Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Testing in OECD 422 combined repeated dose/reproduction toxicity screening study showed no effects on fertility and other reproductive parameters at the highest dose tested. A reproduction and developmental NOAEL of 50 mg/kg/day was established.Further no indications for reproduction toxicity are indicated by results form available 90-day study a developmental study on Etheramine C10i. Possible exposures are very limited due to the characteristics and use of the substance.

Link to relevant study records
Reference
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.
Reason / purpose:
reference to same study
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
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
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
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
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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.
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
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.
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.
Reproductive effects observed:
not specified

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.

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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Screening study for fertility; high quality OECD 422 guideline in compliance to GLP.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
Exposure of humans to Etheramine C10i via inhalation is not likely taking into account the low vapour pressure and high boiling point of the substance as well as low possibility of exposure to aerosols or droplets of an inhalable size. Furthermore, as the substance is classified as corrosive, local effects will be dose-limiting and preclude sufficient uptake for systemic effects to develop.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Substance is corrosive. Effects will be characterized by local corrosive effects that are related to duration, quantity and concentration, rather than by systemic toxicity due to dermal uptake. Dermal absorption is expected to be much lower compared to oral absorption. Related to corrosive properties, exposures are likely to be limited.
Additional information

Etheramine C10i has been evaluated in a Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats by oral gavage followed by a 14-day recovery period, according to OECD 422 and in compliance to GLP.

In a 14-day rangefinderEtheramine C10ishowed small effects were observed on body weight at 50 mg/kg and significant toxicity was noted at 150 mg/kg. The dose levels selected for the full study were5, 15 and 50 mg/kg bw/day applied to groups of 10 males and 10 females Wistar Han rats. 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. An extra 5 males in Groups 1 and 4 were allowed 2 weeks of recovery.

Observations included mortality / viability, clinical signs (daily), functional observations and locomotor activity, body weight and food consumption, clinical pathology and 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 for accuracy, homogeneity and stability.

Parental results:

Based on limited local effects in the stomach following treatment of a corrosive substance with full recovery during 14-day recovery period, a parental NOAEL of 50 mg/kg/day was derived for systemic toxicity.

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.

 

No adverse effects on reproductive organs were identified in the 90 day study in rats on Etheramine C10i. This study was performed according to OECD 408 and does not include detailed reproductive parameters as indicated in OECD 416. Nevertheless, the 90 day study did not indicate any effects on weight of testes, prostate, seminal vesicles, ovaries, and uterus. Histological examination of these tissues also confirmed no treatment-related effects.

 

Considerations on the mode of action show that the most significant treatment-related changes in the studies performed on etheramines involve local irritating/corrosive effects. In physiological circumstances, the etheramines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule is not expected to easily pass membrane structures, and consequently, reproduction toxicity is not a likely concern.

Additionally, QSARs also do not show alerts for reproduction/developmental toxicity. (QSAR Toolbox DART Scheme v.1.2; Derek Nexus v.6.0.1; VEGA - Developmental Toxicity model (CAESAR) v.2.1.7; VEGA - Developmental/Reproductive Toxicity library (PG), v.1.0.0; TOPKAT Developmental Toxicity Potential)

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD 414, GLP) is available on Etheramine C10i: NOAEL for embryo-foetal toxicity was found to be 75 mg/ kg body weight. An additional ZEDTA showed no developmental toxicity/teratogenicity at levels below lethality. Based on molecular properties and mode of action considerations, developmental toxiciyt is also not to be expected. This is further supported by lack of concerns for developmental and reproduction toxicity by QSARs.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 June 2016 - 21 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd 3 November 2015
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with nitrogen
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 0.5 – 5 mg/mL, Test Facility Study No. 498018.

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 171-247 g
- Fasting period before study: no
- Housing: individually housed in Macrolon plastic cages (MIII type, height 18 cm). Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) 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 prior to treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7-22.4
- Humidity (%): 45.9-69.4
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 June 2016 To: 21 July 2016
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the vehicle. Adjustment was made for specific gravity of the test item (0.8518 g/cm3 at 20°C). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at Charles River Den Bosch and on information provided by the Sponsor.
- Concentration in vehicle: 0.8, 2.5 and 7.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
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 (05 July 2016), according to a validated method (Test Facility Study No. 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 under normal laboratory light conditions was also determined (highest concentration only).
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%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum was the day of successful mating; confirmed by vaginal plug).
Duration of treatment / exposure:
From Days 6 to 20 post-coitum, inclusive
Frequency of treatment:
Once daily
Duration of test:
15 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle controls
Dose / conc.:
8 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
75 mg/kg bw/day
No. of animals per sex per dose:
22 females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a combined 28-day/repro-screening study (OECD 422, Test Facility Study No. 498018), in which dose levels of 0, 5, 15 and 50 mg/kg were tested. At 50 mg/kg, hunched posture (both sexes) and lower motor activity (males only) were noted. Clinical biochemistry changes (lower total protein level, higher sodium and inorganic phosphate level) were observed in males at 50 mg/kg. Moreover, rats treated at 50 mg/kg showed treatment-related findings in the stomach, consisting of diffuse lymphogranulocytic inflammation of the forestomach (males only) and hyperplasia of the forestomach (both sexes). These findings were considered not to be adverse in toxicological terms. No reproduction or developmental toxicity was observed by treatment up to 50 mg/kg. In the previous dose range finding study (Test Facility Study No. 498017), rats treated at 150 mg/kg for 14 days showed significant clinical signs, weight loss, reduced food intake, lower thymus and spleen weights and 1/3 female was killed in extremis.

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily for mortality and viability, at least once daily from day 2 post-coitum onwards up to the day prior to necropsy for clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: each ovary and uterine horn, stomach (10 females/group in groups).

