Cyclohexanemethanol, 4-(1-methylethyl)-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 414 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 01-03 December 2015/ signed on 15 February 2016)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cis-4-(isopropyl)cyclohexanemethanol

EC Number:

237-539-8

EC Name:

cis-4-(isopropyl)cyclohexanemethanol

Cas Number:

13828-37-0

Molecular formula:

C10H20O

IUPAC Name:

cis-(4-propan-2-ylcyclohexyl)methanol

Constituent 2

Reference substance name:

trans-(4-propan-2-ylcyclohexyl)methanol

Cas Number:

13674-19-6

Molecular formula:

C10H20O

IUPAC Name:

trans-(4-propan-2-ylcyclohexyl)methanol

Test material form:

liquid

Details on test material:

- Physical state: colourless liquid
- Storage condition of test material: At ambient temperature and protected from light

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1002451619
- Physical state: Colourless liquid
- Expiration date of the lot/batch: 1 January 2017
- Purity test date: 24 November 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature, protected from light

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 71 days old
- Weight at study initiation: 220-295g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female; Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed polypropylene cages were used during pairing.
- Diet: SDS VRF1 Certified pelleted diet. ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days before commencement of pairing

DETAILS OF FOOD AND WATER QUALITY:
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated; minimum of 15 air changes per hour
- Photoperiod: 12 hours light : 12 hours dark

IN-LIFE DATES: From: 8 June 2016 To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: Starting with the lowest concentration, the required amount of test item was placed into a suitable container to which 50% of the final volume of vehicle was added. This was magnetically stirred until all of the test item was uniformly mixed and then made up to the required volume. This was then placed on a high shear homogeniser until homogenous.
- Frequency of preparation: Weekly, and may be prepared in advance of the first day of dosing.
- Storage of formulation: Refrigerated (nominally 2-8 °C).
- The homogeneity and stability of formulations during storage was determined as part of another study, Huntingdon Life Sciences Report No. HIK0016. These investigations demonstrated that formulations in the concentration range of 1 and 250 mg/mL were homogenous and stable for 15 days following refrigerated storage (2-8 ˚C) and for 24 hours following ambient storage.

VEHICLE
- Concentration in vehicle: 7.5, 25 and 75 mg/L
- Amount of vehicle (if gavage): 4 mL/kg bw/day

DOSE VOLUME: 4 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Homogeneity and stability of the test material in the vehicle in the concentration range of 1 and 250 mg/mL had been demonstrated over a period of up to 15 days following refrigerated storage (2-8 ˚C) and for 24 hours following ambient storage in Huntingdon Life Sciences Report No. HIK0016.
Achieved concentration: Samples of each formulation prepared for administration on Day 6 (Phase 1) and Day 19 (Phase 2) after mating were analyzed for achieved concentration of the test item.

Results: The mean concentrations of test substance in test formulations analyzed during the study were within 4% of the nominal concentration, confirming the accuracy of formulation preparation. Differences from mean values were within 1% confirming precise analysis. Procedural recoveries were within 1 % of nominal confirming continued accuracy of the analytical method.

Details on mating procedure:

- Impregnation procedure: co-housed
- If co-housed:
- M/F ratio per cage: 1:1 with identified stock males
- Proof of pregnancy: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm referred to as Day 0 of pregnancy
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals; males were approximately 25-30 weeks at the time of mating.

Duration of treatment / exposure:

