Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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.

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.

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.

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.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 March 2010 - ...................................
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008, Part B.3, 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories, L'Arbresle, France
- Age and weight at study initiation: at the beginning of the treatment period, the males were 10 weeks old and had a mean body weight of 400 g
(range: 375 g to 425 g). The females were 9 weeks old and had a mean body weight of 223 g (range: 207 g to 238 g). The males and the females were sexually mature and were not siblings. The females were previously unmated.
- Housing: The animals were individually housed, except during pairing, in wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing
autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage.
Toward the end of gestation period and during lactation with their litter, the females were individually housed in polycarbonate cages (43.0 x
21.5 x 20.0 cm) containing autoclaved sawdust (SICSA, Alfortville, France). Autoclaved wood shavings (SICSA, Alfortville, France) was provided as
nesting material, a few days before delivery and during the lactation period.
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (7:00 - 19:00).

IN-LIFE DATES: From: 09 March 2010 To: 19 May 2010.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(purified)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item dosage forms were prepared for up to 7 days stored at +4°C and protected from light prior to use. On the day of dosing,
the preparation was allowed to come at room temperature prior to delivery under light protection (brown glass).
The test item was administered as a solution in purified water.
Prior to start the study, a solubility assay was conducted by the CIT pharmacy at 200 mg/mL.

VEHICLE
- Concentration in vehicle: 0, 40, 100 and 200 mg/mL
- Amount of vehicle: 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: each female was placed with the same male until mating occurs or 14 days had elapsed.
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility for seven days.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): in wire-mesh cages.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The GC-FID analytical method for the determination of Hexylene glycol in dosage form samples was provided by the Sponsor and this method was
validated at CIT prior to dosage form analysis.

The validation was based on the ICH Q2(R1) guideline adopted in October 1994, and accordingly the following parameters were checked:
- specificity,
- precision and accuracy,
- injection repeatability,
- linearity,
- Sensitivity Evaluation Test (SET),
- stability of the test item in working solutions.
Duration of treatment / exposure:
The dosage forms were administered daily according to the following schedule:
In the males:
- 4 weeks before pairing,
- during the pairing period (3 weeks),
- until final sacrifice of the females (at least 10 weeks in total).

In the females:
- 4 weeks before pairing,
- during the mating period (3 weeks),
- during gestation,
- during lactation until day 4 post-partum inclusive (or until sacrifice),
- until sacrifice for non-pregnant females.

Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once daily.
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 11 weeks for females and 12 weeks for males.
Remarks:
Doses / Concentrations:
200, 500 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of previous studies:
- CIT/Study No. 15836 TSR: the daily administration of Hexylene glycol to Sprague-Dawley rats at dose-levels of 0, 40, 200 or 1000 mg/kg/day by
gavage for 2 weeks, induced no adverse effects at any dose-level. The notable findings were the increased demand in liver function in both sexes at
1000 mg/kg/day and presence of acidophilic globules in the cortical epithelium of the kidneys in males at 200 and 1000 mg/kg/day (minimal to
severe),
- CIT/Study No. 15837 TCR: the daily administration of Hexylene glycol to Sprague-Dawley rats by oral route at 50, 150 and
450 mg/kg/day for a 13-week treatment period was clinically well tolerated at all dose-levels. The hepatocellular hypertrophy and the changes
indicative of a local irritating effect on the stomach and forestomach observed at 150 and 450 mg/kg/day were not considered as a systemic adverse effect. Consequently, the No Observed Effect Level (NOEL) was 50 mg/kg/day and the No Observed Adverse Effect Level (NOAEL) for the systemic
effect was 450 mg/kg/day,
- COVANCE study No. 121/28-1050: the 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 were there no adverse effects. At 1000 mg/kg body weight/day of
hexylene glycol, marginally lower foetal weights and marginally higher incidences of fetal variations were associated with the reduction in maternal
body weight gain. There were no adverse effects of treatment on pregnancy or the foetus at 30 or 300 mg/kg body weight/day.
Positive control:
None.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 p.c. and days 1 and 5 p.p.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the
pairing period and then after the end of the pairing period until sacrifice.
The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of
pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1-5 p.p.
During the pairing period, the food consumption was measured for neither males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No.
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the
females are mated.
Sperm parameters (parental animals):
Parameters examined in P male (control and high dose groups) parental generations: testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross
anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE: all animals (males were sacrificed at the time of final sacrifice of the females).
Females were sacrificed was follows:
- females: on day 5 p.p.,
- females which had not delivered: on day 25 p.c. (after a body weight recording to check for a possible un-noticed delivery),
- mothers with litter dying entirely.