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [ half per litter]
Statistics:
The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
• Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-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. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:
Pre-implantation loss (%) = [(number of corpora lutea - number of implantation sites)/number of corpora lutea] x 100
Post-implantation loss (%) = [(number of implantation sites - number of live fetuses)/number of implantation sites] x 100
Viable fetuses affected/litter (%) = [(number of viable fetuses affected/litter)/(number of viable fetuses/litter)] x 100
Historical control data:
Historical control data from the same testing lab from 2014-2015 are available.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted by treatment up to 75 mg/kg.
Rales was observed for three females at 75 mg/kg (nos. 74, 79 and 80) for 1-3 days. As this finding was considered to be related to local stomach effects observed in some females at 75 mg/kg (see sections 7.2.5 and 7.2.6), this was probably related to treatment with test item.
Incidental findings noted included alopecia, scabs and salivation. These findings were not considered to be test item related as they occurred in single females, in absence of a dose-related trend and within the range of background findings to be expected for rats of this age and strain treated under the conditions in this study.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 75 mg/kg, mean body weights were lower than controls from Day 9 post-coitum onwards, reaching statistical significance on Day 15 and 21 post-coitum. Mean body weight gains were statistically significantly lower at 75 mg/kg from Day 9 post-coitum onwards, when compared to controls. In addition, weight gain corrected for gravid uterus was statistically significantly lower at 75 mg/kg when compared to controls (5.4 gram versus 26.5 gram).
Mean body weights and (corrected) body weight gains at 8 and 25 mg/kg remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative food consumption were statistically significantly lower at 75 mg/kg during the entire treatment period.
Food consumption was considered to be unaffected by treatment up to 25 mg/kg.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Only subjective appraisal was made, as no treatment-related effect was suspected.
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:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Three females at 75 mg/kg (nos. 70, 74 and 77) showed an irregular surface of the forestomach, which was considered to be test item related. The microscopic correlate was diffuse hyperplasia of the forestomach and/or hyperkeratosis of the limiting ridge. Moreover, thymus reduced in size and Gi-tractus distended with gas were noted for single females at 75 mg/kg (nos. 70 and 75, respectively).
No macroscopic findings were noted in the control, 8 and 25 mg/kg groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings noted for the stomach of the 75 mg/kg group females consisted of:
• Ulceration of the forestomach (marked) in 1/10 females.
• Diffuse hyperplasia of the forestomach in 2/10 females (minimal and slight).
• Hyperkeratosis of the limiting ridge at increased incidence and/or severity (3/10 minimal, 2/10 slight) compared to 1/10 female at 25 mg/kg at minimal degree which was regarded to be within background levels.
These stomach findings were considered to be most likely a local response to exposure with the test item, whereby the marked ulceration can be regarded as adverse.
There were no other test item-related histologic changes. Remaining histologic stomach changes were considered to be incidental findings. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects by treatment up to 75 mg/kg.
One Group 3 female (no. 45) had resorptions only. All other females (with the exception onf one control female and one group 2 female that were not pregnant) were pregnant and had litters with viable fetuses.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on the numbers of pre- or post-implantation loss by treatment up to 75 mg/kg.
The number of pre-implantation loss was slightly higher in the 75 mg/kg group when compared to controls. This change was not statistically significant and as treatment started from implantation onwards (i.e. Day 6 post-coitum), this was not considered to be treatment related.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects by treatment up to 75 mg/kg.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects by treatment up to 75 mg/kg.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no effects by treatment up to 75 mg/kg.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One control female (no. 5) and one Group 2 female (no. 30) were not pregnant. One Group 3 female (no. 45) had resorptions only. All other females were pregnant and had litters with viable fetuses.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
local effects
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
gross pathology
histopathology: non-neoplastic
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean combined (male and female) body weights were 5.3, 5.2, 5.4 and 5.1 gram for the control, 8, 25 and 75 mg/kg groups, respectively.
Fetal body weights (both sexes) were statistically significantly lower at 75 mg/kg when compared to controls. No clear dose related trend was observed and the values at 75 mg/kg were within or only slightly below the range of available historical control data. The level of statistical significance was considered to have arisen as a result of the slightly high concurrent control values, when compared to the available historical control data.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed up to the dose level of 75 mg/kg bw/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female sex ratio was unaffected by treatment up to 75 mg/kg.
Mean sex ratios (males:females) were 51:49, 50:50, 52:48 and 52:48 for the control, 8, 25 and 75 mg/kg groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size for any group.
Mean litter sizes were 10.2, 11.1, 10.2 and 10.6 fetuses/litter for the control, 8, 25 and 75 mg/kg groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 75 mg/kg.
The only malformation observed in groups treated with the test item was a small lower jaw that occurred in one fetus per group at 25 and 75 mg/kg bw/day. Both cases were confirmed skeletally, whereby the mandibles appeared to be fused along the mandibular symphysis. As these cases occurred singly and a small or absent lower jaw was seen previously (skeletally) in historical control data, they were considered chance findings.
Besides the noted jaw findings, one control fetus (A009-04) had a meningocele.
External variations were not seen in any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 75 mg/kg.
The only skeletal malformation observed in this study was fused mandibles. This finding was noted for three fetuses, one per group at 8, 25 and 75 mg/kg bw/day. The same fetuses at 8 and 75 mg/kg bw/day had a small lower jaw as well, and the fetus at 25 mg/kg bw/day had only one socket. These findings occurred singly and do not indicate a treatment relationship.
The variation of slightly to moderately malaligned sternebrae was statistically significantly lower at 25 mg/kg bw/day. The incidence of this finding was 27.7%, 35.8%, 16.7% and 36.9% per litter in controls, 8, 25 and 75 mg/kg bw/day groups, respectively. The concurrent control value was for unknown reasons higher than the available historical control upper limit, while the Group 3 (25 mg/kg bw/day) value was within the applicable data range (4.4%-21.3% per litter). As no dose response could be established for this finding and the occurrence of fewer malaligned sternebrae is not adverse, the lower incidence in Group 3 was not considered to be toxicologically relevant.
Other skeletal variations that were noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered treatment related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 75 mg/kg.
Visceral malformations were only noted for one Group 2 and two Group 3 fetuses. No visceral malformations were noted in the control and highest dose group. In Group 3, a situs inversus was noticed in one fetus during eviscerating, and in another fetus a small left eye was found at soft tissue cephalic examination. In Group 2, the fetus with a small lower jaw had no urinary bladder. Due to the single occurrence and/or as they were noted in historical control fetuses (i.e. small eyes and situs inversus), all three malformations were considered to be chance findings.
Of the visceral variations, the finding of small supernumerary liver lobes was most common. It was observed in 4.5%, 4.4%, 6.7% and 8.6% of fetuses per litter in Groups 1, 2, 3 and 4, respectively. The mean litter incidence of Group 4 was slightly above the maximum historical control value (7.7% per litter), but as it was not statistically significantly higher when compared to the concurrent control group and this variation is most common in historical controls, this was not considered to be treatment related.
The other variations that were noted in this study occurred singly or at low incidences in the absence of a dose-related incidence trend and therefore were not considered to be treatment related.
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at the highest tested dose
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Accuracy of preparations and homogeinity:

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (mean accuracies 93, 94 and 90% with the coefficient of variation 1.6%, not determined and 3.6%, respectively; n = 6 for groups 2 and 4 and 2 for group 3).

Stability:

Analysis of the Group 4 formulation after storage yielded a relative difference of 15%. The value was higher than the criterion range of 10%. The accuracies of the samples after storage were 103% and 104%. The relatively high difference was obtained since the mean accuracy at t=0 was somewhat lower (i.e. 90%). Both at t=0 and t=6 hours the results were in the range of 85-115%. It demonstrated that the formulation of Group 4 set at level 7.51 mg/g could be used for adequately dosing in the storage period of 6 hours. The value of 15% indicated that degradation of the test item had not occurred during storage. Based on this, the formulation was found to be stable during storage at room temperature for at least 6 hours.

