Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September - 28 November 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Ditetrahydrofurylpropane
- Lot/batch No.: 6-OH13S
- Analytical purity: 98.8%
- Substance type: Clear colourless liquid
- Physical state: liquid
- Expiration date of the lot/batch: 19 August 2014
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- Specific Gravity: 1.0
- pH: Not indicated
- Stability in vehicle (water): At least 96 hours
- Solubility in vehicle (water): ~0.1%

Test animals

Species:
rat
Strain:
other: Crl:WI(Han).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight range at start of treatment was 316 - 323 gr (males) or 201 - 204 gr (females).
- Fasting period before study: All males were deprived of food before necropsy. Fasting does not adversely affect interpretation of the examined parameters. Females were not fasted before necropsy.
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon cages (MIII type; height 15 cm) with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). Results of analyses for nutrients and contaminants were examined and archived.
- Water (e.g. ad libitum): Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.
- Acclimation period: At least 5 days prior to start of treatment.
- Randomization: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.

Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3 – 22.2°C
- Humidity (%): 43 - 81%
Temporary deviations from the maximum level of relative humidity occurred in the animal room. Evaluation: Laboratory historical data do not indicate an effect of the deviations.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.

IN-LIFE DATES: From: 28 September - 28 November 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.

Storage conditions of formulations: At ambient temperature.

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Method of gavage: using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method (NOTOX Project 484749). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation.
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Some females (1, 2, 1,1 in Group 1,2,3,4 respectively) were not dosed during littering.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 28-day oral toxicity study with Ditetrahydrofurylpropane (NOTOX Project 484766)

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity. On Day 3 of the mating period all animals were observed under Project number 495179 instead of Project number 495227. Clinical signs recorded under the incorrect number were transferred to the correct number afterwards.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.
Inadvertently, the body weight from one female of Group 1 was determined on Lactation Day 2 instead of lactation Day 1. Given that recording of body weight occurred only one day later than intended, this is considered to provide sufficient information on body weight development of this animal during the lactation phase.

FOOD CONSUMPTION
Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY
(Average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
No

CLINICAL CHEMISTRY
No

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
No
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group).
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS
Yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY
All animals were fasted overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised using iso-flurane vapor (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated. Necropsy was conducted on the following days:
Females which delivered on lactation Days 5 and 6. Females which failed to deliver (Group 1; 1 and Group 4;1) on post-coitum Day 26 (females showed evidence of mating). Males: following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The number of former implantation sites and corpora lutea were recorded for all paired females.

Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Identification marks (not processed) , Cervix, Clitoral gland, Coagulation gland, Epididymides <1>, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes <1>, Uterus, Vagina, All gross lesions.

<1> Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) and Milli-Ro water and transferred to formalin after fixation for at least 24 hours.
<2 > In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

ORGAN WEIGHTS
The following organ weights and terminal body weight were recorded from all F0 –males on the scheduled day of necropsy: Epididymides, Testes.

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). Of the selected 5 males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

The preputial gland, seminal vesicles and prostate from all group 1 and 4 males were processed histologically. No histopathology was required on these tissues (except for those males that failed to sire).

HISTOPATHOLOGY
The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4 for staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all males that failed to sire and all females that failed to deliver healthy pups:
Group 1: one male and female that had no offspring.
Group 4: one male and female that had no offspring.
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testis, uterus, and vagina.

One ovary from one female of Group 4 was not available for histopathology. Reason for missing this tissue is that this tissue was not discernable at trimming. Sufficient data was available for evaluation.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5 or 6.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

One pup of a female of Group 3 was not examined macroscopically. Sufficient macroscopy data of pups were available to allow an adequate evaluation of the study results.

HISTOPATHOLOGY / ORGAN WEIGTHS
No
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex. (Ref. 1)
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. (Ref. 3)
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data. (Ref. 2)

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on histopathology findings.

Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 3 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.

Salivation seen after dosing among all animals at 50 and 150 mg/kg was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.

Alopecia and scales observed among females at 50 and 150 mg/kg occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before allowance for body weight was similar between treated and control animals.
Food efficiency:
no effects observed
Description (incidence and severity):
Food consumption after allowance for body weight was similar between treated and control animals.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Testes and epididymides weights and terminal body weights of treated males were similar to those of control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Necropsy did not reveal any toxicologically relevant alterations.

Incidental findings among control and/or treated animals included red-brown discolouration of the epididymides, a yellowish nodule on the preputial glands, greenish nodules on the epididymides, reduced size of the prostate and seminal vesicles, a tan/red/brown focus on the clitoral glands and alopecia. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings.

Further, the spermatogenic staging profiles were normal for all control and high dose males evaluated.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The spermatogenic staging profiles were normal for all control and high dose males evaluated.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant effects on reproductive parameters were noted.

A trend towards an increase in precoital time was observed at 50 and 150 mg/kg. This was due to a higher precoital time of one pair at 50 mg/kg and two pairs at 150 mg/kg. Litter size of these females was normal, and the respective males showed no spermatogenic abnormalities. Moreover, precoital time of other animals of these dose groups was within the range of precoital times observed among control animals.

Also, the number of corpora lutea and implantation sites showed an apparent trend towards a decrease at 150 mg/kg. The lower mean number of implantation sites was due to one female of Group 4; the mean no. of implantations without this animal was similar to other groups, i.e. 12.8 ± 2.5. Control means of these parameters were considered to be slightly high and mean litter size at this dose level was within the normal range for rats of this age and strain. Also given that mating, fertility and conception indices were unaffected by treatment, no toxicological relevance was ascribed to these apparent differences in precoital time and number of corpora lutea and implantation sites.

Gestation index and duration of gestation were unaffected by treatment.

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No parental toxicity was observed up to the highest dose level tested

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental clinical symptoms of pups consisted of a wound on the right paw, a blue spot on the right shoulder or head and small size. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Two pups at 50 mg/kg and three pups at 150 mg/kg were found dead or missing during lactation. The pup missing at 50 mg/kg was most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered to have been unaffected by treatment.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Three pups found dead (one at 50 mg/kg and two at 150 mg/kg) had no milk in the stomach. No further macroscopic abnormalities were noted among the (surviving) pups.
Histopathological findings:
not examined

Details on results (F1)

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a screening study performed according to OECD guideline 421 and GLP principles, no parental, reproduction or developmental toxicity was observed up to the highest dose level tested (150 mg/kg bw/day). Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 150 mg/kg was derived.
Executive summary:

A reproduction/developmental toxicity screening test with Ditetrahydrofurylpropane in rats by oral gavagewas performed according toOECD guideline 421 and GLP principles. In a 28-day oral toxicity study with Ditetrahydrofurylpropane (NOTOX project 484766), a No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established, based on effects in liver and kidneys at 150 and 500 mg/kg. Based on these results, the dose levels for this reproduction/developmental toxicity screening test were selected to be 15, 50 and 150 mg/kg. After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 15, 50 and 150 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41 -55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. The following parameters were evaluated: mortality / viability, clinical signs, body weights, food consumption, reproduction/developmental parameters, observations pups, macroscopy, organ weights, and histopathology. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.Accuracy, homogeneity and stability of formulations were demonstrated by analyses. No parental, reproduction or developmental toxicity was observedup to the highest dose level tested (150 mg/kg bw/day). Based on the parameters assessed in this study, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 150 mg/kg bw/day was derived.