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Toxicity to reproduction: other studies

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
General information on test item
- Name : Hexylene glycol
- CAS number : 107-41-5
- Molecular weight : 118.1754

Non radiolabelled test item
-Name : Hexylene glycol
- Description: colorless solution
- Storage conditions: at room temperature and protected from humidity and heat, in a dry, cool and well ventilated place
- Container: glass container

- Batch number: A1TX6K010101
- Date of receipt: 07 April 2010
- Analysis date: 25 July 2008
- Purity: 99.95%
- Reanalysis date: 25 April 2013
- Purity: 99.88%
- Expiry date: 25 April 2015

- Batch number: A20CEP010101
- Date of receipt: 15 May 2013
- Analysis date: 25 April 2013
- Purity: 99.88%
- Expiry date: 25 April 2015

Radiolabelled test item
-Name: [14C]-Hexylene glycol
- Batch number: CFQ41689
- Description: colorless liquid
- Container: 2 mL supplied in 1 operculated glass vial, containing 74 MBq at total (1 mCi/mL)
- Specific activity, determined by gravimetric analysis/mass spectrometry: 67 mCi/mmol (2.48 GBq/mmol)
- Molecular weight, at given specific activity: 120.3
- Radiochemical purity: 99.6%
- Date of analysis: 21 January 2013
- Storage conditions: at +4°C and protected from humidity in a dry, cool and ventilated place
- Handling conditions: usual conditions for radioactive compounds
- Container: glass container into plastic container
- Date of receipt: 20 March 2013

Test animals

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

Administration / exposure

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
Doses / concentrations
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.

Results and discussion

Effect levels

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.

Observed effects

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.