Table 1. Summary of the developmental effects observed in the study

Dose level (mg/kg bw/day)

0

8

25

75

Pregnant/total dams

21/22

21/22

22/22

22/22

-early resorptions

-late resorptions

(% per litter)

8.6

0.0

6.1

0.0

8.5

0.0

5.1

0.0

Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses only

0

0

1

0

Pre-implantation loss (number and percent)

14 (5.1%)

11 (3.9%)

22 (7.6%)

28 (9.6%)

Post-implantation loss (number and percent)

21 (8.6%)

14 (6.1%)

12 (8.5%)

12 (5.1%)

Body weight on day 21

316

322

320

294*

Body weight gain day 6-21 (%)

47

49

48

35**

Gravid uterine weight (g)

73.8

77.4

73.8

71.4

Mean live offspring (number)

10.2

11.1

10.2

10.6

Live offspring (percent)

91.4

93.9

91.5

94.9

Mean fetal body weight males (g)

5.5

5.4

5.5

5.2*

Mean fetal body weight females

5.2

5.1

5.3

4.9*

Mean fetal body weight (sexes combined)

5.3

5.2

5.4

5.1*

Malformations (including runts) number and percent of fetuses per litter

 

 

1 (0.5%)

 

 

1 (0.3%)

 

 

2 (1.7%)

 

 

1 (0.4%)

Variations (% per litter)

-external

-soft tissue

-skeletal

 

0

9.9

76.6

 

0

9.4

77.1

 

0

12.6

69.0

 

0

11.6

77.9

*: significantly different from the control group at 0.05

**: significantly different from the control group at 0.01

Table 2. Historical control data

3-(isodecyloxy)-1-propanamine

 

 

 

 

 

 

Project 511810

 

 

 

 

 

 

 

 

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)

 

 

 

 

 

 

 

Gestation Day 21

 

Mean of Study Means

 

 

 

 

 

Study Date Range: 2014 - 2015

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Endpoint

Total

Mean

SD

Median

Min

Max

P5

P95

No of Studies

13

 

 

 

 

 

 

 

Total No. of Animals in the Control Group

304

 

 

 

 

 

 

 

No. of Animals that Died

0

 

 

 

 

 

 

 

No. of Animals that were Euthanized

0

 

 

 

 

 

 

 

No. of Animals that Aborted or Delivered

3

 

 

 

 

 

 

 

Percent Pregnant

 

98.8

2.73

100.0

90.9

100.0

97.1

100.0

No. of Animals Examined at Laparohysterectomy

301

 

 

 

 

 

 

 

No. Nongravid

4

 

 

 

 

 

 

 

No. Gravid

297

 

 

 

 

 

 

 

No. with Only Resorptions

2

 

 

 

 

 

 

 

No. of Dams with Live Fetuses

295

 

 

 

 

 

 

 

Mean No. Viable Fetuses/Dam

 

10.7

0.71

10.6

9.1

11.6

10.3

11.2

Total No. Viable Fetuses

3194

 

 

 

 

 

 

 

Viable Fetuses (%/Litter)

 

95.2

2.63

95.9

88.9

98.4

93.6

96.8

Mean No. Postimplantation Loss/Dam

 

0.5

0.15

0.4

0.2

0.7

0.4

0.6

Total No. Postimplantation Losses

134

 

 

 

 

 

 

 

Postimplantation Loss (%/Litter)

 

4.8

2.63

4.1

1.6

11.1

3.2

6.4

Dead Fetuses (%/Litter)

 

0.0

0.11

0.0

0.0

0.4

0.0

0.1

Early Resorptions (%/Litter)

 

4.7

2.62

4.1

1.6

11.1

3.2

6.3

Late Resorptions (%/Litter)

 

0.0

0.11

0.0

0.0

0.4

0.0

0.1

Mean No. Implantations/Dam

 

11.2

0.69

11.1

9.6

12.0

10.8

11.6

Mean No. Corpora Lutea/Dam

 

11.9

0.71

11.7

10.9

13.2

11.5

12.3

Mean No. Preimplantation Loss/Dam

 

0.7

0.32

0.6

0.2

1.3

0.5

0.9

Total No. Preimplantation Losses

207

 

 

 

 

 

 

 

Preimplantation Loss (%/Litter)

 

6.2

3.43

5.8

2.0

14.5

4.2

8.3

Total No. Male Fetuses

1617

 

 

 

 

 

 

 

Total No. Female Fetuses

1577

 

 

 

 

 

 

 

% Males/Litter

 

50.8

2.12

50.7

46.6

53.7

49.5

52.0

% Female/Litter

 

49.2

2.12

49.3

46.3

53.4

48.0

50.5

Mean Fetal Body Weight (g)

 

5.2

0.08

5.2

5.1

5.3

5.1

5.2

Mean Male Body Weight (g)

 

5.4

0.10

5.4

5.2

5.5

5.3

5.4

Mean Female Body Weight (g)

 

5.1

0.07

5.1

5.0

5.2

5.0

5.1

Mean Male Placenta Weight (g)1

 

0.46

0.01

0.47

0.44

0.47

0.4

0.5

Mean Female Placenta Weight (g)1

 

0.44

0.01

0.44

0.42

0.45

0.4

0.5

1Based on 4 datasets

 

 

 

 

 

 

 

 

3-(isodecyloxy)-1-propanamine

 

 

 

 

 

 

 

Project 511810

 

 

 

 

 

 

 

 

 

Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)

 

 

 

 

 

 

 

 

 

Gestation Day 21

 

 

 

 

 

 

 

 

 

Study Date Range: 2014 - 2015

 

 

 

 

 

 

 

 

 

No. of Studies

13

 

 

 

 

 

 

 

 

Total No. Fetuses/Litters Examined Externally

3194

295

 

 

 

 

 

 

 

Total No. Fetuses/Litters Examined Viscerally

2061

295

 

 

 

 

 

 

 

Total No. Fetuses/Litters Examined Skeletally

2059

295

 

 

 

 

 

 

 

 

Mean of Study Means

 

 

 

 

 

Summary Incidence

 

(% Per Litter Basis)

 

 

 

 

 

(Total No. Affected)

MALFORMATIONS

Mean

SD

Median

Min

Max

P5

P95

Fetuses

Litters

Total External Malformations

 

 

 

 

 

 

 

1

1

Total Visceral Malformations

 

 

 

 

 

 

 

7

7

Total Skeletal Malformations

 

 

 

 

 

 

 

15

15

Total Malformations

 

 

 

 

 

 

 

22

22

EXTERNAL

 

 

 

 

 

 

 

 

 

Exencephaly

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

Eye(s)- Open

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

VISCERAL

 

 

 

 

 

 

 

 

 

Diaphragmatic Hernia

0.0

0.08

0.0

0.0

0.3

0.0

0.1

1

1

Eye(s)- Absent and/or Small

0.1

0.26

0.0

0.0

0.9

0.0

0.2

3

3

Hydrocephaly- External

0.0

0.12

0.0

0.0

0.5

0.0

0.1

1

1

Situs Inversus

0.2

0.34

0.0

0.0

1.0

0.0

0.4

3

3

SKELETAL

 

 

 

 

 

 

 

 

 

Jaw- Upper Jaw Small

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

Jaw- Lower Jaw Absent or Small

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

Limb Bone(s)- Bent

0.3

0.44

0.0

0.0

1.1

0.0

0.5

4

4

Rib Anomaly

0.1

0.31

0.0

0.0

1.1

0.0

0.3

1

1

Skull Anomaly

0.1

0.34

0.0

0.0

1.2

0.0

0.3

2

2

Sternebra(e)- Fused

0.1

0.29

0.0

0.0

1.0

0.0

0.3

2

2

Sternebra(e) Malaligned (Severe)

