Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
31 January 2013 - 17 June 2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age/weight at study initiation: on the first day of treatment, the males were approximately 10 weeks old and had a mean body weight of 388 g (range: 335 g to 438 g) and the females were approximately 9 weeks old had a mean body weight of 236 g (range: 203 g to 270 g)
- Housing: the animals were individually housed, except during pairing and lactation, in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 50±20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 13 February 2013 to 09 April 2013
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The dose formulations were prepared according to the following process, as described in an homogeneity/stability study:
- a small amount of test item was melt using water bath at a temperature of +50°C,
- the vehicle was warmed at +50°C using water bath,
- the appropriate amount of melt test item was weighed in the preparation beaker and the corresponding amount of vehicle was added,
- the test item and the vehicle were mixed using magnetic stirrer in the water bath at +50°C during 10 minutes,
- the obtained suspension was stirred at ambient temperature at least 30 minutes.

No correction factor was applied.
The test item dose formulations were prepared on a weekly basis, stored at room temperature protected from light prior to use and delivered to the study room at room temperature and protected from light.

When not on the day of formulation preparation, test item formulations were warmed under magnetic stirring at +50°C using water bath during at least 30 minutes and then kept under magnetically stirring at ambient temperature for at least 30 minutes before daily delivery to the study room.

VEHICLE
- Choice of vehicle: good suspension in corn oil
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred as day 0 post-coitum
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: assessed in homogeneity study (satisfactory results)
Stability: assessed in stability study (stable after 9 days at room temperature and protected from light)
Duration of treatment / exposure:
In the males:
− 2 weeks before mating,
− during the mating period,
− until sacrifice (at least 5 weeks in total),

In the females:
− 2 weeks before mating,
− during the mating period (1 week),
− during pregnancy,
− during lactation until day 5 post-partum inclusive,
− until sacrifice for females which had not delivered.
Frequency of treatment:
Daily
Details on study schedule:
- No F1 parents (only one generation mated)
- Age at mating of the mated animals in the study: 11-12 weeks
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Controls
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, following the results of a previous 2-week toxicity study. In this study, three groups of three male and three female Sprague-Dawley rats received the test item as a suspension in corn oil at 100, 300 or 1000 mg/kg/day bw for 2 weeks by gavage. There were no unscheduled deaths. Reduced mean body weight gain and mean food consumption were noted in males during the first week of treatment. Clinical signs were ptyalism in all test item-treated animals and hunched posture in two males and one female at the high-dose during the second week of treatment. There were no test item-related macroscopic findings.

- Animal assignment: computerized stratification procedure.
Positive control:
no (not required).
Parental animals: Observations and examinations:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.

CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

DETAILED CLINICAL SIGNS:
- Time schedule: once a week during the treatment period.

BODY WEIGHT:
- Time schedule: Males: on the first day of treatment, then once a week until sacrifice. Females: on the first day of treatment, then once a week until mating (or until sacrifice), on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Time schedule: Males: once a week until sacrifice. Females: once a week until mating, on Days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

- NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: at the end of the treatment period.

HAEMATOLOGY:
- Time schedule: on the day of sacrifice.

CLINICAL CHEMISTRY:
- Time schedule: on the day of sacrifice.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded.
Oestrous cyclicity (parental animals):
Fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until females are mated.
Sperm parameters (parental animals):
Parameters examined in males of parental generation:
- weighing and microscopic examination:yes
- special emphasis to the spermatogenic cycle and testicular interstitial cells (groups 1 and 4).
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: all surviving animals after the end of the mating period
- Female animals: all surviving animals = day 6 post-partum or, for females which had not delivered yet, day 25 post-coitum.

ORGAN WEIGHTS: Yes

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination.

