV%



12.1



18.1



N



10



10



3



Hexylene glycol



250



Mean



39000



33600



SD



5200



5180



CV%



13.3



15.4



N



10



10



4



Hexylene glycol



800



Mean



40200



36200



SD



6670



5970



CV%



16.6



16.5



N



10



10



 


Text table 5: Mean serum T4 concentrations (pg/mL) in F0 and F1 adults


 












































































































































Group



Treatment



Dose


 


(mg/kg/day)



Parameters



F0 Adult Terminal (Male)



F0 Adult Terminal (Female)



F1 Adult Terminal (Male)



F1 Adult Terminal (Female)


 



1



Control


(Vehicle)



0



Mean



54800



30200



58000



39600



SD



16700



8340



9620



9890



CV%



30.5



27.6



16.6



25.0



N



10



10



10



10



2



Hexylene glycol



100



Mean



67300



33000



64200



49500



SD



8660



6360



13800



12700



CV%



12.9



19.3



21.5



25.7



N



10



10



10



10



3



Hexylene glycol



250



Mean



59200



35700



59000



44700



SD



15700



7860



6990



10000



CV%



26.5



22.0



11.8



22.4



N



10



10



10



10



4



Hexylene glycol



800



Mean



48300



34500



48900 *



45600



SD



4790



7680



6730



9910



CV%



9.9



22.3



13.8



21.7



N



10



10



10



10



*              p<0.05


 


Hexylene glycol-Related Effects in Organ Weight Parameters - Terminal Sacrifice (F0)

































































































































































































































 



Hexylene glycol



 



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



250



800



0



100



250



800



Adrenal Glands



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.060



0.062



0.062



0.077**



0.067



0.072



0.075**



0.081**



Body Weight Ratio (%)



0.011



0.012



0.012



0.014**



0.023



0.025



0.026*



0.027**



Kidneys



 



 



 



 



 



 



 



 



Absolute Weight (g)



3.67



3.79



3.83



4.33**



2.15



2.24



2.29*



2.46**



Body Weight Ratio (%)



0.67



0.71*



0.73*



0.80**



0.75



0.77



0.79*



0.83**



Liver



 



 



 



 



 



 



 



 



Absolute Weight (g)



17.5



16.8



17.5



25.7**



11.7



11.8



12.5



13.9*



Body Weight Ratio (%)



3.21



3.14



3.34



4.39**



4.09



4.06



4.28



4.71*



Spleen



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.77



0.77



0,80



0.86*



0.58



0.61



0.62



0.56



Body Weight Ratio (%)



0.14



0.14



0.15



0.16**



0.20



0.21



0.21



0.19



Thymus



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.25



0.24



0.23



0.30*



0.28



0.28



0.29



0.29



Body Weight Ratio (%)



0.05



0.04



0.04



0.06*



0.10



0.10



0.10



0.10



Ovaries



 



 



 



 



 



 



 



 



Absolute Weight (g)



 



 



 



 



0.11



0.12



0.13*



0.13*



Body Weight Ratio (%)



 



 



 



 



0.039



0.040



0.043*



0.043*



 


 


Incidence and Severity of Hexylene glycol-Related Microscopic Findings - Terminal Sacrifice (F0)






















































































































































































































































































Sex



Hexylene glycol



Males



Females



Dose Level (mg/kg/day)



0



100



250



800



0



100



250



800



Adrenal Glands



 



 



 



 



 



 



 



 



Number Examined



25



0



0



23



24



0



0



25



 Hypertrophy, Cortical, Diffuse



 



 



 



 



 



 



 



 



Minimal



0



 



 



5



0



 



 



6



Slight



0



 



 



2



0



 



 



0



 



 



 



 



 



 



 



 



 



Vacuolation, Cortex



 



 



 



 



 



 



 



 



Slight



2



 



 



10



1



 



 



0



 



 



 



 



 



 



 



 



 



Liver



 



 



 



 



 



 



 



 



Number Examined



25



0



0



23



24



2



0



25



  Hypertrophy, Centrilobular



 



 



 



 



 



 



 



 



Minimal



0



 



 



5



1



0



 



7



  Hyperplasia, Bile Duct



 



 



 



 



 



 



 



 



Minimal



1



 



 



5



0



0



 



0



slight



0



 



 



1



0



0



 



0



 



 



 



 



 



 



 



 



 



Kidneys



 



 



 



 



 



 



 



 



Number Examined



25



2



4



23



24



1



1



25



  Accumulation, Hyaline Droplets,



 



 



 



 



 



 



 



 



Slight



3



2



2



12



0



0



0



0



Moderate



0



0



1



7



0



0



0



0



 



 



 



 



 



 



 



 



 



 


: Body weight (g) - group mean values of females before pairing and on GD 0

















































































































Cohort



 



1A + 1B



1A



1B



Bwt, gain



Group



 



Week



Week



Week



GD



during



/Sex



 



10



10



10



0



pairing



Statistics



test



Wi



Wi



Wi



Wi



 



1F



Mean



271



266



275



282



+7



 



 



 



 



 



 



 



2F



Mean



274



275



272



278



+6



 



 



 



 



 



 



 



3F



Mean



278



281



275



278



+3



 



 



 



 



 



 



 



4F



Mean



264



267



261



263**



+2



 



 



 



 



 



 



 



 


 


: Hexylene glycol-Related Effects in Organ Weight Parameters - Terminal Sacrifice (F1)














































































































































































































































 



Hexylene glycol



 



Sex



Males



Females



Dose Level (mg/kg/day)



0



100



250



800



0



100



250



800



Adrenal Glands



 



 



 



 



 



 



 



 



Absolute Weight (g)



0.061



0.061



0.066



0.073**



0.063



0.067



0.072**



0.077**



Body Weight Ratio (%)



0.013



0.014



0.014*



0.016**



0.024



0.025



0.027*



0.030**



Kidneys



 



 



 



 



 



 



 



 



Absolute Weight (g)



3.39



3.48



3.60



3.87**



1.86



1.97



2.00



2.03*



Body Weight Ratio (%)



0.72



0.77**



0.79**



0.86**



0.71



0.73



0.74*



0.78**



Liver



 



 



 



 



 



 



 



 



Absolute Weight (g)



16.6



16.0



17.2



20.1**



9.1



9.9



10.0



11.4**



Body Weight Ratio (%)



3.52



3.54



3.77



4.46**



3.49



3.65



3.69



4.37**



Prostate



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.02



1.09



1.08



1.11



 



 



 



 



Body Weight Ratio (%)



0.22



0.24



0.24



0.25*



 



 



 



 



Seminal Vesicles



 



 



 



 



 



 



 



 



Absolute Weight (g)



1.81



1.91



1.94



1.94



 



 



 



 



Body Weight Ratio (%)



0.39



0.42*



0.42*



0.43*



 



 



 



 



Ovaries



 



 



 



 



 



 



 



 



Absolute Weight (g)



 



 



 



 



0.090



0.103*



0.106*



0.101*



Body Weight Ratio (%)



 



 



 



 



0.034



0.038*



0.039**



0.039**


           

 


 


Incidence and Severity of Hexylene glycol-Related Microscopic Findings - Terminal Sacrifice (F1)
























































































































































































































































































































































Sex



Hexylene glycol



Males



Females



Dose Level (mg/kg/day)



0



100



250



800



0



100



250



800



Adrenal Glands



 



 



 



 



 



 



 



 



Number Examined



20



0



0



20



20



0



0



20



 Hypertrophy, Cortical, Diffuse



 



 



 



 



 



 



 



 



Minimal



0



 



 



10



2



 



 



13



 



 



 



 



 



 



 



 



 



Vacuolation, Cortex



 



 



 



 



 



 



 



 



Slight



2



 



 



9



0



 



 



0



 



 



 



 



 



 



 



 



 



Liver



 



 



 



 



 



 



 



 



Number Examined



20



1



0



20



20



0



0



20



  Hypertrophy, Centrilobular



 



 



 



 



 



 



 



 



Minimal



0



0



 



6



0



 



 



9



  Hyperplasia, Bile Duct



 



 



 



 



 



 



 



 



Minimal



4



0



 



6



0



 



 



0



 



 



 



 



 



 



 



 



 



Kidneys



 



 



 



 



 



 



 



 



Number Examined



20



5



2



20



20



3



2



20



  Accumulation, Hyaline Droplets,



 



 



 



 



 



 



 



 



Slight



1



0



2



9



0



0



0



0



Moderate



0



0



0



6



0



0



0



0



  Basophilia, Tubular, Focal



 



 



 



 



 



 



 



 



Minimal



9



2



0



5



0



1



1



1



Slight



0



0



0



1



0



0



0



0



  Basophilia, Tubular, Multifocal



 



 



 



 



 



 



 



 



Minimal



0



0



1



2



0



0



0



0



Slight



0



0



0



5



0



0



0



0



  Casts, Granular



 



 



 



 



 



 



 



 



Minimal



0



0



0



2



0



0



0



0



Slight



0



0



0



1



0



0



0



0


Conclusions:
Based on the results of this study it is concluded that the No Observed Adverse Effect Level (NOAEL) for systemic toxicity which has the potential to be harmful to human health in the F0 parent animals and the F1 Cohort 1A and 1B adult animals is 800 mg/kg/day. The NOAEL for kidney changes in F0 and F1 males consistent with the accumulation of alpha 2µ-globulin which was deemed adverse for the animals (but this finding is not generally considered relevant to human health) is 100 mg/kg/day. The NOAEL for reproductive performance of the F0 and F1B parent animals and survival and growth of the F1 and F2 offspring was concluded to be 250 mg/kg/day, based on transient reduced survival of the F1 and F2 offspring during the early post-natal period
Executive summary:

The purpose of this study was to assess the influence of Hexylene glycol on reproductive performance when administered continuously 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 and estrous cycles and one cohort was assessed for reproductive performance (as requested in ECHA Decision number: TPE-D-2114453322-58-01/F).


In the F0 generation, three groups of 25 male and 25 female rats received Hexylene glycol at dose levels of 100, 250 or 800 mg/kg/day at a volume dose of 5 mL/kg/day. Males were treated for two weeks before pairing, up to necropsy after the 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, 20 males and 20 females were treated from weaning to scheduled termination in adulthood (relevant to each cohort) at the same dose levels and volume-dose as the F0 generation. A similarly constituted Control group in each generation received the vehicle, purified water, at the same volume dose as the treated groups.


For the F0 generation data were recorded on clinical observations, detailed physical examination and arena 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.


The F1 generation comprised of two cohorts:


For F1 Cohort 1A, data were recorded on clinical condition, detailed physical examination and arena observations, 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, detailed physical examination and arena observations, body weight, food consumption, sexual maturation, estrous cycles, mating performance, fertility, gestation length and gestation index, organ weight and macroscopic 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.


Results


There were four unscheduled deaths (one male at 100 mg/kg/day, one male at 250 mg/kg/day and two males at 800 mg/kg/day) in the F0 generation and one unscheduled death (one Control male) in the F1 generation, none were treatment related.


There were no signs attributed to oral gavage administration and no clinical signs (detailed physical examination and arena observations) attributable to treatment in the F0 or F1 adults.


Body weight gain and food consumption were unaffected by treatment during the two (F0) and 10 (F1) week pre-pairing periods and during gestation or lactation and in the F1 and F2 offspring body weights were unaffected by parental treatment.


Pre-pairing estrous cycles in the F0 and F1 females, estrous cycles at 10 weeks of age (F1) and estrous cycles following weaning of litters and before termination (F0 and F1) were unaffected by treatment.


Mating performance and fertility, gestation length and gestation index in the F0 and F1adults were unaffected by the treatment. The numbers of implantations and total offspring born were unaffected by parental treatment at 100, 250 or 800 mg/kg/day.


In the F1 litters, the live birth index was marginally low, reflecting a higher number of litters with offspring deaths up to Day 1 of age at 800 mg/kg/day and this was considered treatment related. Subsequent survival of the offspring was unaffected. In the F2 litters, a higher number of litters had offspring deaths between Days 1 and 4 of age, resulting in a decreased viability index and a marginally low litter size and this was considered treatment related.