0.0

0.08

0.0

0.0

0.3

0.0

0.1

1

1

Sternoschisis

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

Vertebral Anomaly With or Without Associated Rib Anomaly

0.2

0.53

0.0

0.0

1.9

0.0

0.5

3

3

Vertebral Centra Anomaly

0.1

0.22

0.0

0.0

0.8

0.0

0.2

1

1

3-(isodecyloxy)-1-propanamine

 

 

 

 

 

 

 

Project 511810

APPENDIX 5

 

 

 

 

 

 

 

 

 

Historical Control Data Rat: Crl:WI(Han) (outbred. SPF-Quality)

 

 

 

 

 

 

 

 

 

Gestation Day 21

 

 

 

 

 

 

 

 

 

 

Mean of Study Means

 

 

 

 

Summary Incidence

 

(% Per Litter Basis)

 

 

 

 

 

(Total No. Affected)

VARIATIONS

Mean

SD

Median

Min

Max

P5

P95

Fetuses

Litters

EXTERNAL

 

 

 

 

 

 

 

 

 

None Observed

 

 

 

 

 

 

 

 

 

VISCERAL

 

 

 

 

 

 

 

 

 

Kidney(s)- Renal Papilla(e) Absent and/or Small

0.1

0.25

0.0

0.0

0.9

0.0

0.2

2

2

Liver- Appendix

1.2

0.56

1.3

0.3

2.3

0.9

1.6

23

21

Liver- Discolored

0.1

0.30

0.0

0.0

1.0

0.0

0.3

3

3

Liver- Small Supernumerary Lobe(s)

4.0

1.96

4.0

1.3

7.7

2.8

5.2

69

58

Spleen- Supernumerary

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

Thymus- Partially Undescended Horn(s)

1.3

1.55

0.8

0.0

4.3

0.3

2.2

34

23

Thyroid- Discolored

0.1

0.36

0.0

0.0

1.3

0.0

0.3

1

1

Ureter(s)- Convoluted

1.0

2.39

0.0

0.0

8.7

0.0

2.5

43

28

Ureter(s)- Dilated

0.9

2.33

0.0

0.0

8.5

0.0

2.3

44

19

SKELETAL

 

 

 

 

 

 

 

 

 

7th Cervical Rudimentary Rib(s)

1.7

1.34

1.2

0.0

4.4

0.9

2.5

30

26

7th Cervical Full Rib(s)

0.1

0.36

0.0

0.0

1.1

0.0

0.4

2

2

14th Full Rib(s)

5.7

4.65

5.2

0.0

13.1

2.9

8.5

88

64

14th Rudimentary Rib(s)

44.1

19.84

54.4

19.0

72.0

32.1

56.1

798

250

Metacarpal(s) and/or Metatarsal(s) Unossified

2.2

1.97

1.0

0.0

6.3

1.0

3.4

41

24

Pelvic Girdle- Caudal Shift

6.6

3.77

7.1

1.7

12.8

4.3

8.9

127

71

Rib(s)- Bent

10.6

7.78

10.2

0.8

22.3

5.9

15.3

162

85

Rib(s)- Short

0.0

0.06

0.0

0.0

0.2

0.0

0.0

1

1

Skull- Reduced Ossification

2.7

2.55

1.8

0.0

7.0

1.2

4.3

81

46

Skull- Supernumerary Site

0.0

0.14

0.0

0.0

0.5

0.0

0.1

1

1

Sternebra(e) #1, #2, #3 and/or #4 Unossified

0.2

0.31

0.0

0.0

0.8

0.0

0.3

3

3

Sternebra(e) #5 and/or #6 Unossified

0.9

1.33

0.0

0.0

4.1

0.1

1.7

37

23

Sternebrae- Malaligned (Slight or Moderate)

11.1

5.72

8.9

4.4

21.3

7.6

14.5

188

131

Sternum- Supernumerary Ossification Site

0.1

0.31

0.0

0.0

1.1

0.0

0.3

1

1

Vertebral Centra- Reduced Ossification

0.6

0.88

0.4

0.0

3.0

0.1

1.2

12

12

Conclusions:
In a GLP-compliant guideline study, pregnant Crl:WI(Han) rats were dosed the test substance by oral gavage at 0 (vehicle controls), 8, 25 and 75 mg/kg bw/day from Day 6 to Day 21 post-coitum. The NOAEL for maternal toxicity was set at 25 mg/kg bw/day based on reduced body weight and body weight gain, reduced consumption and local irritation effects in the stomach (ulceration, diffuse forestomach hyperplasia and/or increased incidence and/or severity of hyperkeratosis of the limiting ridge) at the highest dose level. The NOAEL for developmental effects was set at 75 mg/kg bw/day, based on the absence of adverse effects at the highest dose level.
Executive summary:

In a GLP-compliant OECD Guideline 414 study, pregnant rats (22/dose) were administered the test substance solution in water by oral gavage at 0 (vehicle controls), 8, 25 and 75 mg/kg bw/day from Day 6 to Day 21 post-coitum. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The stomach and gross lesions were collected and fixed from all animals at necropsy.

Histopathological examination was performed on the stomach from 10 selected females of all groups.

 

Treatment at 75 mg/kg resulted in statistically significantly lower body weights and body weight gains on Days 15 and 21 post-coitum and from Day 9 post-coitum onwards, respectively. In addition, weight gain corrected for gravid uterus was statistically significantly lower at 75 mg/kg bw/day when compared to controls (5.4 gram versus 26.5 gram). Absolute and relative food consumption were statistically significantly lower at 75 mg/kg bw/day during the entire treatment period. Test item-related morphological findings were present in the stomach of females treated at 75 mg/kg bw/day, consisting of ulceration, diffuse forestomach hyperplasia and/or increased incidence and/or severity of hyperkeratosis of the limiting ridge. Based on these effects the NOAELs for maternal toxicity for both systemic and local effects were set to 25 mg/kg bw/day.

There were no effects on the number of abortions, early or late resorptions, total implantation losses or number of live fetuses. There were no toxicologically relevant or test item related external, visceral and skeletal malformations or variations noted by treatment up to 75 mg/kg. Fetal body weights in both sexes were statistically significantly lower at 75 mg/kg bw/day when compared to controls. The level of statistical significance was considered to have arisen as a result of the slightly high concurrent control value. In addition, as no clear dose related trend was observed and no effects on skeletal developmental were noted, this was considered to be related to the significant lower body weights of the dams rather than a developmental cause. Hence, this minimal change in fetal body weight was considered to be non-adverse.