GROSS NECROPSY: on all parent animals including females that were sacrificed prematurely.
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS:
A microscopic examination was performed on:
- all tissues listed in the tissue procedure table in the males and females of the control and high dose groups (groups 1 and 4) sacrificed at the end of the treatment period and for all animals that die or are sacrificed prematurely,
- the liver in the males and females from the low- and intermediate-dose groups,
- the kidneys and the forestomach in the males from the low- and intermediate-dose groups,
- all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment
period,
- all females sacrificed because of no evidence of mating or no delivery to investigate possible causes.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Postmortem examinations (offspring):
Pups found dead, prematurely sacrificed and pups sacrificed on day 5 post-partum were carefully examined externally for gross external
abnormalities. A macroscopic examination was also performed. No tissues were preserved.
Reproductive indices:
. pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

. post-implantation loss (calculated manually):
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantations

. mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

. fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

. gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females

Offspring viability indices:
. live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

. viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males
Soft feces were noted in a few test item-treated males for 1 or a few days during the first half of the treatment period, and lasted up to 12 days in one high-dose animal (U25166) which showed at the same time a lower body weight gain than the others.
Aggressive behavior was observed in all test item-treated groups at the end of the treatment period from approximately day 61. Since it was seen in controls with a similar incidence, it was not considered to be an effect of the test item treatment. The other clinical signs recorded during the study had similar incidences as the controls and/or are commonly seen in this strain of rat. They were not related to the treatment with the test item.

Females
No relevant clinical signs were recorded for females. Those noted for the test item-treated animals were seen with similar incidences in control animals, were of very low incidence and/or are commonly seen in these species and strain.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No unscheduled deaths occurred in any male group during the study.
Two females (U25241 and U25250) from the high-dose group were prematurely sacrificed during the lactation period [day 2 or 3 post-partum (p.p.), respectively] following the death of their litter (cannibalism of the litter was recorded for female U25241). The first female had no clinical signs before premature sacrifice, while the second experienced piloerection and pallor of the eyes from day 1 p.p.. The death of the litters is discussed below (see § Observation of the pups after birth). At macroscopic post-mortem and microscopic examinations, these high-dose females had incidental findings common in the rats of this strain and age
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males
Minimally higher, but non-statistically significantly, mean body weight gains recorded in group 2 at each interval when compared to the control males led to a statistically significant difference in mean body weight gain over the whole treatment period. Since this was noted in the low-dose male group only and since the final mean body weight was not significantly different from that of the control group (625 g in low-dose animals vs. 590 g in controls), it was considered not to be toxicologically relevant.

Females
Groups 2 and 4 females had a mean body weight gain lower than controls over the pre-mating period, reaching a statistical significant level in group 2. No dose-relationship was observed and mean body weight gain recorded in the mid-dose group was similar to that of the control group, therefore this was considered not to be related to treatment with the test item.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males
Slightly higher mean food consumption was noted in the low- and high-dose groups towards the end of the treatment period. As the amplitude was low and no dose-relationship was observed, this was considered not to be test item treatment-related.

Females
Mean food consumption was comparable in all groups throughout the pre-mating, gestation and lactation periods, with the exception of group 2 females during lactation which had mean food consumption higher than controls. However, this was due to one female (U25221) for which a food consumption of 126 g/day between day 1 and 5 p.p. was recorded.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significantly higher absolute and/or relative kidney and liver weights were noted in the males given 200 or 500 mg/kg/day and in the males and females given 1000 mg/kg/day. These differences were dose-related and correlated to microscopic findings. Consequently, they were considered to be related to test item.
The other organ weight changes were considered not to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent between the sexes.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Unscheduled deaths
No treatment-related findings were noted. Incidental findings were seen in the submitted organs.