The ratio of male to female offspring and the ano-genital distance of the F1 and F2 offspring were considered unaffected by treatment and no F1 male offspring had nipples. One F2 male at 100 mg/kg/day had one nipple; however this was considered not to be toxicologically important.


Absolute body weights of selected F1 males and females at 800 mg/kg/day were slightly but statistically significantly low on Day 1 of the formal F1 generation, when compared with Control.


Sexual maturation of F1 males and females and the period of time to first estrus following maturation was unaffected by treatment.


Hematology, blood chemistry and urinary parameters were not adversely affected by treatment at any dose in the F0 or F1. Adult serum TSH and T4 concentrations were unaffected by treatment in the F0 (adults) or F1 (offspring on Day 22 of age or adults).


Sperm count, motility and motion and morphology; splenic immunophenotyping parameters, ovarian follicle and corpora lutea counts were unaffected by treatment in the F1.


Administration of Hexylene glycol at 800 mg/kg/day resulted in centrilobular hepatocyte hypertrophy, an adaptive change and therefore considered not to be an adverse effect, in the liver of males and females in both F0 and F1 generations and correlated with an increase in organ weight.


Administration of Hexylene glycol resulted in hypertrophy, an adaptive change and therefore considered not to be an adverse effect, of the cortex of the adrenal glands of males and females given 800 mg/kg/day. This correlated with an increase in organ weight and hypertrophy.


Administration of Hexylene glycol to males at 100, 250 or 800 mg/kg in the F0 generation and 250 or 800 mg/kg/day in the F1 generation resulted in cortical vacuolation of the adrenal glands, an adaptive change and therefore considered not to be an adverse effect.


Administration of Hexylene glycol at all dose levels caused an increase in hyaline droplets in the kidney of males. This was considered non-adverse in the F0 generation and in the F1 males given 100 mg/kg/day. Adverse changes were apparent in F1 males given 250 or 800 mg/kg/day (basophilic tubules and granular casts), however these changes are not thought to be significant to man.


Other organ weight changes were considered not to be pathologically significant as there was no associated histopathological change.


Organ weights of the unselected F1 and the F2 offspring on Day 22 of age were unaffected by parental treatment and there were no macroscopic findings related to treatment in the F0 (adults), F1 (offspring and adults), F2 offspring.


Conclusion


Based on the results of this study it is concluded that the No Observed Adverse Effect Level (NOAEL) for systemic toxicity which has the potential to be harmful to human health in the F0 parent animals and the F1 Cohort 1A and 1B adult animals is 800 mg/kg/day. The NOAEL for kidney changes in F0 and F1 males consistent with the accumulation of alpha-2µ-globulin which was deemed adverse for the animals (but this finding is not generally considered relevant to human health) is 100 mg/kg/day. The NOAEL for reproductive performance of the F0 and F1B parent animals and survival and growth of the F1 and F2 offspring was concluded to be 250 mg/kg/day, based on transient reduced survival of the F1 and F2 offspring during the early post-natal period.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study
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

1-Main study for rabbit fetal developmental effect (Chevalier, 2021)


A prenatal development toxicity study by the oral route (gavage) in rabbits was performed with hexylene glycol. The study was performed in compliance with GLP and according to OECD Guideline No. 414 (2018). The test item was administered daily at the dose level of 0, 80, 250 or 600 mg/kg bw/day from Day 6 to Day 28 p.c. inclusive.


Based on the results obtained in this study, it was concluded that:
• The NOEL for maternal parameters and for embryofetal development was set at 250 mg/kg bw/day based on the transient effect in maternal body weight gain and food consumption and the increased incidence of fetal skeletal variation at 600 mg/kg bw/day.
• The No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 600 mg/kg bw/day (the delayed ossifications observed at 250 mg/kg bw/day and above were limited to few structures, classified as variations and not adverse).


2-Preliminary study for rabbit developmental effects (Chevalier , 2020)


Under the experimental conditions of this study where the test item was administered to time-mated female New Zealand White rabbits, the dose level of 1000 mg/kg/day was considered to have exceeded the maximum tolerated dose for an OECD 414 study (mortality, abortion, reduced body weight and food consumption, lower fetal weight).


3- The developmental toxicity of HG has been assessed in a GLP study in rats that was conducted according to OECD test guideline 414 (date not provided) (Clode, 1997). HG was administered by oral gavage to groups of pregnant female rats (Crl: CD(SD) BR strain) on gestation days 6 to 15, inclusive. Dose levels were 0 (water vehicle), 30, 300, or 1000 mg/kg body weight/day and 24 pregnant females were included in each dosing group. Animals were killed on gestation day 20 and uterine/implantation and foetal effects were examined. HG did not affect survival, clinical observations, or necropsy findings in maternal animals. Body weight gain was decreased over the first dosing period (gestation days 6 to 7) in mid- and high-dose maternal animals and returned to control levels thereafter. Food intake was lower in high-dose maternal animals over the gestation day 6 to 7 and 7 to 8 periods and returned to control levels thereafter. No other effects were observed in the maternal animals of any dose group. Mean litter and foetal weights in the high-dose group were marginally but not statistically significantly lower than in the control group. There were no adverse effects of treatment on sex ratio or on the incidences of foetal malformations or external/visceral variations. The incidence of skeletal variations in the high-dose group was slightly higher than in the control group but the nature of the specific changes involved (mainly incomplete ossification of cranial, sternebral, or forepaw structures) suggested only marginal delay in the normal ossification process. Moreover, the variations in the high-dose group were considered to be associated with the reduction in maternal body weight gain. Based on these findings a NOAEL of 300 mg/kg body weight/day was determined for maternal and foetal effects of HG.


 


Another developmental toxicity study was carried out under the FDA guidelines for Reproduction studies (Denny, 1996). Although apparently a reasonably designed study, serious questions have been raised concerning its validity. In this study Sprague-Dawley rats received HG by oral gavage at dose levels of 500, 1200 and 1600 mg/kg/day on gestation days 6-17. There was overt evidence of maternal toxicity with the NOAEL for this parameter being 500 mg/kg based on overt clinical signs of intoxication with reduced weight gain and food consumption at 1200 and 1600 mg/kg. There was no statistically significant increase in total external, visceral and skeletal malformations or variations. However sporadic low occurrences of developmental abnormalities were observed at 1200 and 1600 mg/kg. At 1600 mg/kg there was an increased incidence of skeletal variations (delayed ossification, extra ribs) when analysed on a foetal basis. A NOAEL could not be assigned, as foetuses at the lower dose levels were not examined internally. In view of the unexpected maternal toxicity in this FDA guideline study, attempts were made to repeat the findings at 1200 and 1600 mg/kg but were unsuccessful (G. Daston - personal communication, 1999).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
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 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rabbit
Strain:
other: KBL New Zealand White
Remarks:
Specific Pathogen Free (S.P.F.)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France (Châtillon-sur-Chalaronne, France)
- Age at study initiation: approximately 18-20 weeks
- Weight at study initiation: mean body weight 3566 g (range: 2965 g to 4160 g)
- Fasting period before study: No
- Housing: individually, in noryl cages (Tecniplast, floor area: 4200 cm²/height: 46 cm). Each cage contained dumbbell, music and hay for environmental enrichment.
- Diet: breeding pelleted diet "type 110C", (3409 CRL, KLIBA, Kaiseraugst, Switzerland), ad libitum. In addition, due to drastically reduced food consumption, carrots and hay were distributed to one female in Group 1, one female in Group 2 from Days 22 to 28 p.c. and one female in Group 4 from Days 15 to 28 p.c.
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 5 or 6 days before the beginning of the treatment period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C (set to maintain)
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 5 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 8h dark/16h light

IN-LIFE DATES: From: 28 November 2019 To: 9 January 2020
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Drinking water treated by reverse osmosis ELIX 5 (Millipore SA)
Details on exposure:
FORMULATION PROCEDURE
- Type of test item formulation (visual observation): Solution in the vehicle
- Preparation procedure: According to Report Nos. 36614 VAA (Andre, 2010) and 47334 AHS (Bezard, 2020) describing the preparation procedure for a range of concentrations covering the lowest and highest used in this study
- Frequency of preparation: Up to 9 days, based on test item dose formulation stability and vehicle expiry: at 20 and 200 mg/mL, stability was demonstrated for 9 days at +5°C protected from light. These formulations were also stable when used within 23 hours at room temperature protected from light and magnetically stirred just before sampling (see report n° 36614 VAA, André, December 2010 and report n° 47334 AHS, Bezard, October 2020).
- Storage conditions (control and test item dose formulations): At 2-5°C and protected from light
- Delivery conditions (control and test item dose formulations): At room temperature and protected from light

ADMINISTRATION
The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time. The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage volume of 4 mL/kg/day was used. Control animals (Group 1) received the vehicle only. The dose formulations were maintained under delivery conditions (at room temperature, protected from light) throughout the administration procedure. The control and test item dose formulations were stirred just before administration. The formulations were maintained under continuous magnetic stirring throughout the administration procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analytical technique: Gas chromatography with flame ionization detection (GC FID)
- Principle and validation of the method: Analytical method developed and validated at Charles River Laboratories Evreux (Report Nos. 36614 VAA (Andre, 2010) prior to dose formulation analysis. Checked parameters, acceptance criteria and obtained results are detailed in the validation report
- Determination of test item concentrations in dose formulations: Twice in the study (Days 6 and 28 p.c.). A sample was taken from control and test item dose formulations and analyzed using the validated method.
- Acceptance criterion: Measured concentration = nominal concentration ± 10%.
Details on mating procedure:
Females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as Day 0 p.c.
Duration of treatment / exposure:
Day 6 to Day 28 post coitum (p.c.) inclusive; i.e. from implantation to the day prior to the scheduled hysterectomy
Frequency of treatment:
Once daily
Duration of test:
Day from arrival of animals to the test facility (Day 0 or 1 p.c.) till scheduled euthanasia and hysterectomy (Day 29 p.c.)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
25 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Allocation to groups: during the acclimation period, the animals were allocated to the groups, according to a stratified procedure based on body weight recorded on Day 0 or 1 p.c. provided by the breeder, to ensure comparatively similar mean body weight of the groups.
- Administration route rationale: The oral route was selected since it is one of the routes of administration recommended by the Regulatory Authorities.
- Dose selection rationale: The dose levels were selected in agreement with the Sponsor, on the basis of the results of the following studies:
• Charles River Laboratories Evreux/Study No. 47335 TSL in which Hexylene Glycol was administered at 300 and 1000 mg/kg bw/day for 7 days,
o in this study, no clinical signs and effects on body weight or food consumption were noted at 300 mg/kg bw/day. At 1000 mg/kg bw/day, some clinical signs were observed in one female related to a body weight loss and decreased food consumption. There no findings at necropsy,
o based on these results, the Maximum Tolerated Dose (MTD) was considered to be 1000 mg/kg bw/day.
• Charles River Laboratories Evreux/Study No. 47336 RSL (OECD 414 study) in which Hexylene Glycol was administered at 100, 300 or 1000 mg/kg bw/day in pregnant female rabbits.
In this study, the following results were observed:
o there were 6/8, 8/8, 7/8 and 5/8 pregnant females (with live fetuses) in the groups treated at 0, 100, 300 and 1000 mg/kg bw/day, respectively,
o at 1000 mg/kg bw/day, there were 3/8 pregnant females euthanized on Days 15 p.c. (for humane reasons), or on Days 18 and 19 p.c. (due to abortion). Generally, body weight and food consumption of these females were affected, and signs of poor health was recorded prior to death, in addition to numerous findings at necropsy (mainly deposits in the stomach and colored contents on vagina). At 300 mg/kg bw/day, 1/7 pregnant females had signs of emaciation, piloerection and absence of feces which correlated with effects on body weight and food consumption recorded at this dose. Body weight and food consumption were affected at 300 and 1000 mg/kg bw/day. Effects were dose-related,
o there were no macroscopic findings in females euthanized as scheduled on Days 29 p.c.,
o at 1000 mg/kg bw/day, the gravid uterus weight was slightly lower than controls. There were no test item treatment-related effects on mean gravid uterus weight, mean carcass weight or mean net body weight change at 100 and 300 mg/kg bw/day,
o there were no test item treatment-related findings among hysterectomy data,
o there was a tendency towards a lower fetal weights at 1000 mg/kg bw/day,
o following the external examination of fetuses, there were no malformations at external examination of the fetuses.
The dose level of 1000 mg/kg bw/day was considered highly maternotoxic to be selected in the main study because of the severe changes described above, therefore, 600 mg/kg bw/day was selected as the high dose level. The low-dose and mid-dose were selected using a ratio representing approximately a 2.5-fold interval (i.e. 80 and 250 mg/kg bw/day).
Maternal examinations:
MORBIDITY AND MORTALITY: Yes
- Time schedule: Each animal, once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL SIGNS: Yes
- Time schedule: Each animal, once a day from arrival as part of routine examinations.
- Observations made: clinical signs (including evidence of abortion)

BODY WEIGHT: Yes
- Time schedule: each animal, on Days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 p.c.