Based on this the NOAEL for developmental effects was set at 75 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3-Jul-2017 (RF) - 14-Jul-2017 (end full study)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Not under GLP.
Qualifier:
equivalent or similar to
Guideline:
other: EURL ECVAM ‘Zebrafish Embryo Assay for Developmental Toxicity’.
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 236 Fish Embryo Toxicity (FET)
Version / remarks:
July 2013.
Deviations:
yes
Remarks:
- 2 Embryos per well, Total 40 instead 20 per concentratin. Chemical analysis showed sufficient exposure. - Oxygen saturation was only measured at 48 hours in all new and old solutions.
Principles of method if other than guideline:
The development of the zebrafish embryo is very similar to the embryogenesis in higher vertebrates, including humans, and many molecular pathways are conserved between zebrafish and humans. The Zebrafish Embryo Teratogenicity Developmental Toxicity Assay (ZEDTA) can not only be applied as a screening tool for teratogenicity, but also as a means of investigating specific mechanisms related to the teratogenic potential of certain substances. This assay has been researched thoroughly for these purposes and has achieved high levels of concordance with mammalian developmental toxicity data. See references for some typical examples.
This Zebrafish Embryo Developmental Toxicology Assay (ZEDTA) was conducted according to the method described in EURL ECVAM ‘Zebrafish Embryo Assay for Developmental Toxicity’. Additionally, validity criteria of the study were based on the 96-hour Fish Embryo Toxicity (FET) OECD 236 test guideline.
GLP compliance:
no
Remarks:
Lab was GLP accredited until 2017. Analytical work was still done under GLP.
Limit test:
no
Specific details on test material used for the study:
Soluble at concentrations tested
Species:
other: D. rerio
Strain:
other: Wild type
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wageningen UR Animal sciences group, Wageningen, the Netherlands.
- Age at study initiation: Fertilized embryos were between 2-6 hours old. This was confirmed by microscopic observation. Eggs failing to develop and possibly not fertilized (but added to test at T=0) were replaced with fertilized embryos (also exposed to test material) before 6h had expired.
Embryos arrived and were confirmed microscopically to be between 3-4 hpf (hours post fertilization). As soon as the test solutions were made embryos were divided into bulk batches using glass pipettes at the appropriate concentrations. In doing so, delay in exposure caused by preparation time was avoided. The wells were then filled with each of the stocks made for each chemical at each concentration. After a maximum of one hour the wells were emptied and refilled, after which the embryos (2 per well) were added in the definitive test and (5 per well) in the range finding study and positive controls. Plates were then incubated for the test duration and observed daily.

ENVIRONMENTAL CONDITIONS
- Test conditions: Dutch Standard Water (DSW), pH of 8.2, conductivity of 550-650 µS/cm, and contains: 200 mg of CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3 per liter. The water was made in advance and stored in aerated vats before being used in the test.
- Temperature: 26 ºC.
- Test vessels: Falcon “Multiwell” 24 well flat bottom low evaporation culture plates
- embryos per well: 2; 40 embryos per concentration and 8 embryos as an internal plate control. For the controls 48 embryos (2 per well) were used in all controls.
- Feeding: Test organisms were not fed during the study.
Route of administration:
other: solution in media
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: DSW Media
Details on exposure:
Test substance: Preperation of homogeneous stock solution in DSW (135 mg test substance/100 mL).
Diluted further to required test concentrations. All test wells were filled allowed to stand, discarded and refilled to reduce potential test substance absorption.
Range finding concentrations: 0.0215, 0.215, 1.075, 2.15 and 21.5 mg/L (0.0001, 0.001, 0.005, 0.01, 0.1 mM)
Definitive test concentrations: 0.0172, 0.0860, 0.430, 2.150, 10.750 mg/L (0.00008, 0.0004, 0.002, 0.01, 0.05 mM)

Reference substances:
- 3,4-dichloro aniline (DCA) as referece substance for for quality control at 4 mg/L. Additional acetone solvent control also included
- Valproic acid sodium salt (VPA) as positive reference substance. 1 g/l stock in DSW medium, diluted further to appropriate test concentrations of Concentrations of 1.66, 16.6, 166.2 332.4 mg/ ((0.01, 0.1, 1.0, 2.0 mM)

Solution replenishment: Test solutions were refreshed after 48 hours.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The method has been validated as suitable for this purpose within a relevant concentration range.
Samples were taken at the start and the end of the test as well as old and new solutions before and after solution replenishment. 10 ml pooled samples were taken from each concentration of the test substance and mixed 50:50 with leaching solution to prevent loss by adsorption to test vessels.
Details on mating procedure:
Fertilized zebra fish (D. rerio) wild type eggs were sourced at Wageningen UR Animal sciences group, Wageningen, the Netherlands. Fertilized eggs were between 2-6 hours old when added to the test solutions. This was confirmed by microscopic observation. Eggs failing to develop and possibly not fertilized (but added to test at T=0) were replaced with other fertilized eggs (also exposed to test material) before 6h had expired. This ensured that living embryos were present at the start of test and prevented fertilization success influencing the test data.
The Internal control (IC) replicates were not replaced before 6h (if not fertilized) and were therefore used to calculate the average fertilization % of the embryo batch used.
Duration of treatment / exposure:
96 hours
Frequency of treatment:
Exposures were by solution in media. Test solutions were refreshed after 48 hours.
Duration of test:
96 hours
Dose / conc.:
0 other: mM
Remarks:
Nominal concentration (0.0127 mg/L). Analysis resulted in mean exposure of 81% of nominal
Dose / conc.:
0 other: mM
Remarks:
Nominal concentration (0.086 mg/L). Analysis resulted in mean exposure of 73% of nominal
Dose / conc.:
0.002 other: mM
Remarks:
Nominal concentration (0.43 mg/L). Analysis resulted in mean exposure of 84% of nominal
Dose / conc.:
0.01 other: mM
Remarks:
Nominal concentration (2.15 mg/L). Analysis resulted in mean exposure of 88% of nominal
Dose / conc.:
0.05 other: mM
Remarks:
Nominal concentration (10.750 mg/L). Analysis resulted in mean exposure of 107% of nominal
No. of animals per sex per dose:
40 embryos per concentration.
Control animals:
yes, concurrent vehicle
other: VPA: Positive control; DCA - for quality control test system
Details on study design:
- Dose selection rationale: based on a range finder with concentrations of 0.0215, 0.215, 1.075, 2.150 and 21.500 mg/L ((0.0001, 0.001, 0.005, 0.01, 0.1 mM)
- Other:

Determination of lethality (standar OECD 236 criteria):
- Coagulation of fertilized eggs
- Lack of somite formation
- Lack of detachment of the tail-bud from the yolk sac
- Lack of a heartbeat were used as indicators of lethality

Determination/presence of malformation:
There are an extensive number of observations that can potentially be made during the development of a fish embryo. Severeity was graded between "Severe malformation', 'Low severity malformation' and 'Variations' involving observations such as reduced size / activity / pigment but otherwise normal appearance and proportions.
Low and severe malformations will initially be pooled for the first pass statistical analysis.