Females: Day 6 to 19 after mating, inclusive

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

Animals were killed on Day 20 after mating.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 females/dose
Phase 1: 6 females/dose
Phase 2: 14 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels used in this study, to investigate for effects on embryo-fetal development (OECD414) (0, 30, 100 and 300 mg/kg bw/day), were selected in conjunction with the Sponsor based on the results of a 14-day preliminary study (Huntingdon Life Sciences Study No. HIK0015), a 28-day repeat dose toxicity study (Huntingdon Life Sciences Study No. HIK0016), and a reproductive/developmental toxicity screening study (OECD 421; Huntingdon Life Sciences Study No. HIK0019), all of which were performed via oral gavage administration.
In the 14-day study, dose levels of 50, 150, 300, 500, 750 and 1000 mg/kg bw/day were investigated. The doses of 750 and 1000 mg/kg bw/day were found to exceed the maximum tolerated dose, with a marked decline in clinical condition observed after three or four doses, requiring premature sacrifice of the animals, and these doses were considered unsuitable for further investigation. The dose levels up to and including 500 mg/kg bw/day were well tolerated for the full 14-day period with no unscheduled deaths. Treatment related effects were limited to a suggestion of slightly low weight gain in females receiving 500 mg/kg bw/day and slightly increased liver weight in females at 300 or 500 mg/kg bw/day.
In the 28-day study treatment with the test item at 30, 100 or 300 mg/kg bw/day was well tolerated in females with adverse effects in the liver noted at 300 mg/kg bw/day.
In the OECD 421 study, dose levels of 50, 150 and 300 mg/kg bw/day were well tolerated during the 2-week pre-pairing period and during gestation. Test item-related effects during these periods were limited to a reduction in mean body weight gain during Days 17-20 of gestation for females given 300 mg/kg bw/day. One female given 300 mg/kg bw/day was killed for reasons of animal welfare on Day 20 of gestation following a marked decline in clinical condition, however this premature death was considered to be of uncertain relationship to treatment in view of the isolated incidence of the clinical signs and macroscopic abnormalities detected for this animal, and no factors contributing to death were determined following histopathological evaluation of the retained tissues.
Based on the information available, a dose level of 300 mg/kg/day was considered appropriate to use as the high dose level in Phase 1 of this embryo-fetal development study; higher dose levels were anticipated to cause excessive reduction in weight gain of the females and concomitant confounding effects. Dose levels of 30 and 100 mg/kg/day were selected as the low and intermediate doses to establish a suitable dose response relationship for any treatment-related changes and to give an approximate 3-fold interval between doses.
As no adverse effects of treatment were observed at 300 mg/kg bw/day in Phase 1, an additional 14 females per group were used to form Phase 2 of the study in order to form the full complement of 20 females at each dose level.

- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily at the following times in relation to dose administration: Pre-dose; 1 to 2 hours after completion of dosing; As late as possible in the working day.
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3, 6 and then daily until termination.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 20 after mating; Animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination.
- Necropsy: All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

OTHERS:
Toxicokinetics: Blood samples (nominally 0.5 mL) were collected from animals on Day 6 and Day 19 after mating at the different time points (1, 2, 4, 8 and 24 hours after dosing).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; Gravid uterine weight (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live/dead fetuses: Yes

Fetal examinations:

SACRIFICE:
Method of kill for fetuses: Chilling on a cool plate (approximately 0 °C)

FETAL EXAMINATION AND PROCESSING
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Subjected to a gross internal examination were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

FETAL PATHOLOGY EXAMINATION
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.

Statistics:

See "Any other information on materials and methods incl. tables"

Indices:

Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Historical control data:

See "Attached background material" section

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

The administration of test substance at dose levels up to and including 300 mg/kg bw/day was well tolerated and there were no unscheduled deaths.
There were no test item-related clinical signs apparent at routine physical examination or signs in association with dose administration among females at any dose level investigated.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- During the 24-hour period after the first dose administration, between Day 6 and Day 7 of gestation, females given 300 mg/kg bw/day showed minor but statistically significant mean body weight loss of 2 grams compared to mean weight gain of 2 grams in Controls. Thereafter, mean body weight gain for these females was essentially similar to Control, such that overall mean weight gain from Day 6-20 of gestation was 98% of Control. Mean body weight gain of females given 30 or 100 mg/kg bw/day was unaffected by treatment.
- There was no effect of test substance administration on mean gravid uterine weight or net mean body weight gain at any dose level investigated.
- Body weight change Days 6-20: 109 ± 202, 115 ± 13.4, 111 ± 15.5 and 107 ± 12.3 at 0, 30, 100 and 300 mg/kg bw/day.
- Adjusted body weight change Days 6-20: 26 ± 7.3, 26 ± 10.3, 24 ± 10.4 and 22 ± 8.7 at 0, 30, 100 and 300 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Test substance administration at 300 mg/kg bw/day was associated with slightly low food intake when compared to Controls during Days 6-9 of gestation [19 g/animals/day vs 21 g/animals/day in control group], with statistical significance attained; thereafter, mean food intake for these females was essentially similar to Controls. The transient and slightly reduced food consumption and body weight gain were considered to have likely been caused by the taste of the test item. There was no effect of test substance administration on mean food consumption at 30 or 100 mg/kg bw/day. Full data information are provided in the attached tables of the study report.