FOOD CONSUMPTION: Yes
- Time intervals: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 p.c. Any obvious spillage of food was documented.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice Day 29 p.c.: females were euthanized by an intravenous injection of sodium pentobarbital and were submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.
Macroscopic lesions observed in females were sampled and kept preserved in 10% buffered formalin (or in another appropriate fixative). As remarkable macroscopic lesions were observed in test item-treated group animals, spleen and pancreas of 5 control animals were sampled and preserved.
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 and distribution of dead and live fetuses: Yes
- Number and distribution of early and late resorptions: Yes
- Number and distribution of uterine scars: Yes
- Number and distribution of implantation sites: Yes
The following classification was used to record:
• dead fetus: dead fetus with no degenerative changes
• early resorption: evidence of implant without recognizable embryo
• late resorption: dead embryo or fetus with degenerative changes
• uterine scar: uterine implantation without implant
A gross evaluation of placentas was also undertaken. The placenta weight was recorded for each live fetus and the fetal weight/placental weight ratio was calculated.
Blood sampling:
Not performed
Fetal examinations:
- External examinations: Yes: Each live fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. Dead fetuses were not examined.
- Soft tissue examinations: Yes
- Body: Yes: As soon as possible after euthanasia, all live fetuses in each litter were subjected to a detailed dissection of the soft tissue, which included observation of all the organs and structures of the neck, thorax and abdomen and indication of incomplete testicular descent/cryptorchidism in male fetuses. The fetuses were then eviscerated.
- Body weight and sex: Yes, all animals. Sex was determined by internal examination of the sexual organs.
- Head and brain: Yes: One half of the fetuses were decapitated and the head was fixed in Harrison’s fluid with decalcification. Serial sectioning was performed for evaluation of brain, nasal passages and tongue (and other structures, where appropriate). In the other half of the fetuses, the brain of each fetus was sampled and fixed in Harrison’s fluid. Serial section was made for examination of the organ.
- Skeletal examinations: Yes: The carcasses of the fetuses were fixed with ethyl alcohol. A detailed examination of the skeleton (bones + cartilage) was performed after staining with alizarin red S and alcian blue. This examination included observation of all the bone and cartilage structures of the skull (50% of fetuses), spine, rib cage, pelvis and limbs (100% of fetuses).
- Anogenital distance of all live rodent pups: No
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).
Indices:
Data are recorded and calculated using computerized validated software. Data of non-pregnant females are not included in group mean calculations (body weight, body weight change, food consumption and litter data).

The following parameters were calculated:
For each pregnant female:
• body weight change for different intervals,
• net body weight (presented as carcass weight): Body weight on Day 29 p.c. - gravid uterine weight
• net body weight change: Body weight on Day 29 p.c. - body weight on Day 6 p.c. - gravid uterine weight
For each litter:
• total number of resorptions: Sum of uterine scars + early resorptions + late resorptions
• total number of dead fetuses: Sum of dead fetuses
• % of dead fetuses per litter: (Total number of dead fetuses / Number of implantation sites) x 100
• total number of live fetuses: Sum live male + live female fetuses
• % of live fetuses per litter: (Total number of live fetuses / Number of implantation sites) x 100
• % of pre-implantation loss: ((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) x 100
• % of post-implantation loss: ((Number of implantation sites - Number of live fetuses) / Number of implantation sites) x100
• average fetal body weight: Sum of individual fetal weights / Number of live fetuses
• average placental weight: Sum of individual placental weights / Number of placenta weighed

Parameters calculated for each group are given in section "Any other information on materials and methods incl. tables".
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were observed during the study at any dose level.
All clinical signs recorded in females during the study were considered to be unrelated to the test item treatment as they were present before treatment initiation, and/or both in control and test item-treated animals and/or were observed in isolated animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths occurred at any dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and mean body weight changes recorded in control and test item treated animals are summarized in Table 1 in section "Any other information on results incl. tables".
At 600 mg/kg bw/day, when compared to controls, slight mean lower body weight gain was recorded between Days 6 and 29 p.c. (+360 g vs. +424 g in controls, not statistically significant). The difference was mainly due to an initial lower mean body weight gain recorded between Days 6 and 12 p.c. This difference coincided with effect on food intake (see respectively section). Since the difference in mean body weight changes was not statistically significant and did not impacted terminal body weight recorded on Day 29 p.c., it was not considered as adverse.
At 80 and 250 mg/kg bw/day, no effects on mean body weight or mean body weight change were noted.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption values recorded during the study are summarized in Table 2 in section "Any other information on results incl. tables".
At 600 mg/kg bw/day and when compared with controls, the mean food consumption was nearly 20% lower than that of the control group between Days 9 and 12 p.c..(with p<0.01 during the first interval). From Day 12 p.c. onwards, mean food consumption returned towards control values. Therefore this finding was considered to be non-adverse.
Food consumption of pregnant females given 80 or 250 mg/kg bw/day was not affected.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis 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):
There were no effects on mean gravid uterus weight, carcass weight or net body weight change.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared with controls, there were no test item treatment-related findings in animals euthanized as scheduled, on Day 29 p.c.. The few findings recorded are commonly observed in this species and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
There were no animals that showed signs of premature delivery.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no significant effect on the mean percentage of pre- and post-implementation loss (see Table 3 in section "Any other information on results incl. tables").
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
There was a significant (p<0.05) lower mean number of late resorptions in the low dose group, without dose response relationship. There was no significant effect on the mean number of early resorptions (see Table 3 in section "Any other information on results incl. tables").
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of dead fetuses was 0.0 in the low, mid and high dose groups, which was significant lower (p<0.01) than the 3.3% in the control group. This was within the historical control data (see Table 3 in section "Any other information on results incl. tables").
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At hysterectomy on Day 29 p.c., 23/25, 22/25, 24/25 and 21/25 females were pregnant with live fetuses in the groups treated at 0, 80, 250 and 600 mg/kg bw/day, respectively
Other effects:
not examined
Details on maternal toxic effects:
There were no effects on hysterectomy parameters (mean number of corpora lutea, implantation sites and live fetuses; pre- and post-implantation loss).
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Based on absence of adverse effects
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Transient effect in maternal body weight gain and food consumption were observed at 600 mg/kg bw/day (actual dose received). These effects were considered not adverse.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant effects on mean fetal body weight (see Table 4 in section "Any other information on results incl. tables").
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects on sex ratio (percentages of male fetuses) (see Table 4 in section "Any other information on results incl. tables").
Changes in litter size and weights:
not examined
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
VARIATIONS
There were no variations at external examination of the fetuses. At 80 mg/kg bw/day, one dam had one fetus with paw hyperflexion and malrotated paw.This variation was considered to be incidental and not related to the test item treatment as it was noted in a single fetus from the low dose group.

MALFORMATIONS
Some malformations were considered to be incidental and not related to the test item treatment as they were only observed in the low dose-group exclusively with no dose relationship (cleft palate, paw hyperflexion, malrotated paw, bent tail, in fetus # 10 of litter Q31077 and mandibular micrognathia, open eye, gastroschisis, anencephaly in fetus #7 of litter Q31072, short snout in fetus # 5 of litter Q31061, narrowed tail in fetus # 6 of litter Q31087). In addition, the recorded incidences were often within our Historical Background Data.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
CARTILAGES
There were no test item-related cartilage findings at any dose-levels.
In the 250 and 600 mg/kg bw/day groups and when compared with controls, there were increases in the litter and fetal incidences of unossifed cartilages (i.e. cartilage of hyoid arch, sternebrae and pelvic girdle , cartilage structures listed were present). Because these findings were isolated and only concerned a limited number of bone structures and in absence of any relationship with fetal weight which was not impacted, a relationship to treatment was ruled out. None of these were adverse (all bone structures were present).

VARIATIONS
The incidences of skeletal variations are presented in Table 6. of section "Any other information on results incl. tables".
When compared with controls, there were no test item-related skeletal variations.
The increased incidences of unossification or incomplete ossifications of the skeleton at 250 and/or 600 mg/kg bw/day were observed among few bone structures (hyoid; 5th sternebrae; 13th supernumerary rib, pubis). Considering that the differences recorded at 600 mg/kg bw/day were often statistically significant, a delayed ossification in the above-mentioned bones was considered to be test item-related, but non adverse.

MALFORMATIONS
The incidences of skeletal malformations are presented in Table 7. of section "Any other information on results incl. tables".
When compared with controls, there were no test item-related skeletal malformations since there were mainly observed in the low dose group and with limited incidences (e.g. absent of head-skull parietal or interparietal bones, fused/absence of caudal vertebrae, fused sternebrae, absence of lumbar vertebrae, split palate which were mainly seen in 2 fetuses from 2 different litters, and also observed in control (fused ribs).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
VARIATIONS
No test item-related or relevant soft tissue variations were observed at any dose level.

MALFORMATIONS
The incidences of fetuses and litters with soft tissues malformations are presented in Table 5 of section "Any other information on results incl. tables".
Several malformations were observed in the heart of three fetuses from 2 litters treated at 80 mg/kg/day (ventricular septum defect fetus #7, litter Q31083; thick ventricular septum fetuses #9 and #10, litter Q31066) and in two fetuses from 2 litters given 600 mg/kg/day (ventricular and atrioventricular septum defects with fetus #7, litter Q31118, associated with enlarged ventricular chamber classified as a variation; cardiomegaly in fetus #6, litter Q31119). The presence of hydropericardium in one fetus (litter Q31072) at 80 mg/kg/day was unrelated to the test item since it could be observed in one fetus from the control group.
Findings observed in the kidneys, vessels and ureters at 600 mg/kg/day were not considered to be test item-related in absence of dose relationship (no malformations among fetuses from the mid dose group; the retroesophageal arch of vessels in one fetus #8, litter Q31075 was isolated), the low incidences (dilated aortic arch: fetus #07, litter Q31118; narrow aortic arch, dilated pulmonary trunk and transposition of great vessels in a single fetus #6, litter Q31119; short ureters in two fetuses #5 and 10, litter Q31120); and/or occasionally observed in controls (dilated aortic arch in fetus #2, litter Q31052).
Therefore, there were no test item treatment-related malformations at the fetal soft tissue examination, the lack of dose response of the above-mentioned malformations.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
PLACENTA WEIGHT
The ratio fetal body weight/ placenta in the low and high dose groups was significantly lower (p<0.01 and P<0.05, respectively), however there was no dose response relationship and there were no significant effects on placenta weight (see Table 4 in section "Any other information on results incl. tables").
Details on embryotoxic / teratogenic effects:
A summary of external, soft tissues and skeletal malformations is presented in Table 8 of section "Any other information on results incl. tables".
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on absence of adverse effects
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Remarks on result:
other: Statistically significant increased incidence of delayed ossifications was observed at 600 mg/kg bw/day. The effects were limited to few structures, and classified as variations and not adverse.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: supernumerary rib
skeletal: pelvic girdle
other: hyoid bone
Description (incidence and severity):
Increased incidences of unossification or incomplete ossifications of the skeleton at 250 and/or 600 mg/kg bw/day was observed among few bone structures (hyoid; 5th sternebrae; 13th supernumerary rib, pubis). The delayed ossification was considered to be test item-related, considering that the differences recorded at 600 mg/kg bw/day were often statistically significant. However, the effects were considered non-adverse.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
no

CHEMICAL ANALYSIS OF THE DOSE FORMULATION


The test item concentrations in the administered dose formulations analyzed in weeks 01 and 04 remained within an acceptable range of variations (-6.2% to +0.1%) when compared with the nominal values (nominal value ± 10%).