Maternal examinations:
Not applicable
Ovaries and uterine content:
Not applicable
Fetal examinations:
- Body shape and proportions (in comparison to internal plate control)
- Length/size (in comparison to internal plate control)
- Somites
- Notochord
- Tail
- Fins
- Heart (limited to heartrate and pericardial sac)
- Head
- Face
- Jaws
- Eyes
- Mobility
- Pigmentation/colour
- Abdomen
- Yolk sac
Statistics:
Toxcalc 5.0 software package was used
Indices:
1. Calculations of the NOEC/LOEC and the LC50 for lethality were made as far as the data allowed.
2. Calculations of the NOEC/LOEC ECx for sub lethal/teratogenic effects were made as far as the data allowed (low and severe malformations pooled).
3. Data was evaluated separately i.e. malformation data was mortality corrected and was calculated from surviving not total animals at the start of the test.
4. Nonspecific effects on size, pigmentation and mobility were not considered developmental effects and were excluded from calculations on malformations
5. Historical data from positive control indicated the best concordance with literature data when pooling of all observed non-lethal malformations. Severe and non-severe malformations were therefore scored together as a single endpoint.
Historical control data:
Data from positive control were compared to historical data (part as quality control for the study)
Clinical signs:
not examined
Details on results:
Maternal observations are not relevant in this study.
Key result
Dose descriptor:
NOEC
Effect level:
0 other: mM
Based on:
test mat.
Basis for effect level:
mortality
Remarks on result:
other: See Remarks
Remarks:
Parantal toxiicty as such is not an element in the ZEDTA. However, as this assya also includes FET evaluation according to OECD 236 as measure for acute toxic toxiicty, the evaluations related to lethality (Involving Coagulation of fertilized eggs; Lack of somite formation; Lack of detachment of the tail-bud from the yolk sac; Lack of a heartbeat were used as indicators of lethality) can be regarded as representing teh same as parental toxiicty. The evaluation for developmental toxiicty basically is determined whether sensitivity for developmental effects occurs before lethal efefcts (comparable to severe maternal toxiicty).
Fetal body weight changes:
not examined
Description (incidence and severity):
BW is not a relevant parameter. Only embryonall growth and size is evaluated.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Not applicable.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The LC50 for lethality was determined as 0.003 mM. The LC25 was determined as 0.002mM.
If expressing the lethal endpoints as measured mean values of the test material the LC50 was 0.63 mg/L. The NOEC for lethality was 0.0004mM or 0.063mg/L.
Changes in sex ratio:
not examined
Description (incidence and severity):
Not applicable.
Changes in litter size and weights:
not examined
Description (incidence and severity):
Not applicable.
Changes in postnatal survival:
not examined
Description (incidence and severity):
Study ends at hatching around 96 hour
External malformations:
no effects observed
Description (incidence and severity):
There was no incraese in malformations.
0.003/0.006 (LC50 / EC50)= 0.5 (Selderslaghs et al. method) Conclusion: Non Teratogen
0.002/0.002 (LC25/NOEC) = 1 (Panzica-Kelly et al. method) Conclusion: Non Teratogen

For the positive control VPA:
1.19/0.27 (LC50 / EC50) = 4.4 Conclusion: Teratogenic (Selderslaghs et al.;Cutoff of 2)
1.05/0.1(LC25/NOEC) = 10.5 Conclusion: Teratogenic (Panzica-Kelly et al.; Cutoff 10)
Skeletal malformations:
no effects observed
Description (incidence and severity):
See tables
Visceral malformations:
no effects observed
Description (incidence and severity):
See tables
Other effects:
no effects observed
Description (incidence and severity):
See tables
Details on embryotoxic / teratogenic effects:
See result tables
Key result
Dose descriptor:
NOAEC
Effect level:
0.002 other: mM (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Severe plus non-severe malformations
Remarks on result:
other: To determine TI according Panzica-Kelly et al. method.
Key result
Dose descriptor:
other: LC25
Effect level:
0.002 other: mM (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Lethality
Remarks on result:
other: To determine TI according Panzica-Kelly et al. method.
Key result
Dose descriptor:
other: EC50
Effect level:
0.005 other: mM (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Severe plus non-severe malformations
Remarks on result:
other: To determine TI according Selderslaghs et al. method.
Key result
Dose descriptor:
other: LC50
Effect level:
0.003 other: mM (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Lethality
Remarks on result:
other: To determine TI according Selderslaghs et al. method.
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
Teratogenicity Index (TI) :
- Selderslaghs et al., 2012: TI = 0.5 (Not teratogenic)
- Panzica-Kelly et al., 2012: TI = 1 (Not teratogenic)
Key result
Developmental effects observed:
no

Controls to Valproic Acid (total 6 x 5 = 30)

Test

1

2

3

4

5

6

Mortality %

 

Malformations
Severe 

Malformations
Low severity

Hatching
%

Count

%

Count

%

Count

%

DSW

3HOK
2C

4HOK
1OK

3HOK
2C

4HOK
1BTsH

4HOK
1OK

4HOK
1OK

4

13.3%

0

0

1

3

88

DSW+ Acetone

2HOK
1HS
2C

4HOK
1DSC

3HOK
1C
1BT

4HOK
1C

3HOK
2C

4HOK
1OK

7

23.3%

1

6

1

6

87

3,4 DCA
(4 mg/L)

4M
1C

1ED
1BT
1S
2C

1M
4C

3M
2C

4M
1C

1BT
2M
2C

12

40.0%

14

46

4

13

0

Hatching success calculated from surviving embryos only

Valproic Acid Control (total 5 x 5 = 25)

Test Conc

1

2

3

4

5

6 IC

Mortality

 

M severe

M low severe

Total

Hatching %

Count

%

count

count

0.01 mM

4HOK
1BT

4HOK
1BT

3HOK
1C
1DSC

4C
1HOKS

4HOK
1C

4HOK
1C

7

28.0%

2

0

2

100

0.1  mM

3HOKS(Fins)
2C

2HOK
3C

5HOK

2HOK
2C
1OK

1BT
4OK

4HOK
1C

7

28.0%

1

3

4

66

1.0  mM

3M
2C

2BTM
2M
1C 

2HED
3M

4DSC
1C

2HOKS
2HBT
1C

5HOK

9

36.0%

12

2

14

38

2.0  mM

5DS C

1DSC
1C

5DSC

4DSC
1C

3C
2DSC

4HOK
1M/D

25

100.0%

 

 

 

0

Percentage survival from internal controls: 

85.0%

Hatching success calculated from surviving embryos only. N.D Not determined.

