Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 December 2011 - 20 February 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in the report essentially conform to the following guidelines:
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear light yellow liquid
Details on test material:
- Name of test material (as cited in study report): 3-(isodecyloxy)-1-propanamine
- Substance type: Clear light yellow liquid
- Physical state: liquid
- Purity: 95.3 %w/w
- Lot/batch No.: 890000022898
- Expiration date of the lot/batch: 22 September 2019
- Storage condition of test material: At room temperature in the dark under nitrogen
- Hygroscopic: No
- Volatile: No
- Test substance handling: Flush container with nitrogen after handling
- Density 852 kg/m3
- Stability in water: For at least 160 hours (information from sponsor) Stability for at least 6 hours at room temperature is confirmed over the concentration range 0.5 to 5 mg/mL, Project 498018.
- Solubility in water: Fully

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
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 at start of treatment was 317 gr (males) or 201 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 01 January - 20 February 2012

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. Adjustment was made for density of the test substance. No correction was made for purity of the test substance.
- Storage conditions of formulations: At ambient temperature under nitrogen..
- Dose volume: 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
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 498027). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 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 43-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. One female of Group 2 was 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 d/w.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 15, 50 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
An extra 5 males in Groups 1 and 4 were allowed 2 weeks of recovery.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the dose range finding study (NOTOX Project 498017).
- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
- 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 individually identified by means of subcutaneous injection of Indian ink.
- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology (see also respective paragraphs). Only females with live offspring were selected.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily. The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily detailed clinical observations were made in all animals within 30 minutes after dosing (i.e. based on clinical observations conducted in the dose range finding study (NOTOX Project 498017)). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena (except weeks 4 and 5; see protocol deviations). Arena observations were not performed when the animals were mating, or housed individually. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.
In weeks 4 and 5, it was not recorded that arena observations were conducted. Sufficient (clinical) observations were conducted for adequate interpretation of the study results.

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.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines
Locomotor activity was additionally conducted for one animal of the control group, one animal of Group 2, two animals of Group 3 and one animal of Group 4. As these additional measurements provided additional information, this did not adversely affect the study.
Locomotor activity of males at the end of the recovery period was not determined, although a (potentially) treatment-related effect was noted at the end of treatment. No clinical signs were noted at the end of the recovery period. This suggests that an effect (if any) on motor activity at the end of the recovery period would be expected to be small in nature, and not have any significant impact on the overall conclusion of the study.
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 and animals suspected to be infertile).
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 approximately 21 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
The maximum fasting time of 20 hours before necropsy was exceeded with a maximum of approximately 1 hour. The fasting period was only slightly longer and was therefore considered not to have adversely affected the outcome of the macroscopic and microscopic examination.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining females which failed to deliver and the remaining males: According to test guidelines
- The number of former implantation sites and corpora lutea were recorded for all paired females.
One non-selected female of Group 1 was subjected to a full necropsy as conducted for selected females. Additional information was supplied.

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and Testes

HISTOPATHOLOGY
- According to test guidelines
The urinary bladder from one male of the controlgroup and coagulating glands from one male of Group 4 were not available for histopathological examination. Evaluation: Sufficient histopathological data were available for adequate interpretation of the study results.

Postmortem examinations (offspring):
SACRIFICE
Pups surviving to planned termination were killed by decapitation on lactation Days 5-7 zie rapport.

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.

HISTOPATHOLOGY / ORGAN WEIGHTS
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.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

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.
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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One female at 50 mg/kg was found dead on Day 6. Given the absence of any treatmentrelated findings in this animal, the early stage of treatment in which this mortality occurred and absence of mortality/moribundity among other animals of the same dose group, this death was considered to be unrelated to treatment with the test substance. Histopathologically, no cause of death could be established for this animal.

CLINICAL SIGNS
All males and most females showed hunched posture from weeks 2/3 onwards, which resolved early during the recovery period for males. Salivation seen after dosing among animals at 50 mg/kg, and more incidentally at 15 mg/kg, during the treatment period 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. One female at 5 mg/kg showed a wound on the dorsal area of the head with scabs and swelling. Other incidental findings that were noted included chromodacryorrhoea, piloerection, and ptosis. These findings 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.