Terminal sacrifice
Treatment-related changes were noted in the liver, kidney and forestomach:

. liver
Minimal to slight dose-related hepatocellular hypertrophy was recorded in the males given 200 mg/kg/day and in males and females given 500 or 1000 mg/kg/day (see table below). This correlated with increased liver weights.
In addition, minimal altered cell foci were recorded in males given 500 or 1000 mg/kg/day. It consisted of clear cell focus or foci, associated with basophilic cell foci in one of the males given 1000 mg/kg/day. These findings were considered to be adverse since these changes could be consistent with pre-neoplastic lesions.
In one female given 200 mg/kg/day, a single altered cell focus with clear cells was observed. In view of the absence of this finding in the females given 500 or 1000 mg/kg/day and of the low incidence and severity of this change in this sex, it was considered to be fortuitous rather than to be related to test item.
The significance of the multifocal minimal vacuoles in one male given 1000 mg/kg/day is remained unknown, in view of the low incidence and severity. It was considered to be consistent with fat storage.

. kidney
Minimal to moderate, partially dose-related hyaline droplets were found in the tubular epithelium of the kidneys from males given 200, 500 or 1000 mg/kg/day (see table below). These droplets were brightly eosinophilic granules located in the cytoplasm of proximal tubular cells. This correlated to increased kidney weights.
Slightly increased, in incidence and severity, tubular basophilia was seen in males given 200, 500 or 1000 mg/kg/day. Although this was poorly dose-related and not seen in females, a relationship to the test item cannot be excluded.

. forestomach
Minimal hyperplasia of squamous cells, together with minimal hyperkeratosis, was seen in two males given 500 mg/kg/day and in four males given 1000 mg/kg/day. This was considered to be related to test item.
One group 2 female (U25226) was not pregnant following mating and had blockage in metestrous then in diestrous as seen in reproductive data paragraph. Histologically, follicles and corpora lutea were present, with moderate single cell necrosis in all corpora lutea (consistent with regression).
All other microscopic findings noted in treated animals were considered as incidental changes, as they occurred also in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating
There was no effect of test item treatment on mating at any dose-level.
One pair of animals did not mate in group 2 (female U25227 and male U25147). This was due to the female that stayed in metestrous then in diestrous for the first 15 days of the pairing period. On the 15th day the male was changed for another male from the same dose group that had already mated and on the following day the female’s cycle continued into proestrous and mating occurred shortly after.

Mean pre-coital time was minimally higher in groups given 200 and or 1000 mg/kg/day than in the control group. This was due to two females (U25226 and U25227) in group 2 and one female (U25249) in group 4 which were blocked in metestrous then in diestrous for several days. As this finding was not noted with a dose-relationship and may naturally happen in this species, it was considered to be of fortuitous origin.

Fertility
The fertility index was unaffected by the test item treatment.
One group 2 female (U25226) was not pregnant following mating, in addition to the longer pre-coital time as seen in the previous paragraph (blockage in metestrous then in diestrous). This was not ascribed to the test item treatment since it happened in a single female in the low-dose group.