Control to 3-(Isodecyloxy)-1-propanamine (C10i-EA) (total: 6x2 = 12)

Test Conc 1 2 3 4 5 6 Mortality   M severe M low severe Total Hatching %
Count % count count
DSW 2HOK 1HOK
1HOKCol
2HOK 2HOK 2HOK 1HOK
1C
1 8.3% 0 0 0 100
DSW 1HOK
1C
1MT▲ED
1HOK
2HOK 2HOK 1HOK
1C
2HOK 2 16.7% 1 0 1 90
DSW 1HOK
1C
2HOK 2HOK 2HOK 1HOK
1C
1HOK
1BTs▲
2 16.7% 0 1 1 90
DSW 2HOK 2HOK 2HOK 1HOK
1C
2HOK 1HOK
1BTs
1 8.3% 0 1 1 91
AVG 12.5% 2.4% 7.1% AVG

C10i-EA, 0.00008 mM (total: 5x2 = 10)

Test Conc
mM
1 2 3 4 5 6 Mortality   M severe M low severe Total Hatching %
Count % count count
0.00008 1HOK
1C
1HOK
1BTsH
1HOK
1C
2HOK 1M
1Col BTs▲
2C 2 20.0% 1 2 3 62.5
0.00008 1HOK
1D*
2HOK  1HOKCol
1C
1HOK
1OK
1HOK
1C
1HOK
1MT
3 30.0% 1 0 1 85.7%
0.00008 1HOK
1C
1HOK
1C
1HOK
1C
1HOK
1BTH▲
2HOKCol 2HOK 3 30.0% 1 0 1 100
0.00008 2HOK  1HOK
1BTsH
1HOK
1C
1HOK
1BT
2HOK 2HOK 1 10.0% 1 1 2 89
Percentage survival from internal controls:  75.0% 22.5% 12.9% 22.6%

C10i-EA, 0.0004 mM (total: 5x2 = 10)

Test Conc
mM
1 2 3 4 5 6 Mortality   M severe M low severe Total Hatching %
Count % count count
0.0004 2HOK 2HOK 2HOK 2HOK 2HOK 2HOK 0 0.0% 0 0 0 100
0.0004 1MEDT▲
1C
1HOK
1C
2HOKS 1HOKS
1BT▲
2HOKCol Mob 1HOK
1C
2 20.0% 2 0 2 75
0.0004 2HOKS 1HOKS
1C
2HOK 2HOK 1HOK
1BTs
1HOK
1C
1 10.0% 0 1 1 89
0.0004 1OK
1HOK
1OK
1HOK
1HOKCOL
1C
1HOK
1CVM ED▲
2HOK 1HOK
1C
1 10.0% 1 0 1 67
Percentage survival from internal controls:  62.5% 10.0% 8.3% 11.1%

C10i-EA, 0.002 mM (total: 5x2 = 10)

Test Conc
mM
1 2 3 4 5 6 Mortality   M severe M low severe Total Hatching %
Count % count count
0.002 1HOK
1C
1HOK
1C
1C
1MT
1HOK
1BT(Meye)▲
2HOK 2HOK 3 30.0% 2 0 2 71
0.002 2HOK 2HOK 1HOK
1C
2C 2HOK 1HOK
1C
3 30.0% 0 0 0 100
0.002 2HOK 2HOK 1C
1HOK
1C
1HOK
1HOK
1DS C
1OK
1BTs
3 30.0% 0 0 0 100
0.002 2C 1HOK
1OK
1HOK
1BTs
2BT▲ 2C 1OK
1Del
4 40.0% 2 1 3 33
Percentage survival from internal controls:  87.5% 32.5% 14.8% 18.5%

C10i-EA, 0.01 mM (total: 5x2 = 10)

Test Conc
mM
1 2 3 4 5 6 Mortality   M severe M low severe Total Hatching %
Count % count count
0.01 2C 2C 2C 2C 2C 1HOK
1OK
10 100.0% 0 0 0 0
0.01 2C 2DSC 1C
1DSC
2C 1DSC
1C
1HOK
1C
10 100.0% 0 0 0 0
0.01 2C 1C
1DSC
1HBT
1C▲
2C 1DSC
1MT▲
1HOK
1C
8 80.0% 2 0 2 20
0.01 1HBTs
1C
1C
1DSC
1C
1DSC
1C
1DSC
1C
1DSC
1HOK
1C
9 90.0% 1 0 1 10
Percentage survival from internal controls:  62.5% 92.5% 100.0% 100.0%

C10i-EA, 0.05 mM (total: 5x2 = 10)

Test Conc
mM

1

2

3

4

5

6

Mortality

 

M severe

M low severe

Total

Hatching %

Count

%

count

count

0.05

2C

2C

2C

2C

2C

2OK

10

100.0%

 

 

 

 

0.05

2C

2C

2C

2C

2C

2OK

10

100.0%

 

 

 

 

0.05

2C

2C

2C

2C

2C

1OK
1C

10

100.0%

 

 

 

 

0.05

2C

2C

2C

2C

2C

2OK

10

100.0%

 

 

 

 

Percentage hatched from internal controls: 

87.5%

100.0%

Conclusions:
According to two validated methods used to evaluate Teratogenic potential with this assay, the test substance is concluded to be not teratogenic.
Executive summary:

3-(Isodecyloxy)-1-propanamine (C10i-EA) was evaluated for developmental toxicity in Zebrafish Embryo Developmental Toxicology Assay (ZEDTA).

This ZEDTA was conducted according to the method described in EURL ECVAM ‘Zebrafish Embryo Assay for Developmental Toxicity’. Additionally, validity criteria of the study were based on the 96-hour Fish Embryo Toxicity (FET) OECD 236 test guideline.

Method:

Two approaches were used for interpretation of the test data with regards to developmental effects via the calculation of a teratogenic index (TI) based on nominal concentrations: One using the LC50/EC50-malformations ratio, concluding to a positive result when the ratio is ≥ 2 (Selderslaghs et al. method) and the other using the LC25/NOEC-malformations ratio, concluding to a positive result when the ratio is ≥ 10 (Panzica-Kelly et al. method). The last approach is also suggested in the ECVAM method.

Chemical analysis with a validated method was conducted to quantify the substance exposure. Measured values however, are relevant for the acute lethal toxicity endpoints but not for interpretation of the developmental toxicity data. These data are therefore only reported based on nominal concentrations (in mM).

Dutch Standard Water (DSW) was used for testing. The following concentrations of 3-(Isodecyloxy)-1-propanamine (C10i-EA) were prpared 0.00008, 0.0004, 0.002, 0.01, 0.05 mM (0.0172, 0.0860, 0.430, 2.150, 10.750 mg/L), for the quaility control DCA: 4 mg/L, and for positive control VPA: Concentrations of 0.01, 0.1, 1.0, 2.0 mM (1.66, 16.6, 166.2 332.4 mg/L). Replenishment after 48 hours from a newly made stock solution was carried out.

Samples for concentration analysis were taken at the start and the end of the test as well as old and new solutions before and after solution replenishment.

Fertilized eggs were between 2-6 hours old when added to the test solutions. This was confirmed by microscopic observation. Eggs failing to develop and possibly not fertilized (but added to test at T=0) were replaced with other fertilized eggs (also exposed to test material) before 6h had expired. This ensured that living embryos were present at the start of test and prevented fertilization success influencing the test data.

The Internal control (IC) replicates were not replaced before 6h (if not fertilized) and were therefore used to calculate the average fertilization % of the embryo batch used.

Tests were performed in 24-well plates, with 2 embryos per well, and 40 embryos per concentration and 8 embryos as an internal plate control. For the controls 48 embryos (2 per well) were used in all controls.

Every 24 hours, observations were recorded. Effects on the embryo in comparison to the controls were noted. At the end of the exposure period, acute lethal toxicity was determined based on a positive outcome in any of the four apical observations as detailed in the OECD 236 guideline.