No test item was detected in the control dose formulation.


 


BODY WEIGHTS AND BODY WEIGHT GAINS


Mean body weights and mean body weight changes recorded in control and test item treated animals are summarized in Table 1.


Table 1: Body Weights (kg) and Body Weight Changes (g)











































































































































Dose level (mg/kg bw/day)



0



80



250



600



Body weight



 



 



 



 



Day 6 p.c.



3.58



3.59



3.56



3.60



Day 9 p.c.



3.64



3.64



3.60



3.60



Day 12 p.c.



3.66



3.69



3.64



3.61



Day 15 p.c.



3.73



3.75



3.70



3.68



Day 19 p.c.



3.77



3.78



3.71



3.73



Day 24 p.c.



3.86



3.86



3.82



3.82



Day 29 p.c.



4.00



3.99



3.97



3.96



% from controls



-



0



-1



-1



Body weight change



 



 



 



 



Days 6 - 9 p.c.



+58



+50



+43



0



Days 9 - 12 p.c.



+20



+41



+35



+12



Days 12 - 15 p.c.



+78



+60



+59



+65



Days 15 - 19 p.c.



+36



+38



+19



+57



Days 19 - 24 p.c.



+93



+74



+103



+90



Days 24 - 29 p.c.



+140



+134



+148



+136



Days 6 - 29 p.c.



+424



+396



+407



+360



% from controls



-



-7



-4



-15



-: not applicable.


No statistically significant differences versus controls.


Rounded values with two digits


Bold: test item-related difference


 


FOOD CONSUMPTION


Mean food consumption values recorded during the study are summarized in Table 2.


Table 2: Mean Food Consumption (g/animal/day)























































Dose level (mg/kg bw/day)



0



80



250



600



Days 6 - 9 p.c.



164


(-)



156


(-5%)



153


(-7%)



131**


(-20%)



Days 9 - 12 p.c.



150


(-)



148


(-1%)



143


(-5%)



123


(-18%)



Days 12 - 15 p.c.



126


(-)



115


(-9%)



113


(-10%)



113


(-10%)



Days 15 - 19 p.c.



132


(-)



130


(-2%)



124


(-6%)



142


(+8%)



Days 19 - 24 p.c.



132


(-)



128


(-3%)



132


(0%)



140


(+6%)



Days 24 - 29 p.c.



117


(-)



110


(-6%)



118


(+1%)



123


(+5%)



Statistically significant : **: p<0.01.


Bold: test item-related difference


In brackets: variation from controls


-: not applicable


 


HYSTERECTOMY DATA


Hysterectomy data are presented in Table 3.


Table 3: Hysterectomy Data






























































































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Number of females with live fetuses at termination



23



22



24



21



159+42



Mean number of corpora lutea per animal



11.7



11.9



11.6



11.9



[11.4-12.8]



Mean number of implantation sites per animal



10.4



10.4



10.4



10.1



[9.5-11.4]



Mean percentage of pre-implantation loss (%)



10.0



11.6



10.2



14.9



[10.5-17.9]



Mean number of live fetuses per animal



8.9



9.8**



9.5



8.9



[8.5-10.6]



Dead fetuses (%)



3.3



0.0**



0.0**



0.0**



[0.00-1.00]



Mean number of implantation scars



0.0



0.0



0.0



0.0



/



Mean number of early resorptions



0.3



0.4



0.5



0.7



/



Mean number of late resorptions



0.8



0.3*



0.5



0.6



/



Mean percentage of post-implantation loss (%)



13.2



6.3



8.7



11.8



[3.9-20.5]



Statistically significant: *: p<0.05 and **: p<0.01.


HCD: Historical Control Data (NZW Rabbits, 2015-2016, n = 8 studies and 2016-2017, n = 2 studies).


[  ]: range of study means (min and max).


/: not present in HCD.


 


FETAL BODY WEIGHT, SEX RATIO, PLACENTAL WEIGHT


Mean fetal body and placenta weight as well as sex ratio are presented in Table 4.


Table 4: Mean Fetal Body Weight (g), Mean Percentages of Male Fetuses (%) and Placental Weight (g)






























































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Fetal body weight



 



 



 



 



 



- male fetuses



43.6



39.4*



41.4



40.2



[34.5-39.4]



- female fetuses



41.1



40.3



40.7



40.4



[33.9-38.5]



Mean fetal placenta weight



5.45



5.67



5.68



5.77



[3.87-8.21]a



Ratio fetal BW/ placenta



7.88



7.07**



7.37



7.22*



/



Mean percentage of male fetuses



47.0



52.4



45.3



44.9



[47.4-58.3]



Statistically significant: *: p<0.05 and **: p<0.01.


HCD: Historical Control Data (NZW Rabbits, 2015-2016, n = 8 studies and 2016-2017, n = 2 studies).


a: only HCD with n = 2 studies.


[  ]: range of study means (min and max).


/: not present in HCD.


 


FETAL SOFT TISSUE EXAMINATIONS


The incidences of fetuses and litters with soft tissues malformations are presented in Table 5.


Table 5: Mean litter (L) and Fetal (F) Incidences of Main Soft Tissue Malformations (%)














































































































































































































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Number of litters



22



22



24



21



159+42



Number of fetuses



205



215



227



186



1558+391



Heart



 



 



 



 



 



. ventricular septum defect, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



4.8 (0.5)



/



. ventricular septum thick, L (F)



0.0 (0.0)



4.5 (0.9)



0.0 (0.0)



0.0 (0.0)



/



. hydropericardium, L (F)



4.5 (0.5)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



. atrioventricular septum defect, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



. atrial septum defect, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



. cardiomegaly, L (F)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



/



Dose level (mg/kg bw/day)



0



80



250



600



HCD



Number of litters



22



22



24



21



159+42



Number of fetuses



205



215



227



186



1558+391



Kidneys



 



 



 



 



 



. malpositioned kidney, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



4.8 (1.1)



/



Vessels



 



 



 



 



 



. dilated aortic arch, L (F)



4.5 (0.5)



4.5 (0.5)



0.0 (0.0)



4.8 (0.5)



0.0-4.0 (0.0-0.4)



. narrowed aortic arch, L (F)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



/



. dilated pulmonary trunk, L (F)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



0.0-5.3 (0.0-0.5)



. transposition of great vessels, L (F)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



/



. retroesophageal aortic arch, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



Ureter



 



 



 



 



 



. short ureter, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



4.8 (1.1)



/



Litters affected, n (%)



3 (13.6)



4 (18.2)



1 (4.2)



3 (14.3)



8 (5.0)



Fetus affected, n (%)



4 (2.0)



6 (2.8)



1 (0.4)



5 (2.7)



8 (0.5)



HCD: Historical Control Data (NZW Rabbits, 2015-2016, n = 8 studies and 2016-2017, n = 2 studies).


/: not present in HCD.


 


FETAL SKELETAL VARIATIONS


The incidences of skeletal variations are presented in Table 6.


Table 6: Litter (L) and Fetal (F) Incidences (%) of Skeletal Variations














































































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Dams with live fetuses, n



22



22



24



21



159+42



Live fetuses, n



205



215



227



186



1558+391



. hyoid: incomplete ossification of centrum, L(F)



4.5
(0.5)



18.2
(2.3)



33.3*
(6.2#)



28.6*
(4.8**)



0.0-13.5
(0.0-2.2)



. hyoid: unossified centrum, L(F)



9.1
(1.0)



13.6
(2.3)



12.5
(3.1)



19.0
(7.5**)



0.0-5.6
(0.0-0.5)



. hyoid: unossified arch, L(F)



0.0
(0.0)



4.5
(0.5)



16.7
(2.2)



14.3
(5.4#)



0.0-4.2
(0.0-0.4)



. sternebrae: unossified of 5th, L(F)



63.6
(23.4)



72.7 (18.1)



45.8
(16.7)



85.7
(38.2**)



5.6-52.6
(0.6-9.9)



. supernumerary 13th rib, L(F)



100.0 (42.4)



86.4
(48.4)



91.7
(49.8)



95.2
(53.8*)



94.4-100
(54.8-81.2)



. pubis: incomplete ossification L(F)



0.0
(0.0)



18.2 (2.8*)



20.8
(4.0**)



14.3
(3.8**)



0-27.8
(0.0-3.7)



n: number.


HCD: Historical Control Data (NZW Rabbits, 2015-2016, n = 8 studies and 2016-2017, n = 2 studies): *: p<0.05; **: p<0.01; #: p<0.001.


 


FETAL SKELETAL MALFORMATIONS


The incidences of skeletal malformations are presented in Table 7.


Table 7: Litter (L) and Fetal (F) Incidences (%) of Skeletal Malformations






























































































































































































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Dams with live fetuses, n



22



22



24



21



159+42



Live fetuses, n



205



215



227



186



1558+391



Head-skull



 



 



 



 



 



. parietal absent, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



. interparietal absent, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



0.0-18.2 (0.0-2.1)



Head others



 



 



 



 



 



. palate: split, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



0.0-2.7 (0.0-0.3)



Lumbar vertebrae



 



 



 



 



 



. absent, L(F)



9.1 (1.0)



0.0 (0.0)



4.2 (0.4)



4.8 (0. 5)



0.0 (0.0)



Caudal vertebrae



 



 



 



 



 



. misaligned, L(F)



0.0 (0.0)



4.5 (0.5)



4.2 (0.4)



0.0 (0.0)



0.0 (0.0)



. fused, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



0.0-8.3 (0.0-0.9)



. absent, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



0.0-4.2 (0.0-0.4)



Dose level (mg/kg bw/day)



0



80



250



600



HCD



Dams with live fetuses, n



22



22



24



21



159+42



Live fetuses, n



205



215



227



186



1558+391



Sternebrae



 



 



 



 



 



. fused, L(F)



4.5 (0.5)



13.6 (1.4)



0.0 (0.0)



0.0 (0.0)



5.6-27.3 (0.5-3.6)



Rib



 



 



 



 



 



. fused, L(F)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



0.0-8.3 (0.0-0.9)



Litters affected, n (%)



4 (18.2)



4 (18.2)



2 (8.3)



2 (9.5)



46 (28.9)-19 (45.2)



Fetus affected, n (%)



4 (2.0)



5 (2.3)



2 (0.9)



2 (1.1)



56 (3.6)-27 (6.9)



n: number.


HCD: Historical Control Data (NZW Rabbits, 2015-2016, n = 8 studies and 2016-2017, n = 2 studies).


 


SUMMARY FETAL INCIDENCES OF EXTERNAL, SOFT TISSUE AND SKELETAL MALFORMATIONS


A summary of external, soft tissues and skeletal malformationsis presented in Table 8.


Table 8: Litter and Fetal Incidences of External, Soft Tissue and Skeletal Malformations
















































Group



1



2



3



4



Dose-level (mg/kg bw/day)



0



80



250



600



Pregnant females with live fetuses, n



22



22



24



21



Litter affected, n (%)



8 (36.4)



8 (36.4)



3 (12.5)



4 (19.0)



Live fetuses, n



205



215



227



186



Fetus affected n (%)



9 (4.4)



9 (4.2)



3 (1.3)



6 (3.2)



 

Conclusions:
A prenatal development toxicity study by the oral route (gavage) in rabbits was performed with hexylene glycol. The study was performed in compliance with GLP and according to OECD Guideline No. 414 (2018). The test item was administered daily at the dose level of 0, 80, 250 or 600 mg/kg bw/day from Day 6 to Day 28 p.c. inclusive.

