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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
2-methylpentane-2,4-diol
Cas Number:
107-41-5
IUPAC Name:
2-methylpentane-2,4-diol
Test material form:
liquid
Details on test material:
Description: Colorless
Storage: At room temperature, protected from humidity and heat

Test animals

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

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

IN-LIFE DATES: From: 28 November 2019 To: 9 January 2020

Administration / exposure

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

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

Examinations

Maternal examinations:
MORBIDITY AND MORTALITY: Yes
- Time schedule: Each animal, once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

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

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

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

WATER CONSUMPTION: No

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

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

Parameters calculated for each group are given in section "Any other information on materials and methods incl. tables".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs were observed during the study at any dose level.
All clinical signs recorded in females during the study were considered to be unrelated to the test item treatment as they were present before treatment initiation, and/or both in control and test item-treated animals and/or were observed in isolated animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths occurred at any dose level.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and mean body weight changes recorded in control and test item treated animals are summarized in Table 1 in section "Any other information on results incl. tables".
At 600 mg/kg bw/day, when compared to controls, slight mean lower body weight gain was recorded between Days 6 and 29 p.c. (+360 g vs. +424 g in controls, not statistically significant). The difference was mainly due to an initial lower mean body weight gain recorded between Days 6 and 12 p.c. This difference coincided with effect on food intake (see respectively section). Since the difference in mean body weight changes was not statistically significant and did not impacted terminal body weight recorded on Day 29 p.c., it was not considered as adverse.
At 80 and 250 mg/kg bw/day, no effects on mean body weight or mean body weight change were noted.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption values recorded during the study are summarized in Table 2 in section "Any other information on results incl. tables".
At 600 mg/kg bw/day and when compared with controls, the mean food consumption was nearly 20% lower than that of the control group between Days 9 and 12 p.c..(with p<0.01 during the first interval). From Day 12 p.c. onwards, mean food consumption returned towards control values. Therefore this finding was considered to be non-adverse.
Food consumption of pregnant females given 80 or 250 mg/kg bw/day was not affected.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no effects on mean gravid uterus weight, carcass weight or net body weight change.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared with controls, there were no test item treatment-related findings in animals euthanized as scheduled, on Day 29 p.c.. The few findings recorded are commonly observed in this species and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no animals that showed signs of premature delivery.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no significant effect on the mean percentage of pre- and post-implementation loss (see Table 3 in section "Any other information on results incl. tables").
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
There was a significant (p<0.05) lower mean number of late resorptions in the low dose group, without dose response relationship. There was no significant effect on the mean number of early resorptions (see Table 3 in section "Any other information on results incl. tables").
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of dead fetuses was 0.0 in the low, mid and high dose groups, which was significant lower (p<0.01) than the 3.3% in the control group. This was within the historical control data (see Table 3 in section "Any other information on results incl. tables").
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At hysterectomy on Day 29 p.c., 23/25, 22/25, 24/25 and 21/25 females were pregnant with live fetuses in the groups treated at 0, 80, 250 and 600 mg/kg bw/day, respectively
Other effects:
not examined
Details on maternal toxic effects:
There were no effects on hysterectomy parameters (mean number of corpora lutea, implantation sites and live fetuses; pre- and post-implantation loss).

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: Based on absence of adverse effects
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Transient effect in maternal body weight gain and food consumption were observed at 600 mg/kg bw/day (actual dose received). These effects were considered not adverse.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant effects on mean fetal body weight (see Table 4 in section "Any other information on results incl. tables").
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no effects on sex ratio (percentages of male fetuses) (see Table 4 in section "Any other information on results incl. tables").
Changes in litter size and weights:
not examined
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
VARIATIONS
There were no variations at external examination of the fetuses. At 80 mg/kg bw/day, one dam had one fetus with paw hyperflexion and malrotated paw.This variation was considered to be incidental and not related to the test item treatment as it was noted in a single fetus from the low dose group.