Also all non-lethal findings were recorded at this stage. After final observations at 96-hours, a 96-hour NOEC / ECx level for develepmantal effects/malformations and lethality were generated to allow for TI (Teratogenicity index) calculations according to the Selderslaghs et al and/or Kelly et al methods.

Results:

Chemical analysis with a validated method was conducted to demonstrate sufficient substance exposure. Measured mean concentrations ranged from 72 -107% of the nominal concentrations.

The biological validity criteria were primarily met for this study and the analytical validity criteria were concluded acceptable for a robust determination of the test concentrations,

Assay validity was further confirmed by adequate performance with the quality control Dichloroanaline and the correct conclusion for the positive control Valproic acid.

All of the required endpoints could be calculated.

The results of this study are therefore considered a reliable representation of the effects of 3-(Isodecyloxy)-1-propanamine to developing D.Rerio embryos.

Teratogenic index (TI) for 3-(Isodecyloxy)-1-propanamine:

- TI, LC50/EC50 = 0.5 (Selderslaghs et al. method, positive if TI ≥ 2); Conclusion: Non Teratogen

- TI, LC25/NOEC = 1 (Panzica-Kelly et al. method, positive if TI ≥ 10); Conclusion: Non Teratogen

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Full study for developmental toxicity; high quality OECD 414 guideline in compliance to GLP.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
Exposure of humans to Etheramine C10i via inhalation is not likely taking into account the low vapour pressure and high boiling point of the substance as well as low possibility of exposure to aerosols or droplets of an inhalable size. Furthermore, as the substance is classified as corrosive, local effects will be dose-limiting and preclude sufficient uptake for systemic effects to develop.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Quality of whole database:
Substance is corrosive. Effects will be characterized by local corrosive effects that are related to duration, quantity and concentration, rather than by systemic toxicity due to dermal uptake. Dermal absorption is expected to be much lower compared to oral absorption. Related to corrosive properties, exposures are likely to be limited.
Additional information

Etheramine C10i has been evaluated in a Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats by oral gavage followed by a 14-day recovery period, according to OECD 422 and in compliance to GLP.

Based on limited local effects in the stomach following treatment of a corrosive substance with full recovery during 14-day recovery period, a parental NOAEL of 50 mg/kg/day was derived for systemic toxicity.

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.

 

In a GLP-compliant OECD Guideline 414 study, pregnant rats (22/dose) were administered Etheramine C10i dissolved in water by oral gavage at 0 (vehicle controls), 8, 25 and 75 mg/kg bw/day from Day 6 to Day 21 post-coitum. All animals surviving to Day 21 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. The stomach and gross lesions were collected and fixed from all animals at necropsy.

Histopathological examination was performed on the stomach from 10 selected females of all groups.

Treatment at 75 mg/kg resulted in statistically significantly lower body weights and body weight gains on Days 15 and 21 post-coitum and from Day 9 post-coitum onwards, respectively. In addition, weight gain corrected for gravid uterus was statistically significantly lower at 75 mg/kg bw/day when compared to controls (5.4 gram versus 26.5 gram). Absolute and relative food consumption were statistically significantly lower at 75 mg/kg bw/day during the entire treatment period. Test item-related morphological findings were present in the stomach of females treated at 75 mg/kg bw/day, consisting of ulceration, diffuse forestomach hyperplasia and/or increased incidence and/or severity of hyperkeratosis of the limiting ridge. Based on these effects the NOAELs for maternal toxicity for both systemic and local effects were set to 25 mg/kg bw/day.

There were no effects on the number of abortions, early or late resorptions, total implantation losses or number of live fetuses. There were no toxicologically relevant or test item related external, visceral and skeletal malformations or variations noted by treatment up to 75 mg/kg. Fetal body weights in both sexes were statistically significantly lower at 75 mg/kg bw/day when compared to controls. The level of statistical significance was considered to have arisen as a result of the slightly high concurrent control value. In addition, as no clear dose related trend was observed and no effects on skeletal developmental were noted, this was considered to be related to the significant lower body weights of the dams rather than a developmental cause. Hence, this minimal change in fetal body weight was considered to be non-adverse.

Based on this the NOAEL for developmental effects was set at 75 mg/kg bw/day.

Further evaluation for developmental toxicity in a non-rodent species involves testing of Etheramine C10i in the Zebrafish Embryo Developmental Toxicology Assay (ZEDTA). The development of the zebrafish embryo is very similar to the embryogenesis in higher vertebrates, including humans, and many molecular pathways are conserved between zebrafish and humans. The Zebrafish Embryo Teratogenicity Assay can not only be applied as a screening tool for teratogenicity, but also as a means of investigating specific mechanisms related to the teratogenic potential of certain substances. This assay has been researched thoroughly for these purposes and has achieved high levels of concordance with mammalian developmental toxicity data.

This ZEDTA was conducted according to the method described in EURL ECVAM ‘Zebrafish Embryo Assay for Developmental Toxicity’. Additionally, validity criteria of the study were based on the 96-hour Fish Embryo Toxicity (FET) OECD 236 test guideline.

Two approaches were used for interpretation of the test data with regards to developmental effects via the calculation of a teratogenic index (TI) based on nominal concentrations: One using the LC50/EC50-malformations ratio, concluding to a positive result when the ratio is ≥ 2 (Selderslaghs et al. method) and the other using the LC25/NOEC-malformations ratio, concluding to a positive result when the ratio is ≥ 10 (Panzica-Kelly et al. method). The last approach is also suggested in the ECVAM method.

Chemical analysis with a validated method was conducted to demonstrate sufficient substance exposure.

Results: The biological validity criteria were primarily met for this study and the analytical validity criteria were concluded acceptable for a robust determination of the test concentrations,

Assay validity was further confirmed by adequate performance with the quality control Dichloroanaline and the correct conclusion for the positive control Valproic acid.

Teratogenic index (TI) for Etheramine C10i:

- TI, LC50/EC50 = 0.5 (Selderslaghs et al. method, positive if TI ≥ 2); Conclusion: Non Teratogen

- TI, LC25/NOEC = 1 (Panzica-Kelly et al. method, positive if TI 10); Conclusion: Non Teratogen

An additional consideration is that specific teratogenic effects or specific organ interactions from Etheramine C10i are unlikely. Etheramine structure consists of an apolar alkyl chain and a polar primary amine group on the other end of the molecule.In physiological circumstances the nitrogen is positively charged, resulting to a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces such as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Cytotoxicity at the local site of contact through disruption of cell membrane will is considered the most prominent mechanism of action for toxic effects.

Mode of Action Analysis / Human Relevance Framework

The most significant treatment-related changes in the studies performed on etheramines involve local irritating/corrosive effects. Results from in vivo studies support this, where the critical effects are based on local effects in the stomach, despite the fact that profiling and available information in support of TK further indicate general good absorption.

In physiological circumstances, the etheramines have a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar tails easily dissolve in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule is not expected to easily pass membrane structures, and consequently, reproduction toxicity is not a likely concern.

Justification for classification or non-classification

Available data do not indicate a concern for reproduction or developmental toxicity.