Food efficiency:

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:

no effects observed

Description (incidence and severity):

There was no effect of test substance administration on mean gravid uterine weight at any dose level investigated.
- Gravid uterine weight: 84 ± 20.8, 89 ± 10.9, 87 ± 10.4 and 85 ± 9.7 at 0, 30, 100 and 300 mg/kg bw/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related macroscopic abnormalities detected at scheduled termination on Day 20 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

- Corpora lutea (mean): 17.5 ± 2.65, 17.0 ± 2.50, 17.2 ± 2.48 and 17.6 ± 2.37 (/20) at 0, 30, 100 and 300 mg/kg bw/day.
- Implantations (mean): 15.4 ± 2.44, 16.3 ± 2.10; 15 ± 1.85 and 16.2 ± 1.66 (/20) at 0, 30, 100 and 300 mg/kg bw/day.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- Pre-implantation loss (mean): 2.1, 0.7, 1.6 and 1.4 (/20 females) at 0, 30, 100 and 300 mg/kg bw/day.
- Pre-implantation loss (%): 11.2, 4.7, 8.5 and 8.4 at 0, 30, 100 and 300 mg/kg bw/day.
- Post-implantation loss (mean): 1.3, 0.8, 0.9 and 1.1 (/20 females) at 0, 30, 100 and 300 mg/kg bw/day.
- Post-implantation loss (%): 9.6, 4.6, 5.3 and 6.9 at 0, 30, 100 and 300 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

None observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

- Early resorptions (mean): 1.3, 0.8, 0.7 and 1.1 (/20) at 0, 30, 100 and 300 mg/kg bw/day.
- Late resorptions (mean): 0.1, 0.0, 0.2 and 0.0 (/20) at 0, 30, 100 and 300 mg/kg bw/day.
- Total: 1.3, 0.8, 0.9, 1.1 (/20) at 0, 30, 100 and 300 mg/kg bw/day.

Dead fetuses:

no effects observed

Description (incidence and severity):

- Live young males (mean): 7.3 ± 2.31, 7.7 ± 2.62, 8.0 ± 1.93 and 7.0 ± 1.86 at 0, 30, 100 and 300 mg/kg bw/day.
- Live young females (mean): 6.9 ± 2.30, 7.8 ± 2.09, 6.8 ± 2.05 and 8.1 ± 1.61 at 0, 30, 100 and 300 mg/kg bw/day.
- Total Live young (mean): 14.1 ± 3.73, 15.5 ± 2.16, 14.7 ± 1.69 and 15.1 ± 1.96 at 0, 30, 100 and 300 mg/kg bw/day.
- Total Live young (%): 91.6, 95.1, 94.2 and 93.2 at 0, 30, 100 and 300 mg/kg bw/day.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable. Animals were killed on Day 20 after mating.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Not applicable. Animals were killed on Day 20 after mating.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

20/20 females were pregnant at all dose levels.

Other effects:

not specified

Details on maternal toxic effects:

Litter Responses: At scheduled termination on Day 20 after mating, all females were found to be pregnant with live young.
Reproductive Assessment: There was no evidence that maternal treatment with test substance had any adverse effect on litter data, as assessed by the mean numbers of corpora lutea, implantations, resorptions, live young and pre- and post implantation losses, at any dose level investigated.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

early or late resorptions

food consumption and compound intake

gross pathology

maternal abnormalities

mortality

pre and post implantation loss

Remarks on result:

other: No adverse effects were observed.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At scheduled termination on Day 20 of gestation, mean fetal weights in the 300 mg/kg/day group were marginally, but statistically significantly, lower than Control (93% of Control for male fetuses, 94% of Control for female fetuses and 93% of Control for overall mean fetal weight); values were below the lower limit of the Historical Control Data (HCD) range (HCD range 3.68-3.92g for males, 3.52-3.77g for females and 3.59-3.84g for overall fetal weight). Mean placental and litter weights at this dose level were essentially similar to Control due to the slightly higher litter size in this group, and it must be noted that the slightly higher litter size (15.1 vs 14.1 in control group) would contribute towards the slightly lower mean fetal weights and as a consequence the magnitude of difference in mean fetal weights were deemed not to be adverse.
There was no effect of maternal treatment with the test substance on mean placental, litter or fetal weights at 30 or 100 mg/kg/day.
- Male fetal weight: 3.78 ± 0.312, 3.76 ± 0.251, 3.75 ± 0.358 and 3.52* ± 0.307 at 0, 30, 100 and 300 mg/kg bw/day.
- Female fetal weight: 3.60 ± 0.271, 3.59 ± 0.227, 3.57 ± 0.324 and 3.38* ± 0.367 at 0, 30, 100 and 300 mg/kg bw/day.
- Overall fetal weight: 3.69 ± 0.286, 3.67 ± 0.239, 3.66 ± 0.328 and 3.44** ± 0.266 at 0, 30, 100 and 300 mg/kg bw/day.