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

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

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

MALFORMATIONS
The incidences of fetuses and litters with soft tissues malformations are presented in Table 5 of section "Any other information on results incl. tables".
Several malformations were observed in the heart of three fetuses from 2 litters treated at 80 mg/kg/day (ventricular septum defect fetus #7, litter Q31083; thick ventricular septum fetuses #9 and #10, litter Q31066) and in two fetuses from 2 litters given 600 mg/kg/day (ventricular and atrioventricular septum defects with fetus #7, litter Q31118, associated with enlarged ventricular chamber classified as a variation; cardiomegaly in fetus #6, litter Q31119). The presence of hydropericardium in one fetus (litter Q31072) at 80 mg/kg/day was unrelated to the test item since it could be observed in one fetus from the control group.
Findings observed in the kidneys, vessels and ureters at 600 mg/kg/day were not considered to be test item-related in absence of dose relationship (no malformations among fetuses from the mid dose group; the retroesophageal arch of vessels in one fetus #8, litter Q31075 was isolated), the low incidences (dilated aortic arch: fetus #07, litter Q31118; narrow aortic arch, dilated pulmonary trunk and transposition of great vessels in a single fetus #6, litter Q31119; short ureters in two fetuses #5 and 10, litter Q31120); and/or occasionally observed in controls (dilated aortic arch in fetus #2, litter Q31052).
Therefore, there were no test item treatment-related malformations at the fetal soft tissue examination, the lack of dose response of the above-mentioned malformations.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
PLACENTA WEIGHT
The ratio fetal body weight/ placenta in the low and high dose groups was significantly lower (p<0.01 and P<0.05, respectively), however there was no dose response relationship and there were no significant effects on placenta weight (see Table 4 in section "Any other information on results incl. tables").
Details on embryotoxic / teratogenic effects:
A summary of external, soft tissues and skeletal malformations is presented in Table 8 of section "Any other information on results incl. tables".

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on absence of adverse effects
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Remarks on result:
other: Statistically significant increased incidence of delayed ossifications was observed at 600 mg/kg bw/day. The effects were limited to few structures, and classified as variations and not adverse.

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: supernumerary rib
skeletal: pelvic girdle
other: hyoid bone
Description (incidence and severity):
Increased incidences of unossification or incomplete ossifications of the skeleton at 250 and/or 600 mg/kg bw/day was observed among few bone structures (hyoid; 5th sternebrae; 13th supernumerary rib, pubis). The delayed ossification was considered to be test item-related, considering that the differences recorded at 600 mg/kg bw/day were often statistically significant. However, the effects were considered non-adverse.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

CHEMICAL ANALYSIS OF THE DOSE FORMULATION


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


No test item was detected in the control dose formulation.


 


BODY WEIGHTS AND BODY WEIGHT GAINS


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


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











































































































































Dose level (mg/kg bw/day)



0



80



250



600



Body weight



 



 



 



 



Day 6 p.c.



3.58



3.59



3.56



3.60



Day 9 p.c.



3.64



3.64



3.60



3.60



Day 12 p.c.



3.66



3.69



3.64



3.61



Day 15 p.c.



3.73



3.75



3.70



3.68



Day 19 p.c.



3.77



3.78



3.71



3.73



Day 24 p.c.



3.86



3.86



3.82



3.82



Day 29 p.c.



4.00



3.99



3.97



3.96



% from controls



-



0



-1



-1



Body weight change



 



 



 



 



Days 6 - 9 p.c.



+58



+50



+43



0



Days 9 - 12 p.c.



+20



+41



+35



+12



Days 12 - 15 p.c.



+78



+60



+59



+65



Days 15 - 19 p.c.



+36



+38



+19



+57



Days 19 - 24 p.c.



+93



+74



+103



+90



Days 24 - 29 p.c.



+140



+134



+148



+136



Days 6 - 29 p.c.



+424



+396



+407



+360



% from controls



-



-7



-4



-15



-: not applicable.


No statistically significant differences versus controls.


Rounded values with two digits


Bold: test item-related difference


 


FOOD CONSUMPTION


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


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























































Dose level (mg/kg bw/day)



0



80



250



600



Days 6 - 9 p.c.



164


(-)



156


(-5%)



153


(-7%)



131**


(-20%)



Days 9 - 12 p.c.