FUNCTIONAL OBSERVATIONS
A statistically significant lower motor activity (total movements and ambulations) was recorded for males at 15 and 50 mg/kg. Females at 50 mg/kg showed a statistically significant higher motor activity (total movements), but a clear dose related trend was absent, and for ambulations no dose-related trend was noted for the statistically significant higher means at 5 and 50 mg/kg. Therefore, these changes in motor activity for females were considered not to be of toxicological relevance. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. Any statistically significant changes in body weight gain were considered to be of no toxicological relevance since these changes were slight in nature, occurred in the absence of a dose-related trend, and/or were absent at the end of the treatment period, or had resolved during the recovery period. These changes in body weight gain were present for males at 15 mg/kg on Day 7 and 14 of the mating phase, for males at 50 mg/kg on Day 7 and 14 of the recovery phase, and for females at 50 mg/kg on Days 17 and 20 of the post-coitum phase.

FOOD CONSUMPTION
No toxicologically relevant changes in food consumption before or after correction for body weight were noted. Any statistically significant changes in food consumption before or after correction for body weight were considered to be of no toxicological relevance since these changes were slight in nature, occurred in the absence of a dose-related trend, and resolved during the treatment period. These changes consisted of a lower (relative) food consumption in males at 15 and 50 mg/kg over Days 1-8 and/or 8-16 of the pre-mating period, and a higher (relative) food consumption of females at 5, 15 and 50 mg/kg over Days 14-17 of the post-coitum phase.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes in haematological parameters were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related trend, remained within the range considered normal for rats of this age and strain, and/or were absent at the end of the treatment or recovery period. These changes consisted of a lower white blood cell count, higher red blod cell count, higher haemoglobin, haematocrit and lower mean corpuscular haemoglobin concentration (MCHC) level in males at 5 mg/kg at the end of treatment, lower haemoglobin level in males at 50 mg/kg at the end of the recovery phase, higher relative monocyte counts in females at 15 mg/kg and lower platelet counts in females at 5 mg/kg.

CLINICAL BIOCHEMISTRY
Males at 50 mg/kg showed a statistically significant lower total protein level, higher sodium level and higher inorganic phosphate level at the end of the treatment period (all means being essentially within the normal range of values expected for rats of this age and strain). At the end of the recovery period, inorganic phosphate levels remained higher with statistical significance, whilst total protein appeared lower and sodium level appeared higher than controls (without statistical significance, but mean sodium level slightly exceeded the range considered normal for rats of this age and strain).

MACROSCOPIC EXAMINATION
Necropsy did not reveal any toxicologically relevant alterations. The female at 50 mg/kg found dead on Day 6 of treatment showed an advanced stage of autolysis, and an enlarged liver and adrenal glands (correlating microscopically to congestion and autolysis, respectively). The incidence of other macroscopic findings among control and treated animals 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, and included red foci on the glandular mucosa of the stomach (correlating microscopically to congestion), red foci on the lungs, thymus, kidneys or caecum, a yellowish hard nodule on the epididymal adipose tissue, a yellowish soft nodule on the tail of the epididymides, fluid in the uterus, scab formation on the skin of the head.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. The statistically significant higher prostate weight and prostate to body weight ratio of males at 5 and 15 mg/kg at the end of treatment was considered not to be a sign of toxicity as a treatment-related trend was absent and means remained within the range considered normal for rats of this age and strain.

MICROSCOPIC EXAMINATION
There were treatment related microscopic findings in the stomach of the 50 mg/kg treated rats:
- Diffuse lymphogranulocytic inflammation of the forestomach up to slight degree in 3/5 males at 50 mg/kg.
- Increase in incidence and severity of hyperplasia of the forestomach in males at 50 mg/kg: Minimal degree in 2/5 males and 1/5 females, slight degree in 1/5 males and 3/5 females and moderate degree in 1/5 males in the 50 mg/kg treated rats, compared to minimal degree in 1/5 control females and 2/5 males at 5 mg/kg.
The recovery of the stomach lesions was complete in the males.

REPRODUCTIVE DATA
The number of implantation sites showed an apparent trend towards an increase over the dose groups, which at 50 mg/kg achieved a level of statistical significance and exceeded the range of values considered to be normal for rats of this age and strain. Mating, fertility and conception indices, precoital time, and number of corpora lutea were unaffected by treatment.

GESTATION
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PARTURITION/MATERNAL CARE
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)

Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects were observed up to the highest dose level tested. In the range finding study severe effects were noted at 150 mg/kg.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

EARLY POSTNATAL PUP DEVELOPMENT
A statistically significant higher mean number of living pups per litter was recorded at 50 mg/kg.