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): At scheduled termination on Day 20 of gestation, mean fetal weights in the 300 mg/kg/day group were marginally, but statistically significantly, lower than Control (93% of Control for male fetuses, 94% of Control for female fetuses and 93% of Control for overall mean fetal weight); values were below the lower limit of the Historical Control Data (HCD) range (HCD range 3.68-3.92g for males, 3.52-3.77g for females and 3.59-3.84g for overall fetal weight). Mean placental and litter weights at this dose level were essentially similar to Control due to the slightly higher litter size in this group, and it must be noted that the slightly higher litter size would contribute towards the slightly lower mean fetal weights and as a consequence the magnitude of difference in mean fetal weights were deemed not to be adverse.
There was no adverse effect of maternal treatment with the test substance on mean placental, litter or fetal weights at 30 or 100 mg/kg/day.
- Male fetal weight: 3.78 ± 0.312, 3.76 ± 0.251, 3.75 ± 0.358 and 3.52* ± 0.307 at 0, 30, 100 and 300 mg/kg bw/day.
- Female fetal weight: 3.60 ± 0.271, 3.59 ± 0.227, 3.57 ± 0.324 and 3.38* ± 0.367 at 0, 30, 100 and 300 mg/kg bw/day.
- Overall fetal weight: 3.69 ± 0.286, 3.67 ± 0.239, 3.66 ± 0.328 and 3.44** ± 0.266 at 0, 30, 100 and 300 mg/kg bw/day.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- Live young males (mean): 7.3 ± 2.31, 7.7 ± 2.62, 8.0 ± 1.93 and 7.0 ± 1.86 at 0, 30, 100 and 300 mg/kg bw/day.
- Live young females (mean): 6.9 ± 2.30, 7.8 ± 2.09, 6.8 ± 2.05 and 8.1 ± 1.61 at 0, 30, 100 and 300 mg/kg bw/day.
- Total Live young (mean): 14.1 ± 3.73, 15.5 ± 2.16, 14.7 ± 1.69 and 15.1 ± 1.96 at 0, 30, 100 and 300 mg/kg bw/day.
- Total Live young (%): 91.6, 95.1, 94.2 and 93.2 at 0, 30, 100 and 300 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio, as assessed by the percentage of males per litter, was in line with expectations and unaffected by maternal treatment.
- Sex ratio (%): 51.4, 49.2, 54.2 and 46.1 at 0, 30, 100 and 300 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- LItter size (mean): 14.10 ± 3.726, 15.50 ± 2.164, 14.70 ± 1.689 and 15.05 ± 1.959 at 0, 30, 100 and 300 mg/kg bw/day.
- Litter weight (mean): 52.01 ± 14.458, 56.61 ± 6.859, 53.70 ± 6.152 and 51.64 ± 6.729 at 0, 30, 100 and 300 mg/kg bw/day.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

At 300 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of delayed/incomplete ossification/ unossified pelvic bones, metacarpals and metatarsals [Pelvic bones: 6, 10, 13 and 41 / Metacarpals: 0, 0, 2, 40 / Metatarsals: 1, 2, 1 and 7 at 0, 30, 100 and 300 mg/kg bw/day], and an increased fetal incidence of delayed/incomplete ossification/ unossified sacrocaudal vertebrae and sternebrae (other than the 5th and/or 6th sternebrae) [Sacrocaudal vertebrae: 8, 12, 15, 30 / Sternebrae other: 3, 11, 7 and 34 at 0, 30, 100 and 300 mg/kg bw/day] when compared to concurrent control and the HCD range. There was also a slightly increased incidence of delayed/incomplete ossification/ unossified cranial centres [4, 4, 13 and 19 at 0, 30, 100 and 300 mg/kg bw/day] when compared to concurrent Controls, however the fetal and litter incidences of this finding were within the HCD range. There were no test item-related fetal findings apparent in litters derived from females given 30 or 100 mg/kg/day.
It was noted that at 30 and 300 mg/kg/day there was an increased incidence of short supernumerary 14th rib compared to concurrent control [4, 14, 4, 20 at 0, 30, 100 and 300 mg/kg bw/day], with the fetal and litter incidence exceeding the HCD range in the 300 mg/kg/day group. In the absence of a clear dose related response, with no similar increased incidence evident in the 100 mg/kg/day group, this finding was considered to be an isolated incidental finding, with no relationship to maternal treatment with the test substance.

Visceral malformations:

no effects observed

Other effects:

not specified

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

skeletal malformations

Remarks on result:

other: No adverse effects were observed.