Based on the results obtained in this study, it was concluded that:
• The NOEL for maternal parameters and for embryofetal development was set at 250 mg/kg bw/day based on the transient effect in maternal body weight gain and food consumption and the increased incidence of fetal skeletal variation at 600 mg/kg bw/day.
• The No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 600 mg/kg bw/day (the delayed ossifications observed at 250 mg/kg bw/day and above were limited to few structures, classified as variations and not adverse).
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, Hexylene glycol, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 28 post-coitum (p.c.) inclusive).


Three groups of 25 time-mated female New-Zealand White rabbits received the test item (Hexylene glycol; batch No. I66002404), once daily by the oral route (gavage) at dose levels of 80, 250 or 600 mg/kg/day from Day 6 to Day 28 p.c. inclusive. A control group of 25 time-mated females received the vehicle (drinking water treated by reverse osmosis), under the same experimental conditions. A constant dosage volume of 4 mL/kg/day was used. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. Placentas were observed and weighed. The fetuses were weighed and sexed by internal examination of the gonads. The fetuses were subjected to detailed external, soft tissue and skeletal (bones + cartilages) examinations.


At hysterectomy on Day 29 p.c., 22/25, 22/25, 24/25 and 21/25 females were pregnant with live fetuses in the groups treated at 0, 80, 250 and 600 mg/kg/day, respectively.


No test item-related deaths or clinical signs were recorded.


At 600 mg/kg/day, a slight reduction of body weight gain (-15%, not significant) associated with statistically significantly lower food consumption (up to -20%, during the first days of dosing) were recorded transiently during Days 6-12 p.c. of the study.  This transient finding was not considered adverse.


Body weight and food consumption were not affected at 80 and 250 mg/kg/day.


There were no test item-related macroscopic findings.There were no effects on mean gravid uterus weight, carcass weight or net body weight change.


There were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites and live fetuses, or pre- and post-implantation loss), no effects on mean fetal body weight or the percentage of male fetuses (sex ratio).


There were no test item treatment-related variations or malformations at cartilage, external or soft tissue examination of the fetuses.


At the examination of the skeleton, there was a tendency towards a delayed ossification (variations of few bones such as the hyoid, 5th sternebrae; 13th supernumerary rib and pubis). No test item-malformations of the skeleton were observed.


Based on the results obtained in this study:


-The NOEL for maternal parameters and for embryofetal development was set at
250 mg/kg/day based on the transient effect in maternal body weight gain and food consumption and the increased incidence of fetal skeletal variation at 600 mg/kg/day.


-The No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 600 mg/kg/day (the delayed ossifications observed at 250 mg/kg/day and above were limited to few structures, classified as variations and not adverse).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines.
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd. Margate
- Age at study initiation: 9 weeks
- Weight at study initiation: between 188.8 to 256.5 g
- Fasting period before study: None
- Housing: in groups of 4 in stainless steel wire mesh cages suspended over cardboard-lined trays.
- Diet (e.g. ad libitum): SQC Rat and Mouse Breeder Diet No 3, Expanded, Special Diets Services Ltd, Witham was provided ad libitum
- Water (e.g. ad libitum): Mains drinking water was available ad libitum
- Acclimation period: None


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: solutions of the test article in the vehicle were prepared daily for each group. The test article was weighed into a beaker, some vehicle was added and the mixture was stirred until homogenous. It was transferred to a measuring cylinder and made up to final volume. The formulation was transferred to a bottle and stirred again until homogeneous. Before dosing the formulations were stored at room temperature in sealed containers.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses for achieved concentrations were conducted on samples of the dosing formulations perpared for each group on Days 1 and 17 of the dosing period. In addition, analyses were preformed on samples from group 2 on day 8. The reserve samples of the formulations for groups 3 and 4 on day 17 and additional formulations for groups 3 and 4 were prepared after the end of dosing.
Details on mating procedure:
Mating was conducted at the suppliers laboratory. It was confirmed by the presence of a vaginal plug or sperm in a vaginal smear. The day on which mating was observed was designated as Day 0 of gestation and females were delivered to the laboratory by Day 3 of gestation.
Duration of treatment / exposure:
10 days (day 6 to day 15 of gestation)
Frequency of treatment:
daily
Duration of test:
17 days (day 3 to day 20 of gestation)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 females/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selected by sponser following review of results of a 14-day toxicity study in rats
- Rationale for animal assignment (if not random): random
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations; mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on days 4, 6, 7, 8, 9, 12, 15, 17 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: liver, kidney, adrenals, and spleen


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: pregnancy status
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

- Individual foetal weights were recorded, and foetal abnormalities were recorded
Statistics:
Data were processed where appropriate, to give litter mean values, group mean values, and standard deviations. For each parameter analysed, one of 3 procedures was used;

1. ANOVA. Pairwise comparisons were made using Dunnet's test. Post-dose regression test was performed to determine whether there was a linear relationship between increasing dose and response. Levene's test for equality of variances was also performed.

2. Kruskal-Wallis ANOVA, the Terpstra-Jonckheere test for a dose related trend and the Wilcoxon rank sum test for pairwise comparisons.

3. Cochran-Armitage test for dose response and the Fisher-Irwin Exact test for pairwise comparisons were performed. Where no significant dose response test was seen a Bonferroni adjustment was applied to the pairwise comparisions.
Indices:
Not reported
Historical control data:
Not reported
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Gross pathological findings:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slight increase in pre-implantation loss at the high dose-level, as dosing occurs after implantation, this effect was doubtful.
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
The majority of rats in the high dose group lost weight over the first day of dosing so that the mean body weight gain was significantly lower than controls between days 6 and 7 of gestation. After day 7 of gestation, group mean weight gain was similar to control animals. Group mean intake of the high dose animals was significantly lower than controls between days 6 to 8 of gestation. At necropsy, there were minor incidences of large pale livers, which were not considered to be an adverse effect of treatment because this is a common finding in this strain of rat. The pregnancy rate was 91.7 to 95.8% for all groups.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a slight, non-significant, reduction in mean litter weight in the high dose group compared to control. This is partially explained by the slightly lower litter size in this group; however, group mean foetal weight in the high dose group was also slightly lower than control. The latter finding was probably a consequence of the marginally lower maternai body weight of the high dose group animais.
External malformations:
no effects observed
Description (incidence and severity):
Malformations were observed in all dose groups. The nature and incidence of the abnormalities observed were unrelated to materna! treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The overall incidence of skeletal variations in the treated groups was also slightly higher than control, and in the high dose group significantly more litters had 100 % of foetuses with skeletal variations compared to contrai (p < 0.05, Fisher-Irwin test).
These variations were mainly incomplete ossification of cranial, stemebral or forepaw structures. There was a significantly increasing dose response in the proportion of foetuses showing incomplete ossification of the cranial bones (p < 0.05, Cochran-Armitage test), and in the high dose group there was a significant increase in the proportion of foetuses showing incomplete ossification of the occipital, unossified hyoid arch and extra thoraco-lumber ribs (all p < 0.05, Fisher-Irwin test).
Since these differences from control were generally of a very low order of magnitude and only reached occasional statistical significance in the high dose group, it is probable that there was some marginal delay in the normal ossification process associated with the lower maternai body weight gain in the high dose group.
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The mean number of corpora lutea in the high dose group was slightly higher and the mean number of implantations was slightly lower than the control values. This resulted in a significant increase in percentage pre-implantation loss. There was a slight, non-significant reduction in mean litter weight in the high dose group compared to control. Malformations were observed in all dose groups. The nature and incidence was unrelated to maternal treatment. The overall incidence of external/visceral variations was slightly higher in the treated groups compared to the control due to a higher proportion of foetuses showing subcutaneous hemorrhage of the trunk and limbs. The overall incidence of skeletal variations in the treated groups was also slightly higher than control; which were incomplete ossification of cranial, sternebral, or forepaw structures.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: slight delay in ossification, greater number of fetuses with extra thoraco-lumbar ribs, and slight decrease (not statistically significant) in foetal body weight at 1000 mg/kg bw/d. No teratogenic effects were observed.
Abnormalities:
not specified
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Conclusions:
Oral administration of 1000 mg/kg body weight/day of hexylene glycol to pregnant female rats was associated with slightly reduced body weight gain and food intake. At 300 mg/kg body weight/day of hexylene glycol, there was a transient reduction in body weight gain and only at 30 mg/kg body weight/day of hexylene glycol there were no adverse effects. At 1000 mg/kg body weight/day of hexylene glycol, marginally lower foetal weights and marginally higher incidences of foetal variations were associated with the reduction in maternal weight gain. There were no adverse effects of treatment on pregnancy or the foetus at 30 or 300 mg/kg body weight/day.
Executive summary:

The developmental toxicity of hexylene glycol has been assessed in a GLP study in rats that was conducted according to OECD test guideline 414 (date not provided) (Clode, 1997). Hexylene glycol was administered by oral gavage to groups of pregnant female rats (Crl:CD(SD)BR strain) on gestation days 6 to 15, inclusive. Dose levels were 0 (water vehicle), 30, 300, or 1000 mg/kg body weight/day and 24 pregnant females were included in each dosing group. Animals were killed on gestation day 20 and uterine/implantation and foetal effects were examined.


Hexylene glycol did not affect survival, clinical observations, or necropsy findings in maternal animals. Body weight gain was decreased over the first dosing period (gestation days 6 to 7) in mid- and high-dose maternal animals and returned to control levels thereafter. Food intake was lower in high-dose maternal animals over the gestation day 6 to 7 and 7 to 8 periods and returned to control levels thereafter. No other effects were observed in the maternal animals of any dose group. Mean litter and foetal weights in the high-dose group were marginally but not statistically significantly lower than in the control group. There were no adverse effects of treatment on sex ratio or on the incidences of foetal malformations or external/visceral variations. The incidence of skeletal variations in the high-dose group was slightly higher than in the control group but the nature of the specific changes involved (mainly incomplete ossification of cranial, sternebral, or forepaw structures) suggested only marginal delay in the normal ossification process. Moreover, the variations in the high-dose group were considered to be associated with the reduction in maternal body weight gain. Based on these findings a NOAEL of 300 mg/kg body weight/day was determined for maternal and foetal effects of hexylene glycol (Clode, 1997).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study
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

Developmental toxicity study in rabbit (2021)


The objective of this study was to evaluate the potential toxic effects of the test item, Hexylene glycol, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 28 post-coitum (p.c.) inclusive).


Three groups of 25 time-mated female New-Zealand White rabbits received the test item (Hexylene glycol; batch No. I66002404), once daily by the oral route (gavage) at dose levels of 80, 250 or 600 mg/kg/day from Day 6 to Day 28 p.c. inclusive. A control group of 25 time-mated females received the vehicle (drinking water treated by reverse osmosis), under the same experimental conditions. A constant dosage volume of 4 mL/kg/day was used. The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c., the females were euthanized and a macroscopic post-mortem examination was performed. Hysterectomies were performed and the numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. Placentas were observed and weighed. The fetuses were weighed and sexed by internal examination of the gonads. The fetuses were subjected to detailed external, soft tissue and skeletal (bones + cartilages) examinations.


At hysterectomy on Day 29 p.c., 22/25, 22/25, 24/25 and 21/25 females were pregnant with live fetuses in the groups treated at 0, 80, 250 and 600 mg/kg/day, respectively.


No test item-related deaths or clinical signs were recorded.


At 600 mg/kg/day, a slight reduction of body weight gain (-15%, not significant) associated with statistically significantly lower food consumption (up to -20%, during the first days of dosing) were recorded transiently during Days 6-12 p.c. of the study.  This transient finding was not considered adverse.


Body weight and food consumption were not affected at 80 and 250 mg/kg/day.


There were no test item-related macroscopic findings.There were no effects on mean gravid uterus weight, carcass weight or net body weight change.


There were no significant effects on hysterectomy parameters (mean number of corpora lutea, implantation sites and live fetuses, or pre- and post-implantation loss), no effects on mean fetal body weight or the percentage of male fetuses (sex ratio).


There were no test item treatment-related variations or malformations at cartilage, external or soft tissue examination of the fetuses.


At the examination of the skeleton, there was a tendency towards a delayed ossification (variations of few bones such as the hyoid, 5th sternebrae; 13th supernumerary rib and pubis). No test item-malformations of the skeleton were observed.