MORTALITY PUPS
One pup of the control group, one pup at 5 mg/kg, three pups at 15 mg/kg, and eight pups at 50 mg/kg were found dead or missing during lactation. Pups missing were most likely cannibalised. The minor increase in postnatal loss and subsequent lower viability index at 50 mg/kg was considered to be due to a higher number of dead pups for litter no. 87, and to be of no toxicological significance. Number of dead pups at first litter check and sex ratio were considered unaffected by treatment.

CLINICAL SIGNS PUPS
One pup of at 50 mg/kg found missing on Day 2 of lactation showed a lean appearance at first litter check. Absence of milk was noted in the stomach of all five pups found dead at 50 mg/kg. 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.

BODY WEIGHT PUPS
Body weight of male and female pups at 50 mg/kg was lower than controls on Days 1 and 4, achieving a level of statistical significance (except for male pups on Day 1). Weight gain over Days 1-4 did not show a dose-related trend.

MACROSCOPY PUPS
Macroscopic findings of pups that were found dead consisted of a beginning stage of autolysis and/or absence of milk in the stomach. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects were observed up to the highest dose level tested. In the range finding study severe effects were noted at 150 mg/kg.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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 90% and 110%). 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%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Applicant's summary and conclusion

Conclusions:
In an oral OECD422 study, no toxicologically relevant effects were noted in any of the examined parameters up to the highest dose level tested (50 mg/kg). Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg/day was derived.
Executive summary:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of 3-(isodecyloxy)-1-propanamine in rats by oral gavage followed by a 14-day recovery period.

 

Guidelines

The study was based on the following guidelines:

1) OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996.

2) OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

3) OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

4) OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

5) EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

6) OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

7) OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Rationale for dose levels

Based on the results of a 14-day dose range finding study, the dose levels for this combined 28-day oral gavage study with reproduction/ developmental toxicity screening test were selected to be 5, 15 and 50 mg/kg.

Study outline

After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 5, 15 and 50 mg/kg/day. An extra 5 males in Groups 1 and 4 were allowed 2 weeks of recovery. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 43-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Evaluated parameters

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (Week 4 (males); end of lactation (females)), body weight and food consumption (at least at weekly intervals), clinical pathology (Week 4 and end of recovery (males); end of lactation (females)), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed once during the study to assess accuracy, homogeneity and stability.

 

Results/discussion

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results:

Findings during treatment consisted of hunched posture among most animals at 50 mg/kg from weeks 2/3 onwards (resolving early during the recovery period), and lower motor activity for males at 15 and 50 mg/kg. Motor activity at the end of the recovery phase was not assessed, but given that no clinical signs were noted at the end of the recovery period, an effect (if any) on motor activity would be expected to be small in nature, and not have any significant impact on the overall conclusion of the study.

Clinical biochemistry changes in males at 50 mg/kg consisted of a lower total protein level, and higher sodium and inorganic phosphate level at the end of the treatment and recovery period. These changes were slight in nature, did not always achieve a level of statistical significance and were essentially within the range considered normal for rats of this age and strain. Moreover, since supportive morphological changes were absent, these clinical biochemistry changes were considered not to be adverse in toxicological terms.

Histopathological assessment showed treatment-related findings in the stomach of both sexes at 50 mg/kg, consisting of diffuse lymphogranulocytic inflammation of the forestomach up to slight degree in 3/5 males at 50 mg/kg, and an increased incidence and severity of hyperplasia of the forestomach in males and females at 50 mg/kg. Since these morphological changes were generally mild in nature, and food intake and body weight gain values were unaffected by treatment, these morphological changes were considered not to be adverse in toxicological terms.

No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (functional observations (except motor activity), body weight, food consumption, haematological investigations, macroscopic examination and organ weights).

Reproductive results:

At 50 mg/kg, an increased number of implantation sites was recorded. This finding was considered not to represent a toxicologically relevant effect as the increase was slight in nature, and the opposite effect (i.e. a decrease) would be expected in case of reproductive toxicity.

No toxicologically significant changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and number of corpora lutea).

Developmental results:

The lower pup body weights of male and female pups at 50 mg/kg on Day 4 was also apparent on Day 1, and pup body weight gain over Days 1-4 was similar across the groups. This lower pup body weight was considered to have occurred secondary to the higher mean number of living pups per litter and corresponding higher number of implantation sites at this dose. These changes were therefore

considered not to be adverse in toxicological terms.

No toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs and macroscopy).

 

Conclusion

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg/day was derived.