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

The objective of this study was to assess the influence of maternal exposure to the test substance on embryo-fetal survival and development in the Crl:CD(SD) rat when administered by oral gavage at doses of 30, 100 or 300 mg/kg/day from implantation on Day 6 of gestation throughout organogenesis and the fetal growth phases of pregnancy to Day 19 of gestation. 

Treatment was well tolerated with no unscheduled deaths. Clinical condition, body weight performance, gravid uterine weight, adjusted body weight gain, food consumption were not adversely affected by the test substance at any dose level investigated, and there were no test item-related macroscopic abnormalities detected at scheduled termination. Test item-related maternal effects were limited to the 300 mg/kg/day group, manifest as minor weight loss following the first dose administration only and slightly low food consumption during Days 6-9 of gestation, however these minor effects were considered not to be adverse at the magnitude observed.

There was no effect of maternal treatment with the test substance on the number of implantations, early or late resorptions, live young, sex ratio or on placental and litter weights at any dose level investigated. At 300 mg/kg/day, mean fetal weights were 6-7% lower than Control although the magnitude of the slight reduction was considered not to be adverse. The lower fetal weight was associated with an increased incidence of delayed/incomplete ossification or unossified pelvic bones, metacarpals, metatarsals, sacrocaudal vertebrae and sternebrae. Changes in the degree of ossification represent a transient stage in fetal development and are therefore considered not to be adverse (Palmer, 1977; Carney and Kimmel, 2007). In this case the differences in mean fetal weight and consequent minor differences in the degree of ossification were considered to be associated with the slightly reduced food consumption and reduced bodyweight gain of the dams during Day 6-9 of gestation.

The transient and slightly reduced food consumption and weight gain were considered to have likely been caused by the taste of the test item. 

Based on the results of this embryo-fetal toxicity study, it was concluded that the dose level of 300 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.

Applicant's summary and conclusion

Conclusions:

Based on the results of this embryo-fetal toxicity study, it was concluded that the dose level of 300 mg/kg bw/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of twenty female rats received test substance, formulated in corn oil, at doses of 30, 100 or 300 mg/kg bw/day by the oral gavage route of administration at a dose volume of 4 mL/kg bw/day, from Day 6 to Day 19 after mating, inclusive. A similarly constituted Control group received the vehicle, at the same volume dose over the same period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. During the study blood samples were taken from selected animals in each group on Days 6 and 19 after mating and were used for toxicokinetic evaluation. Clinical observations, post-dosing observations, body weight and food consumption measurements were performed. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Treatment of female rats with the test substance was well tolerated and there were no unscheduled deaths or test item-related signs observed.

Body weight performance, gravid uterine weight, adjusted body weight gain and food consumption were not adversely affected by the test substance at dose levels up to and including 300 mg/kg/day, and there were no test item-related macroscopic abnormalities detected at scheduled termination.  Test item-related maternal effects were limited to the 300 mg/kg/day group, manifest as minor weight loss following the first dose administration only and slightly low food consumption during Days 6-9 of gestation, however effects were minor and considered not to be adverse at the magnitude observed.

There was no effect of maternal treatment with the test substance on litter data, as assessed by the number of implantations, resorptions, live young, sex ratio and pre- and post-implantation losses.  Placental and litter weights were similar in all groups.  At 300 mg/kg/day, mean male, female and overall fetal weights were 6 and 7% lower than Control respectively and were considered possibly to be related to the slightly reduced food consumption and bodyweight gain of the dams during Days 6-9 of gestation; fetal weights were unaffected by the test substance at 30 or 100 mg/kg/day.  

At 300 mg/kg/day there was an increased fetal and litter incidence of the minor abnormalities of delayed/incomplete ossification/ unossified pelvic bones, metacarpals and metatarsals, and an increased fetal incidence of delayed/incomplete ossification/ unossified sacrocaudal vertebrae and sternebrae (other than 5th/6th) when compared to concurrent control and Historical Control Data (HCD).  There was also a slightly increased incidence of delayed/incomplete ossification/ unossified cranial centres when compared to concurrent Controls, however the fetal and litter incidences of this finding were within the HCD range and were considered possibly to be associated with the slightly reduced food consumption and bodyweight gain of the dams during Days 6-9 of gestation.  There were no test item-related fetal findings apparent in litters derived from females given 30 or 100 mg/kg/day.

Conclusion

Based on the results of this embryo-fetal toxicity study, it was concluded that the dose level of 300 mg/kg/day represented the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and for embryo-fetal survival, growth and development.