150


(-)



148


(-1%)



143


(-5%)



123


(-18%)



Days 12 - 15 p.c.



126


(-)



115


(-9%)



113


(-10%)



113


(-10%)



Days 15 - 19 p.c.



132


(-)



130


(-2%)



124


(-6%)



142


(+8%)



Days 19 - 24 p.c.



132


(-)



128


(-3%)



132


(0%)



140


(+6%)



Days 24 - 29 p.c.



117


(-)



110


(-6%)



118


(+1%)



123


(+5%)



Statistically significant : **: p<0.01.


Bold: test item-related difference


In brackets: variation from controls


-: not applicable


 


HYSTERECTOMY DATA


Hysterectomy data are presented in Table 3.


Table 3: Hysterectomy Data






























































































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Number of females with live fetuses at termination



23



22



24



21



159+42



Mean number of corpora lutea per animal



11.7



11.9



11.6



11.9



[11.4-12.8]



Mean number of implantation sites per animal



10.4



10.4



10.4



10.1



[9.5-11.4]



Mean percentage of pre-implantation loss (%)



10.0



11.6



10.2



14.9



[10.5-17.9]



Mean number of live fetuses per animal



8.9



9.8**



9.5



8.9



[8.5-10.6]



Dead fetuses (%)



3.3



0.0**



0.0**



0.0**



[0.00-1.00]



Mean number of implantation scars



0.0



0.0



0.0



0.0



/



Mean number of early resorptions



0.3



0.4



0.5



0.7



/



Mean number of late resorptions



0.8



0.3*



0.5



0.6



/



Mean percentage of post-implantation loss (%)



13.2



6.3



8.7



11.8



[3.9-20.5]



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


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


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


/: not present in HCD.


 


FETAL BODY WEIGHT, SEX RATIO, PLACENTAL WEIGHT


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


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






























































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Fetal body weight



 



 



 



 



 



- male fetuses



43.6



39.4*



41.4



40.2



[34.5-39.4]



- female fetuses



41.1



40.3



40.7



40.4



[33.9-38.5]



Mean fetal placenta weight



5.45



5.67



5.68



5.77



[3.87-8.21]a



Ratio fetal BW/ placenta



7.88



7.07**



7.37



7.22*



/



Mean percentage of male fetuses



47.0



52.4



45.3



44.9



[47.4-58.3]



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


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


a: only HCD with n = 2 studies.


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


/: not present in HCD.


 


FETAL SOFT TISSUE EXAMINATIONS


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


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














































































































































































































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Number of litters



22



22



24



21



159+42



Number of fetuses



205



215



227



186



1558+391



Heart



 



 



 



 



 



. ventricular septum defect, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



4.8 (0.5)



/



. ventricular septum thick, L (F)



0.0 (0.0)



4.5 (0.9)



0.0 (0.0)



0.0 (0.0)



/



. hydropericardium, L (F)



4.5 (0.5)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



. atrioventricular septum defect, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



. atrial septum defect, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



. cardiomegaly, L (F)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



/



Dose level (mg/kg bw/day)



0



80



250



600



HCD



Number of litters



22



22



24



21



159+42



Number of fetuses



205



215



227



186



1558+391



Kidneys



 



 



 



 



 



. malpositioned kidney, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



4.8 (1.1)



/



Vessels



 



 



 



 



 



. dilated aortic arch, L (F)



4.5 (0.5)



4.5 (0.5)



0.0 (0.0)



4.8 (0.5)



0.0-4.0 (0.0-0.4)



. narrowed aortic arch, L (F)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



/



. dilated pulmonary trunk, L (F)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



0.0-5.3 (0.0-0.5)



. transposition of great vessels, L (F)



0.0 (0.0)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



/



. retroesophageal aortic arch, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



Ureter



 



 



 



 



 



. short ureter, L (F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



4.8 (1.1)



/



Litters affected, n (%)



3 (13.6)



4 (18.2)



1 (4.2)



3 (14.3)



8 (5.0)



Fetus affected, n (%)



4 (2.0)



6 (2.8)



1 (0.4)



5 (2.7)



8 (0.5)



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


/: not present in HCD.