Based on the results obtained in this study:


-The NOEL for maternal parameters and for embryofetal development was set at
250 mg/kg/day based on the transient effect in maternal body weight gain and food consumption and the increased incidence of fetal skeletal variation at 600 mg/kg/day.


-The No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo-fetal development was considered to be 600 mg/kg/day (the delayed ossifications observed at 250 mg/kg/day and above were limited to few structures, classified as variations and not adverse).


 


Preliminary study in rabbit (2020)


Three groups of eight mated female New Zealand White rabbits received formulations of the test item, daily from Days 6 to 28 p.c., by the oral route (gavage), at dose levels of 100, 300 or 1000 mg/kg/day. Another group of eight mated females received the vehicle only (drinking water), under the same experimental conditions and acted as a control group. A constant dosage volume of 5 mL/kg/administration was used.


The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c., surviving females were euthanized and submitted to a macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, early and late resorptions, live and dead fetuses were recorded. The fetuses and placentas were weighed. The fetuses were examined for external abnormalities.


There were 6/8, 8/8, 7/8 and 5/8 pregnant females (with live fetuses) in the groups treated at 0, 100, 300 and 1000 mg/kg/day, respectively.


 At 1000 mg/kg/day, there were 3/8 pregnant females euthanized on Day 15 p.c. for humane reasons, or on Days 18 and 19 p.c. due to abortion. Body weight and food consumption of these females were affected along with ante-mortem signs of poor health. Numerous findings at necropsy were observed and consisted of deposits in the stomach and colored contents on vagina. These effects were considered to be treatment related.


Body weight gain was 13% lower than controls during the dosing period, and coincided with reduced food consumption.


 At 300 mg/kg/day, 1/7 pregnant females had signs of emaciation, piloerection and absence of feces which coincided with the reduction of its food consumption and body weight.


There were no macroscopic findings in females euthanized as scheduled on Day 29 p.c.


At 1000 mg/kg/day, the gravid uterus weight was 7% slightly lower than controls and correlated with the lower fetal body weight (-8%). There were no test item treatment-related effects on mean gravid uterus weight, mean carcass weight or mean net body weight change at 100 and 300 mg/kg/day.


There were no test item treatment-related findings among hysterectomy data.


 Following the external examination of fetuses, there were neither variations nor malformations at external examination of the fetuses that could be attributed to the test item treatment with certainty (presence of curled tail in 1 fetus, and the omphalocele in another fetus from another litter).


 Under the experimental conditions of this study where the test item was administered to time-mated female New Zealand White rabbits, the dose level of 1000 mg/kg/day was considered to have exceeded the maximum tolerated dose for an OECD 414 study (mortality, abortion, reduced body weight and food consumption, lower fetal weight).


 


Developmental toxicity study in rat (1997)


The developmental toxicity of hexylene glycol has been assessed in a GLP study in rats that was conducted according to OECD test guideline 414 (date not provided) (Clode, 1997). Hexylene glycol was administered by oral gavage to groups of pregnant female rats (Crl:CD(SD)BR strain) on gestation days 6 to 15, inclusive. Dose levels were 0 (water vehicle), 30, 300, or 1000 mg/kg body weight/day and 24 pregnant females were included in each dosing group. Animals were killed on gestation day 20 and uterine/implantation and foetal effects were examined.


Hexylene glycol did not affect survival, clinical observations, or necropsy findings in maternal animals. Body weight gain was decreased over the first dosing period (gestation days 6 to 7) in mid- and high-dose maternal animals and returned to control levels thereafter. Food intake was lower in high-dose maternal animals over the gestation day 6 to 7 and 7 to 8 periods and returned to control levels thereafter. No other effects were observed in the maternal animals of any dose group. Mean litter and foetal weights in the high-dose group were marginally but not statistically significantly lower than in the control group. There were no adverse effects of treatment on sex ratio or on the incidences of foetal malformations or external/visceral variations. The incidence of skeletal variations in the high-dose group was slightly higher than in the control group but the nature of the specific changes involved (mainly incomplete ossification of cranial, sternebral, or forepaw structures) suggested only marginal delay in the normal ossification process. Moreover, the variations in the high-dose group were considered to be associated with the reduction in maternal body weight gain. Based on these findings a NOAEL of 300 mg/kg body weight/day was determined for maternal and foetal effects of hexylene glycol (Clode, 1997).

Toxicity to reproduction: other studies

Description of key information

In a 90-day oral toxicity study, the NOAEL for the male and female reproductive organs toxicity was the highest dose level tested, 450 mg/kg/day. When compared with controls, no or no clear treatment-related effect of HG administered at 1000 mg/kg/day by gavage from day 6 post-coitum (p. c.) to day 4 post-partum (p. p.), was observed on littering duration, mean progesterone and prolactine serum levels. The significant decrease of the pup viability index observed on day 4 p. p. in non cross-fostered litters was not observed in cross-fostered litters. However, the limited number of cross-fostered litters analyzed precludes the drawing of a definitive conclusion. The administration of HG during the gestation and on day 1 p. p. caused clinical signs in dams, difficulties to deliver, and pup mortality. Compared to the control group, the increase of the length of littering and the high value of post-implantation losses recorded in the HG group given 1000 mg/kg/day could not be attributed to the test item treatment with certainty. Radioactivity was detected in all pups sacrificed on day 1 p. p., demonstrating the transfer of test item from the plasma through the milk, although the amounts detected were very low.

No effects on the gonads were observed in a good quality 90-day oral gavage study in rats (Fabreguette, 1999) which were administered HG at doses up to 450 mg/kg/day by oral gavage. Reproductive organs examined were the testes, prostate, seminal vesicles, epididymes, ovaries, vagina and uterus.

Because of the adverse findings, increased pup mortality and reduced pup body weight gain observed at 1000 mg/kg/day in the OECD 421 study (Allen, 2010), a study was conducted to provide general information concerning the effects of HG on rat gestation, parturition and early lactation, and on the growth and development of the offspring up to day 5post-partum (p. p.)(Spezia, 2011). Two groups (group 3 and group 4) of 10 mated female Sprague-Dawley rats received HG by daily oral (gavage) administration during gestation and lactation (day 6post-coitumto day 4post-partum). The dose-level was 1000 mg/kg/day. Two other groups (group 1 and group 2) of 10 mated females received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions and acted as control groups. The dosing volume was 5 mL/kg/day. At birth three litters from control group 2 were exchanged with three litters from test item-treated group 4 (cross-fostering procedure). This permitted the evaluation of any adverse effects after pre-natal (group 4), post-natal (group 2) or prenatal and post-natal (group 3) exposure of pups on growth and developmental parameters. HG concentration in the administered dosage form remained within the acceptable range of variation. In dams and when compared with controls, HG administered at 1000 mg/kg/day by gavage from day 6post-coitum(p. c.)to day 4post-partum(p. p.), could be associated with increased duration of littering, increased mean progesterone and decreased mean prolactine serum levels. However, given the uncertainty in littering time differences and in biological variations of hormones, the toxicological significance of such findings remained doubtful. On histopathological examination, all females had mammary glands in lactation but the limited number of litters analyzed precludes the drawing of a definitive conclusion. In pups, there were no effects on mean litter size or sex ratio and there were no external abnormalities. In non-cross fostered litter, there was a statistically significant increase in the number of pups found dead in the treated group (15.1% vs. 4.0% in the control group) on days 1-4p. p., resulting in a statistically significant decrease in the viability index on day 4p. p. (84.9% vs. 96.0% in control). Pups had no clinical signs or abnormal behavior. There were no effects on body weight on days 1 and 5p. p. In cross-fostered litters, there were no significant differences in the number of pups found dead in the treated group when compared to respective controls. Pups had no clinical signs or abnormal behavior. There was a slightly higher pup mean body weight on days 1p. p. (vs. in controls). At pups necropsy on day 5p. p., there were no treatment-related findings. Overall, there were no biologically significant findings in cross-fostered pups but the limited numbers of litters exchanged precludes the drawing of a definitive conclusion. When compared with controls, no or no clear treatment-related effect of HG administered at 1000 mg/kg/day by gavage from day 6post-coitum(p. c.)to day 4post-partum(p. p.), was observed on littering duration, mean progesterone and prolactine serum levels. The significant decrease of the pup viability index observed on day 4p. p.in non cross-fostered litters was not observed in cross-fostered litters. However, the limited number of cross-fostered litters analyzed precludes the drawing of a definitive conclusion.

The exposure of pups to HG and/or its metabolites was evaluated via dams on day 1post-partum (p. p.)(Chevalier, 2014). One group of 20 time-mated female Sprague-Dawley rats received the test item, HG, from day 6post-coitum(p. c.) until 20 or 21p. c. with non-radiolabelled HG, and on day 1post-partum(p. p.) with [14C]-HG, by oral route (gavage) at the dose-level of 1000 mg/kg/day. One additional group of five rats received the vehicle, drinking water, under the same experimental conditions, from day 6 until day 1 post-partum (p. p.).A constant dose-volume of 5 mL/kg was used. The test item concentrations in the formulations prepared for use on day 6p. c. and on day 20p. c., the total radioactivity of the radiolabelled test item solution and the radiochemical purity of radiolabelled solution were measured. Clinical signs and mortality were checked daily. Body weight of dams was recorded at designated intervals throughout the study. On the day of parturition which was closely monitored, the pups were identified, examined, weighed, sexed and carefully examined for presence of milk in the stomach. Once the parturition was completed, the radiolabelled test item was given orally to the dams, presence of milk was checked 1, 3 and 5 hours post-dosing, and blood was collected from the dams before sacrifice for determination of radioactivity in plasma. Dams were sacrificed, examined macroscopically, the number of corpora lutea and implantation sites were recorded and the mammary glands were preserved. After their sacrifice on PND 1, all pups were examined to detect gross external and macroscopic abnormalities. Then, two male and two female pups per litter were selected for determination of total radioactivity by liquid scintillation counting. HG concentrations in the administered dose formulations prepared for use on day 6p. c. and on day 20p. c. remained within an acceptable range of approximately -6% when compared to the nominal values, the total radioactivity of the radiolabelled test item solution was -3% when compared to the nominal value and the radiochemical purity of the radiolabelled dose formulation was close to 97%. A total of 4/5 and 19/20 females were pregnant in the control and 1000 mg/kg/day group, respectively. At 1000 mg/kg/day, two females were sacrificed during pregnancy on day 22p. c. due to difficulties to deliver and one dam was prematurely sacrificed during lactation (on day 1p. p.) since the litter was entirely dead. During gestation, staggering gait was observed in all females, and during lactation day 1p. p., almost all the females treated at 1000 mg/kg/day had piloerection and loss of balance. Signs of hypoactivity, ptyalism and half-closed eyes were noted in one or two females. Clinical signs and incidence of premature sacrifices were HG-related. Seventeen pregnant females gave birth to live pups. Repeated administrations of HG at this dose provoked implantation losses. The percentage of dead pups in the HG treated group was 8.3vs. none in the control group, it was mainly due to the dead litter on day 1p. p. (13 pups). The length of gestation in test item-treated females was similar to the control animals. There was a tendency towards increase of the length of parturition when compared to the control values. Specifically, females treated at 1000 mg/kg/day required 3.5 hours for complete parturitionversus2.3 hours for the control group. A total of three litters treated at 1000 mg/kg/day had few pups with hematoma on head, abdomen and/or on the back including one pup with increased size of head. These observations were considered to be HG-related. Radioactivity was detected in the plasma of dams (approx. 12% of the administered dose) and data were characterized by low interanimal variability. In the 64 pups analyzed (16 litters), radioactivity was also detected on day 1p. p.. Data showed that approximately 134 ± 59 Bq/g or 0.087 ± 0.039% of the radioactive dose administered was measured in 4 pups/litter. A great inter variability was observed between litters as well as high intra variability between pups from a same litter. Compared to the radioactivity in the plasma of dams (on Bq/g basis), only 3% were found in the pups. This result confirmed the test item passage through the plasma to the milk compartment, although in limited quantities on day 1p. p. The administration of HG during the gestation and on day 1p. p. caused clinical signs in dams, difficulties to deliver, and pup mortality. Compared to the control group, the increase of the length of littering and the high value of post-implantation losses recorded in the Hexylene group given 1000 mg/kg/day could not be attributed to the test item treatment with certainty. Radioactivity was detected in all pups sacrificed on day 1p. p., demonstrating the transfer of test item from the plasma through the milk, although the amounts detected were very low (ca. 0.09% of the dose).

