Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

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

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Hexylene glycol
- Substance type: mono-constituant
- Physical state: colorless solution
- Analytical purity: 99.95%
- Purity test date: 22 July 2008
- Lot/batch No.: A1TX6K010101
- Expiration dates of the lot/batch: first and second receipts: 22 July 2010; third receipt: 31 July 2010
- Storage conditions of test material: at room temperature, protected from humidity and heat, in a dry, cool and well-ventilated place.

Test animals

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.

Administration / exposure

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.
Doses / concentrations
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.

Examinations

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.
Estrous 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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

Reproductive function: estrous 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.

Effect levels (P0)

open allclose all
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

Results: F1 generation

General toxicity (F1)

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

Effect levels (F1)

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

Overall reproductive toxicity

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

Applicant's summary and conclusion

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.