 


FETAL SKELETAL VARIATIONS


The incidences of skeletal variations are presented in Table 6.


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














































































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Dams with live fetuses, n



22



22



24



21



159+42



Live fetuses, n



205



215



227



186



1558+391



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



4.5
(0.5)



18.2
(2.3)



33.3*
(6.2#)



28.6*
(4.8**)



0.0-13.5
(0.0-2.2)



. hyoid: unossified centrum, L(F)



9.1
(1.0)



13.6
(2.3)



12.5
(3.1)



19.0
(7.5**)



0.0-5.6
(0.0-0.5)



. hyoid: unossified arch, L(F)



0.0
(0.0)



4.5
(0.5)



16.7
(2.2)



14.3
(5.4#)



0.0-4.2
(0.0-0.4)



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



63.6
(23.4)



72.7 (18.1)



45.8
(16.7)



85.7
(38.2**)



5.6-52.6
(0.6-9.9)



. supernumerary 13th rib, L(F)



100.0 (42.4)



86.4
(48.4)



91.7
(49.8)



95.2
(53.8*)



94.4-100
(54.8-81.2)



. pubis: incomplete ossification L(F)



0.0
(0.0)



18.2 (2.8*)



20.8
(4.0**)



14.3
(3.8**)



0-27.8
(0.0-3.7)



n: number.


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


 


FETAL SKELETAL MALFORMATIONS


The incidences of skeletal malformations are presented in Table 7.


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






























































































































































































Dose level (mg/kg bw/day)



0



80



250



600



HCD



Dams with live fetuses, n



22



22



24



21



159+42



Live fetuses, n



205



215



227



186



1558+391



Head-skull



 



 



 



 



 



. parietal absent, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



/



. interparietal absent, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



0.0-18.2 (0.0-2.1)



Head others



 



 



 



 



 



. palate: split, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



0.0-2.7 (0.0-0.3)



Lumbar vertebrae



 



 



 



 



 



. absent, L(F)



9.1 (1.0)



0.0 (0.0)



4.2 (0.4)



4.8 (0. 5)



0.0 (0.0)



Caudal vertebrae



 



 



 



 



 



. misaligned, L(F)



0.0 (0.0)



4.5 (0.5)



4.2 (0.4)



0.0 (0.0)



0.0 (0.0)



. fused, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



0.0-8.3 (0.0-0.9)



. absent, L(F)



0.0 (0.0)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



0.0-4.2 (0.0-0.4)



Dose level (mg/kg bw/day)



0



80



250



600



HCD



Dams with live fetuses, n



22



22



24



21



159+42



Live fetuses, n



205



215



227



186



1558+391



Sternebrae



 



 



 



 



 



. fused, L(F)



4.5 (0.5)



13.6 (1.4)



0.0 (0.0)



0.0 (0.0)



5.6-27.3 (0.5-3.6)



Rib



 



 



 



 



 



. fused, L(F)



4.5 (0.5)



0.0 (0.0)



0.0 (0.0)



4.8 (0.5)



0.0-8.3 (0.0-0.9)



Litters affected, n (%)



4 (18.2)



4 (18.2)



2 (8.3)



2 (9.5)



46 (28.9)-19 (45.2)



Fetus affected, n (%)



4 (2.0)



5 (2.3)



2 (0.9)



2 (1.1)



56 (3.6)-27 (6.9)



n: number.


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


 


SUMMARY FETAL INCIDENCES OF EXTERNAL, SOFT TISSUE AND SKELETAL MALFORMATIONS


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


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
















































Group



1



2



3



4



Dose-level (mg/kg bw/day)



0



80



250



600



Pregnant females with live fetuses, n



22



22



24



21



Litter affected, n (%)



8 (36.4)



8 (36.4)



3 (12.5)



4 (19.0)



Live fetuses, n



205



215



227



186



Fetus affected n (%)



9 (4.4)



9 (4.2)



3 (1.3)



6 (3.2)



 

Applicant's summary and conclusion

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

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

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


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


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


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


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


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


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


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


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


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


Based on the results obtained in this study:


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


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

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