Link to relevant study records

Referenceopen allclose all

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline available
Principles of method if other than guideline:
Evaluation of the reproductive parameters rats treated from day 6 post-coitum (p.c.) until 20 or 21 p.c. with non radiolabelled Hexylene glycol and on day 1 p.p. with [14C]-Hexylene glycol.
GLP compliance:
no
Type of method:
in vivo
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: 258 g (range: 227 g to 296 g)
- Fasting period before study: no
- Housing: individually housed in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted maintenance diet, ad libitum
- Water: filtered tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Non radiolabelled test item formulations:
The test item solutions were prepared and administered from day 6 p.c. (post-coitum) to the last day of gestation (i.e. day 20 or 21 p.c.). Based on the stability demonstrated in a previous study, the test item dose formulations were prepared and stored up to 7 days at +4°C and protected from light "prior-to-use”. After preparation, the formulation was divided into daily aliquots and stored at +4°C. On the day of dosing, the dose formulation was allowed to come at room temperature prior to delivery under light protection (amber glass beaker).

Radiolabelled test item formulation:
The isotopic dose formulation was prepared once on day 1 p.p. and delivered at room temperature and protected from light (amber glass beaker). As 2 days of parturition were anticipated, one formulation was prepared and further divided into two aliquots. The aliquot not delivered to the animal room was stored at +4°C and protected from light "prior-to-use".
Specifically, [14C]-Hexylene glycol, was diluted as appropriate with purified water so that females received approximately 4 MBq/kg by gavage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Non radiolabelled Hexylene glycol:
GC-FID analytical method
The test item concentrations in the administered dose formulations prepared for use on day 6 p.c. and on day 20 p.c. remained within an acceptable range of -6.0% to -4.9% when compared to the nominal values.

- Radiolabelled dose formulation:
Total radioactivity: High Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method.
The total radioactivity of the radiolabelled test item solution was found at 36.1 MBq/mL (-2.5% when compared to the nominal value 37.0 MBq/mL).
The radiochemical purity of radiolabelled solution received was satisfactory at 97.7%.

Radiochemical purity: HPLC/UV/on line radioactivity detection.
The total radioactivity of the radiolabelled test item solution was found within -3.1% to -1.8% when compared to the nominal value (0.8 MBq/mL).
The radiochemical purity of the radiolabelled dose formulation was found at 97.6%.
Duration of treatment / exposure:
during gestation from days 6 to 21 or 22 p.c. with non radiolabelled formulation, and after completion of parturition on day 1 p.p. with radiolabelled formulation
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20 treated, 5 control
Control animals:
yes, concurrent vehicle
Details on study design:
CLINICAL EXAMINATIONS OF FEMALES
Morbidity and mortality
Each female was checked for mortality and morbidity once a day before the treatment period and at least twice a day during treatment period including weekends and public holidays.
Females showing signs of poor clinical condition, especially if death appears imminent, were humanely sacrificed and subjected to a macroscopic post-mortem examination (see § Females prematurely sacrificed).

Clinical signs
From arrival, each female was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least twice a day (the first daily observation being approximately 1.5 hour post-dosing), at approximately the same time, for the recording of clinical signs.

Body weight
The body weight of each female was recorded on days 2, 6, 14 and 20 p.c. and day 1 p.p..


Parturition
Parturition was closely monitored on day 21 (or day 22) p.c. from 15:00 to day 23 p.c. 8h30. The length of littering was calculated (1st pup to last pup delivered). Females were examined every 30 minutes.
The length of gestation was calculated and the exact time of total delivery was recorded.

OBSERVATION OF THE PROGENY DURING THE post-partum PERIOD
Each pup was individually identified on day 1 post-partum by a subcutaneous injection of Indian ink.

Litter size
The total litter size, sex (see § Radioactive biological sample analysis) and number of pups were recorded as soon as possible after birth. Any gross external malformations in the pups were noted.
The litters were observed daily in order to note the number of live, dead and cannibalized pups.

Presence of milk in the pups’ stomach
Once the radiolabelled formulation was administered to the dam, the presence of milk in the stomach of pups was visually assessed and then recorded at the following time-points:
once the parturition was completed,
1, 3 and 5 hours post administration of the radiolabelled formulation.

Presence of milk in the stomach of control pups was also monitored according to the same procedure mentioned above. Pups from group 1 were sacrificed afterwards (see § Sacrifice, Pups).

Clinical signs
The pups were observed daily for clinical signs, external abnormalities or abnormal behavior.

Body weight
The body weight of each pup was recorded on day 1 p.p..

PATHOLOGY
Sacrifice
Pups
All surviving pups from dams having evident complete parturition (see § Duration) were sacrificed by an intraperitoneal injection of sodium pentobarbital at least 5 hours post-dosing (6 hours and 20 minutes ± 43 minutes) of dams given [14C]-Hexylene glycol solution on day 1 p.p..

Before sacrifice of any pups, the technician had sent to the Study Director the exact time of delivery and the number of pups showing milk presence. Subsequently, the Study Director took the decision to sacrifice or not the pups.
Sacrifice of pups on day 1 p.p. was placed under the following conditions:
dosing of the dam occurred 5 hours before,
at least two cases of milk presence per pup.

Pups from dams of group 1 were sacrificed after the last observation of milk presence (see § Presence of milk in the pups’ stomach).

Dams
Dams were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination:
females: on day 1 p.p., at the same time as their own litters,
mother B22197 with litter dying entirely: on day 1 p.p..

Females prematurely sacrificed
Prematurely sacrificed females during pregnancy
Females B22194 and B22198 which had difficulties to deliver were sacrificed, on day 22 p.c., by inhalation of carbon dioxide gas followed by cervical dislocation. They were submitted for a macroscopic post mortem examination of the principal thoracic and abdominal organs. The pregnancy status was determined and the numbers of corpora lutea and implantation sites were recorded. For apparently non-pregnant females (B21184 and B21191), the presence of implantation scars on the uterine horns was checked using the ammonium sulphide staining technique.
No tissues were preserved.

Prematurely sacrificed during lactation (see § Dams)
Female B21197 was deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination. The female was submitted for a macroscopic post mortem examination of the principal thoracic and abdominal organs. In addition, the number of corpora lutea and implantation sites were recorded.
Mammary glands were preserved.

Pups prematurely sacrificed or found dead
An external examination followed by a macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead pups. No tissues were preserved.

Macroscopic post-mortem examination
Female examination
A complete macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all females including any that are sacrificed prematurely. In all females, the number of corpora lutea and implantation sites was recorded.
For apparently non-pregnant females the presence of implantation scars on the uterine horns was checked using the ammonium sulphide staining technique.

Pup examinations
Pups found dead were carefully examined externally for gross external abnormalities and a visceral macroscopic examination was performed. Presence of milk in the stomach was documented.
No tissues were preserved.
Statistics:
Mean values were compared by one-way analysis of variance and the Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by the Fisher exact probability test.
Dose descriptor:
dose level:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Clinical signs in dams, difficulties to deliver, and pup mortality. The increase of the length of littering and the high value of post-implantation losses could not be attributed to the treatment with certainty.
CLINICAL EXAMINATIONS

Mortality and pregnancy status
A total of 4/5 and 19/20 females were pregnant in the control and 1000 mg/kg/day group, respectively. In the latter group, three dams were prematurely sacrificed. A control female was prematurely sacrificed because it was not pregnant.
At 1000 mg/kg/day, one female was prematurely sacrificed because it was not pregnant. Two females were sacrificed during the gestation period on day 22 p.c. due to difficulties to deliver. A female had staggering gait on days 7 and 8 p.c. (like all test item-treated females from the group), piloerection and round back from day 21 p.c., and 13 dead fetuses plus one early resorption were observed in uterine horns at necropsy. A female showed staggering gait (on days 7 and 8 p.c.), piloerection, round back, coldness to the touch and pallor of extremities before its sacrifice. At macroscopic post-mortem examination, four alive and three dead fetuses were found in the uterine horns. One dam was prematurely sacrificed during lactation (on day 1 p.p.) since the litter died entirely after birth. Female showed staggering gait with similar frequency.

Clinical signs
The following clinical signs detailed during the gestation and the lactation period, were test item-related.

Gestation period (non radiolabelled formulation)
Staggering gait was observed in all females given 1000 mg/kg/day on days 7 and 8 p.c..

Lactation period (radiolabelled formulation)
On day 1 p.p., almost all the females treated at 1000 mg/kg/day had piloerection and loss of balance (this sign corroborated the staggering gait noted during gestation period). Signs of hypoactivity, ptyalism and half-closed eyes were noted in one or two females.

Body weight and body weight change (g)
The dams gained weight normally throughout the dosing period, and on day 20 p.c. body weight and body weight change of females treated at 1000 mg/kg/day was similar to the control animals (+120 g

POST-MORTEM EXAMINATIONS: maternal necropsy findings

The presence of one or two placentae in the uterus of two females given 1000 mg/kg/day was considered to be of incidental occurrence.

REPRODUCTIVE AND LITTER DATA: reproductive and delivery data

The delivery data are summarized below in Table 1.

At 1000 mg/kg/day, two females had difficulties to deliver and one female had entirely dead litter at necropsy (13 dead fetuses). All other pregnant females (17) gave birth to live pups. The percentage of dead pups recorded on day 1 p.p. was above the historical control value, HCD (which was calculated over 5 days), and there were no death in any pups from the four litter of the control group. This difference was mainly due to one female which had 13 dead pups on day 1 p.p..

In addition, when compared to the HCD, there was a tendency towards a minimally increase of the length of parturition when compared to the control values. Specifically, females treated at 1000 mg/kg/day required 3.5 hours for complete parturition versus 2.3 hours for the control group. Although the difference was minimal, a relationship to treatment could not be ruled out as this value coincided with difficulties to deliver recorded in some animals (see above).
deliver recorded in some animals (see above).

OBSERVATIONS OF THE PUPS AFTER BIRTH

Pup body weight
The mean pup body weight of group treated at 1000 mg/kg/day was similar to control.

Sex ratio
There were no effects on the sex ratio.

Milk presence
For all live pups presence of milk in the stomach was observed on day 1 p.p. at time 1, 3 and 5 hours after treatment of females.

Clinical signs and external examination of pups
No clinical signs were recorded in any pups from the control group whereas three litters treated (five pups in total) at 1000 mg/kg/day had pups with hematoma on head, abdomen and/or on the back, including one pup with increased size of head. These clinical signs are already observed in historical control data but in a lesser extent.

Table 1: Summary of reproductive and delivery data

 

Dose-level (mg/kg/day)

0

1000

Reference Control Data (September 2009 to January 2012)

Number of females surviving until delivery

4

17

. mean number ofcorpora lutea

17.3

16.2

14.9 – 19.2

. mean number of implantation sites

14.0

14.1

14.0 -17.5

. mean number of live pups delivered/female

14.0

12.7

13.2 -16.3

. mean post-implantation loss (%) per animal

0

10

4.5 – 15.6

. duration of gestation (days)

21.0

21.2

21.0 – 21.7

. percentage of dead pups (%)

(cannibalized + died pups)

0

8.8(a)

5.1% (b)

. mean littering length (hours)

2.3

3.5

Not applicable

PND: post-natal day,

Bold characters: mean values outside historical range,

(a): excluding female B21194 sacrificed before delivery, since % are calculated for females which delivered

(b): mean percentage calculated over the period PND 1-5,

The length of gestation in test item-treated females was similar to the control animals.