Delivery data
All pregnant females gave birth to live pups. The minimal differences observed in mean number of pups per litter between the control group and the groups given the test item were due to one control female (U25215) which delivered four pups only.
No difficulties at delivery were observed in any female. The length of gestation in test item-treated females was similar to the control animals.
No effects of test item treatment were observed on pre- and post-implantation loss.
The increase of mean post-implantation loss noted in the intermediate-dose group was due to one female (U25236) which had 56.3% post-implantation loss.
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No adverse effect
Key result
Dose descriptor:
NOEL
Remarks:
reproductive performance (mating and fertility)
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect
Dose descriptor:
NOEL
Remarks:
parental toxicity
Sex:
male
Remarks on result:
not determinable
Dose descriptor:
NOEL
Remarks:
parental toxicity
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
With the exception of the pups which were prematurely sacrificed, few pups had clinical signs. Individuals at all dose-levels had emaciation and/or pallor however there were several pups in the control group observed to be emaciated so there was no indication that this was related to treatment of the dams with the test item.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
In the group treated at 1000 mg/kg/day, 58.6% of the live born pups (92 out of 157) were found dead or cannibalized or were prematurely sacrificed because of poor clinical condition by post-natal day 5. The majority of the deaths occurred on post-natal days 1 and 2. Deaths occurred in all litters and in two litters no pups remained alive on post-natal day 5. For one of these litters the female (U25250) was observed to have pilo-erection and pallor of the eyes and all the pups died. For the other litter, the female had no clinical signs.
At 500 mg/kg/day, mean implantation loss (i.e. fetal/pup loss) up to weighing of the pups on PND 1 was slightly increased. This value includes live born pups subsequently found dead or cannibalized as well as embryo-fetal losses. Mean values were particularly affected by one dam (No. V25236) with marked implantation loss. However, in a screening study with a limited group size, an association with treatment cannot be excluded.
At 200 mg/kg/day, mean implantation loss (fetal/pup loss) and pup mortality up to PND 5 was similar to that of the control group.
Found dead or cannibalized pups generally did not have clinical signs. Signs recorded in the few prematurely sacrificed pups from the 500 and 100 mg/kg/day groups (hypoactivity, loud breathing and coldness) were considered to be not particularly related to the test item treatment but rather indicators of imminent death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, there was a statistically significant reduction in mean body weight gain between PND 1 and 5, when compared to the control pups. This impacted the mean final body weight on PND 5 in this group (-15% when compared with controls). This effect was considered to be treatment-related.
The slightly lower mean body weight gain and PND 5 body weights recorded at 200 and 500 mg/kg/day compared with control pups were considered not to be an effect of treatment of the dams but to be related to the slightly higher mean litter size and thus to the increased intra-litter competition in these groups. This conclusion is supported by the calculated values for mean litter weight on PND 5 which do not indicate an effect of treatment of the dams at 200 and 500 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no relevant external abnormalities in the pups from any groups.
A large number of the high-dose pups were autolyzed at the time of necropsy. No other post mortem macroscopic abnormalities were ascribed to treatment of the parents with the test item at any dose-level since the findings were isolated (two high-dose pups had misshapen kidneys or reddish focus on a kidney and one mid-dose pup had dilated renal pelvis and ureter).
Histopathological findings:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Conclusions:
In all dose groups, there were no marked clinical signs and no effects on body weight or food consumption in males or females.None of the mating or fertility parameters were adversely affected by treatment. At 1000 mg/kg/day, there was a marked increase in pup mortality and body weight gain of the surviving pups was significantly reduced between PND 1 and 5. Increased kidney and liver weights were noted in the parent males and females. At microscopic examination, adverse altered liver cell foci and minimal hyperplasia of squamous cells of the forestomach together with hyperkeratosis were seen in some males. Hepatocellular hypertrophy was recorded in both males and females and hyaline droplets along with basophilic tubules were found in male kidneys. At 500 mg/kg/day, there was a slight increase in implantation loss (i.e. fetal/pup loss) up to PND 1. Although an association with treatment cannot be excluded, it was considered that the increase was slight and, in a screening study with a limited number of animals per group, this effect cannot be definitely attributed to treatment.Increased kidney and liver weights were noted only in the parent males and, at microscopic examination, adverse altered liver cell foci and minimal hyperplasia and hyperkeratosis of the forestomach were seen in some males. Hepatocellular hypertrophy was recorded in both males and females and hyaline droplets along with basophilic tubules were found in male kidneys. At 200 mg/kg/day, mean implantation loss (fetal/pup loss), pup mortality and body weight gain of the pups up to PND 5were comparable with that of the control group. Increased kidney and liver weights were noted in the males and, at microscopic examination, hepatocellular hypertrophy and hyaline droplets along with basophilic tubules in the kidneys were found in males only.
Executive summary:

The potential toxic effects of Hexylene glycol 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.). 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, Hexylene glycol (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 4 p.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 macroscopic post-mortem findings 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 Hexylene glycol 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.

 

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 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

A GLP study that was conducted according to OECD test guideline 414 identified a NOAEL of 300 mg/kg body weight/day (the middle dose tested) for both maternal and foetal effects following oral administration of hexylene glycol to female rats during gestation days 6 to 15. No teratogenic effects were observed up to 1000 mg/kg/day.

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
Reference
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:
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 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)
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
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
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
Developmental effects observed:
not specified
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

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
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 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. certificate)
Type of method:
in vivo
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals 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

A high rate of pup’s mortality was observed in a reproduction/developmental toxicity screening test (OECD TG no. 421) at the dose level of 1000 mg/kg/day. According to the mecanistic studies performed, this effect don't seem to be a direct effect on the pups but seems secondary to the maternal toxicity. Accordingly, no classification is warranted for reproductive and developmental toxicity according to the criteria for classification of Regulation (EC) No 1272/2008.