Conclusions:
The administration of Hexylene glycol during the gestation and on day 1 p.p. caused clinical signs in dams, difficulties to deliver, and pup mortality. Compared to the control group, the increase of the length of littering and the high value of post-implantation losses recorded in the Hexylene group given 1000 mg/kg/day could not be attributed to the test item treatment with certainty.
Executive summary:

One group of 20 time-mated female Sprague-Dawley rats received the test item, Hexylene glycol, from day 6 post-coitum (p.c.) until 20 or 21 p.c. with non-radiolabelled Hexylene glycol, and on day 1 post-partum (p.p.) with [14C]-Hexylene glycol, by oral route (gavage) at the dose-level of 1000 mg/kg/day. One additional group of five rats received the vehicle, drinking water, under the same experimental conditions, from day 6 until day 1  post-partum (p.p.). A constant dose-volume of 5 mL/kg was used. The test item concentrations in the formulations prepared for use on day 6  p.c.  and on day 20  p.c., the total radioactivity of the radiolabelled test item solution and the radiochemical purity of radiolabelled solution were measured. Clinical signs and mortality were checked daily. Body weight of dams was recorded at designated intervals throughout the study. On the day of parturition which was closely monitored, the pups were identified, examined, weighed, sexed and carefully examined for presence of milk in the stomach. Once the parturition was completed, the radiolabelled test item was given orally to the dams, presence of milk was checked 1, 3 and 5 hours post-dosing, and blood was collected from the dams before sacrifice for determination of radioactivity in plasma (see section 7.1.1). Dams were sacrificed, examined macroscopically, the number of  corpora lutea  and implantation sites were recorded and the mammary glands were preserved. After their sacrifice on PND 1, all pups  were examined to detect gross external and macroscopic abnormalities. Then, two male and two female pups per litter were selected for determination of total radioactivity by liquid scintillation counting (see section 7.1.1).

The test item concentrations in the administered dose formulations prepared for use on day 6  p.c.  and on day 20 p.c. remained within an acceptable range of approximately -6% when compared to the nominal values, the total radioactivity of the radiolabelled test item solution was -3% when compared to the nominal value and the radiochemical purity of the radiolabelled dose formulation was close to 97%. A total of 4/5 and 19/20 females were pregnant in the control and 1000 mg/kg/day group, respectively. At 1000 mg/kg/day, two females were sacrificed during pregnancy on day 22  p.c.  due to difficulties to deliver and one dam was prematurely sacrificed during lactation (on day 1  p.p.) since the litter was entirely dead. During gestation, staggering gait was observed in all females, and during lactation day 1  p.p., almost all the females treated at 1000 mg/kg/day had piloerection and loss of balance. Signs of hypoactivity, ptyalism and half-closed eyes were noted in one or two females. Clinical signs and incidence of premature sacrifices were test item-related. Seventeen pregnant females gave birth to live pups. Repeated administrations of hexylene glycol at this dose provoked implantation losses. The percentage of dead pups in the hexylene glycol treated group was 8.3  vs.  none in the control group, it was mainly due to the dead litter on day 1  p.p.  (13 pups). The length of gestation in test item-treated females was similar to the control animals. There was a tendency towards increase of the length of parturition when compared to the control values. Specifically, females treated at 1000 mg/kg/day required 3.5 hours for complete parturition  versus  2.3 hours for the control group. A total of three litters treated at 1000 mg/kg/day had few pups with hematoma on head, abdomen and/or on the back including one pup with increased size of head. These observations were considered to be test item-related.

The administration of Hexylene glycol during the gestation and on day 1  p.p.  caused clinical signs in dams, difficulties to deliver, and pup mortality. Compared to the control group, the increase of the length of littering and the high value of post-implantation losses recorded in the Hexylene group given 1000 mg/kg/day could not be attributed to the test item treatment with certainty.

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
15 February to 23 May 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP (no applicable testing guideline); adequate coherence between data, comments and conclusions.
Qualifier:
no guideline required
Principles of method if other than guideline:
Investigative study to provide general information concerning possible effects on rat gestation, parturition, early lactation and growth and development of the offspring up to day 5 post-partum, with a cross-fostering protocol.
GLP compliance:
yes (incl. QA statement)
Type of method:
in vivo
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - mated mothers
- Source: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: mated mothers: 10-12 weeks
- Weight at study initiation: 226 to 312 g
- Fasting period before study: no
- Housing: individually housed in wire-mesh cages with a metal tray containing autoclaved sawdust
- Diet / Water: free access to SSNIFF R/M-H pelleted maintenance diet and to tap water

- Acclimation period: 4 or 5 days

ENVIRONMENTAL CONDITIONS
- temperature: 22 ± 2°C
- relative humidity: 50 ± 20%
- light/dark cycle: 12h/12h (7:00 - 19:00)
- ventilation: about 12 cycles/hour of filtered, non-recycled air.


IN-LIFE DATES: From 18 March to 13 April 2011
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
drinking water treated by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Solution in the vehicle based on a solubility assay.
Based on stability data, solutions were prepared for up to 7 days and stored at +4°C and protected from light.

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle: 5 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Validated Gas Chromatography with FID detection (GC-FID) analytical method.
Stability of solutions at 2 and 200 mg/mL was demonstrated over 9-day storage at +4°C, protected from light.
Adequate consistence between nominal and measured test item concentration (within ±10%).
Duration of treatment / exposure:
mid-gestation (day 6 post-coitum) to beginning of lactation (day 4 post-partum, incl.): total of 19-20 days
Frequency of treatment:
once a day
Duration of test:
see above "duration of exposure"
Remarks:
Doses / Concentrations:
0 and 1000 mg/kg/day
Basis:
actual ingested
(two groups per dose for cross-fostering protocol)
No. of animals per sex per dose:
10 females in each of four groups (groups defined by dose x cross-fostering status)
Control animals:
yes, concurrent vehicle
Details on study design:
Mated females were exposed as indicated. At birth, all pups from group 4 were exchanged (cross-fostered) with all pups from group 2.

Summary on possible pup exposure (via dams):
- group 1: possible prenatal exposure to vehicle + possible postnatal exposure to vehicle
- group 2*: possible prenatal exposure to vehicle + possible postnatal exposure to test item
- group 3: possible prenatal exposure to treatment + possible postnatal exposure to test item
- group 4*: possible prenatal exposure to treatment + possible postnatal exposure to vehicle
* as numbered before cross-fostering

This enabled to assess any adverse effects after pre-natal (group 4), post-natal (group 2) or prenatal and post-natal (group 3) exposure of pups on growth and development.
Dose descriptor:
dose level:
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: no or no clear treatment-related effect on littering duration, mean progesterone and prolactine serum levels. No significant decrease of the pup viability index on day 4 p. p. non cross-fostered litters
The test item concentration in the administered dosage form remained within the acceptable range of variation.
In dams and when compared with controls, the test item (Hexylene glycol, purity 99.84%) administered at 1000 mg/kg/day by gavage from day 6 post-coitum(p.c.) to day 4post-partum(p.p.), could be associated with increased duration of littering, increased mean progesterone and decreased mean prolactine serum levels. However, given the uncertainty in littering time differences and in biological variations of hormones, the toxicological significance of such findings remained doubtful. On histopathological examination, all females had mammary glands in lactation but the limited number of litters analyzed precludes the drawing of a definitive conclusion.
In pups, there were no effects on mean litter size or sex ratio and there were no external abnormalities.
In non-cross fostered litter, there was a statistically significant increase in the number of pups found dead in the treated group (15.1% vs. 4.0% in the control group) on days 1-4 p.p., resulting in a statistically significant decrease in the viability index on day 4 p.p. (84.9% vs. 96.0% in control).
Pups had no clinical signs or abnormal behavior. There were no effects on body weight on days 1 and 5 p.p.
In cross-fostered litters, there were no significant differences in the number of pups found dead in the treated group when compared to respective controls. Pups had no clinical signs or abnormal behavior. There was a slightly higher pup mean body weight on days 1 p.p. (vs. in controls). At pups necropsy on day 5 p.p., there were no treatment-related findings. Overall, there were no biologically significant findings in cross-fostered pups but the limited numbers of litters exchanged precludes the drawing of a definitive conclusion.
Executive summary:

Because of the adverse findings, increased pup mortality and reduced pup body weight gain observed at 1000 mg/kg/day in the OECD 421 study (Allen, 2010), a study was conducted to provide general information concerning the effects of the test item, Hexylene glycol, on rat gestation, parturition and early lactation, and on the growth and development of the offspring up to day 5post-partum (p.p.)(Spezia, 2011). Two groups (group 3 and group 4) of 10 mated female Sprague-Dawley rats received the test item, Hexylene glycol, by daily oral (gavage) administration during gestation and lactation (day 6 post-coitum to day 4 post-partum). The dose-level was 1000 mg/kg/day. Two other groups (group 1 and group 2) of 10 mated females received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions and acted as control groups. The dosing volume was 5 mL/kg/day. At birth three litters from control group 2 were exchanged with three litters from test item-treated group 4 (cross-fostering procedure). This permitted the evaluation of any adverse effects after pre-natal (group 4), post-natal (group 2) or prenatal and post-natal (group 3) exposure of pups on growth and developmental parameters.

The test item concentration in the administered dosage form remained within the acceptable range of variation. In dams and when compared with controls, the test item (Hexylene glycol, purity 99.84%) administered at 1000 mg/kg/day by gavage from day 6post-coitum(p.c.) to day 4 post-partum(p.p.), could be associated with increased duration of littering, increased mean progesterone and decreased mean prolactine serum levels. However, given the uncertainty in littering time differences and in biological variations of hormones, the toxicological significance of such findings remained doubtful. On histopathological examination, all females had mammary glands in lactation but the limited number of litters analyzed precludes the drawing of a definitive conclusion. In pups, there were no effects on mean litter size or sex ratio and there were no external abnormalities. In non-cross fostered litter, there was a statistically significant increase in the number of pups found dead in the treated group (15.1% vs. 4.0% in the control group) on days 1-4 p.p., resulting in a statistically significant decrease in the viability index on day 4 p.p. (84.9% vs. 96.0% in control). Pups had no clinical signs or abnormal behavior. There were no effects on body weight on days 1 and 5 p.p. In cross-fostered litters, there were no significant differences in the number of pups found dead in the treated group when compared to respective controls. Pups had no clinical signs or abnormal behavior. There was a slightly higher pup mean body weight on days 1 p.p. (vs. in controls). At pups necropsy on day 5 p.p., there were no treatment-related findings. Overall, there were no biologically significant findings in cross-fostered pups but the limited numbers of litters exchanged precludes the drawing of a definitive conclusion.

When compared with controls, no or no clear treatment-related effect of Hexylene glycol administered at 1000 mg/kg/day by gavage from day 6 post-coitum (p.c.) to day 4 post-partum (p.p.), was observed on littering duration, mean progesterone and prolactine serum levels. The significant decrease of the pup viability index observed on day 4p.p. in non cross-fostered litters was not observed in cross-fostered litters. However, the limited number of cross-fostered litters analyzed precludes the drawing of a definitive conclusion.

Justification for classification or non-classification

Assessment and comparison with classification criteria for fertility:


No effects on reproductive performance were observed in the available extended one generation study at cocentration up to 800 mg/kg/day. Mating performance, fertility,  reproduction parameters and oestrous cycles for both the F0 and F1 adult generations were unaffected by treatment. Effects on reproductive organs were not observed in the extended one generation study. No classification on fertility is therfore warranted.


Assessment and comparison with classification criteria for developmental effects:


The rat and rabbit developmental studies did not show any teratogenicity effect, although a marginal increase of fetal skeletal variations but non adverse was noted at the top dose dose-level in both studies.


In the extended one generation study, the only finding observed was a minor decrease of pups survival at birth in F1 generation and at PND 4 in the F2 generation at the top dose-level of 800 mg/kg/day. The survival was thereafter unaffected as well as pup development during the whole lactation period. Athough this finding occured at a lower incidence (-7% decrease for live birth index in F1 vs controls and -3% decrease survival index at PND 4 in F2 vs controls), it has already been observed in preliminary studies with the test item (OECD 421 (CIT,2010) and preliminary study to OECD 443 (Covance 2021)), therefore a classification as reproduction category 2 for development is considered warranted.

Additional information