HISTOPATHOLOGY:
- on all tissues listed in the table below for the first five control and high dose animals (groups 1 and 4) sacrificed as scheduled,
- on all macroscopic lesions,
- all females sacrificed because of no delivery to investigate possible causes,
- liver (both sexes), thymus (females only), seminal vesicles (males only) and bone marrow (females only) from the first five sacrificed as scheduled males and the first five females sacrificed on day 6 p.p. of the low- and intermediate-dose groups (groups 2 and 3).
Postmortem examinations (offspring):
SACRIFICE: on day 5 post-partum

GROSS NECROPSY: on all pups (surviving and found dead)

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No
Reproductive indices:
Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)
Offspring viability indices:
Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on day 4 p.p. = 100 * (Number of surviving pups on day 4 p.p. / Number of live born pups)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism, considered of minor toxicological significance, was observed in all animals at 300 and 1000 mg/kg/day bw from the first or second week of treatment and in most of the animals at 100 mg/kg/day bw but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg/day bw but more at 1000 mg/kg/day bw were considered to be of limited toxicological significance. Incidental findings consisted of half-closed eyes, reflux at dosing, cutaneous lesion and abnormal growth of teeth.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no deaths considered as test item-related. One female given 1000 mg/kg/day bw was prematurely sacrificed on day 23 p.c. due to difficulty to deliver. Piloerection, round back, cold to the touch, abdominal breathing, reddish vaginal discharge, chromodacryorrhea and chromorhinorrhea were observed on day 22 and/or 23 p.c. Blood in the bedding, fetuses (mostly dead) and a few placentae were found in the bedding on day 23 p.c. This death was considered to be incidental. There were no other unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no relevant effects on mean body weights and mean body weight gains. The low mean body weight gain in high-dose males for the interval days 1 to 8 when compared to controls was considered to be of no toxicological significance (no statistical level and transient decrease).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In males, mean food consumption at 1000 mg/kg/day bw was statistically significantly reduced in the first week of treatment when compared with controls (which correlated with a non-statistically significantly lower mean body weight gain at that time). It became similar to controls in the second week of dosing. This slight and transient effect was considered to be of limited toxicological significance. Mean food consumption at 100 and 300 mg/kg/day bw was not affected. Mean food consumption in females was considered to be unaffected by the test item treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
APPT values were not considered to be affected (one control data in males was higher than the others hence the shortened mean values in the test item-treated groups; there was no dose-relationship in females). For the slightly higher mean white blood cell counts when compared with controls, a relationship with the test item treatment was considered to be doubtful (no clear dose-relationship, individual values generally included in historical control data and no statistical level).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean cholesterol level was higher in test item-treated female groups in a dose-related manner, reaching statistical and toxicological significance at 300 and 1000 mg/kg/day bw. All individual values in both groups were included in historical control data and in absence of adverse correlates in the study, this was considered to be non-adverse. There was no dose relationship in males, but an effect of the test item may be suspected in two males treated at 1000 mg/kg/day bw which had a high cholesterol level (2.3 and 2.4 mmol/L). Mean sodium concentration was also statistically significant higher in females treated at 1000 mg/kg/day bw when compared with controls. Males or other electrolytes in females were not affected and the variation was not severe. Therefore, this finding was not considered to be of toxicological significance.An effect of the test item treatment on mean albumin concentration at 300 mg/kg/day bw in both sexes was considered unlikely as there was no dose-relationship. The increase observed in triglyceride levels at 300 and 1000 mg/kg/day bw in both sexes was considered to be incidental as there was no statistical level reached for the group means and no dose-relationship seen in individual data. Moreover, the increase at 1000 mg/kg/day bw was due to isolated animals per sex only.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no relevant findings on FOB in test item-treated groups when compared to the control group. An effect of the test item treatment in males and females at 300 and/or 1000 mg/kg/day bw on mean motor activity data was considered to be equivocal but of limited toxicological significance. The vast majority of the individual data were similar to what can usually be seen in this type of study.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related changes were observed in the liver and the thymus.

Liver
Minimal hepatocellular hypertrophy was seen in 1/5 males given the test item at 300 mg/kg/day bw and 4/6 males and 2/5 females given 1000 mg/kg/day bw. This non-adverse change was considered to be test item-related and correlated with the higher weight noted at necropsy. Other changes seen in the liver, including the minimal foci of subcapsular necrosis seen in 1/5 control females, 1/5 males at 300 mg/kg/day bw and 2/5 females at 1000 mg/kg/day bw were considered to be fortuitous and without any relationship with the test item.

Thymus
Minimal or slight lymphoid atrophy was seen in 2/5 females given 1000 mg/kg/day bw, correlating with the lower weight at necropsy. Any relationship with the test item was considered to be likely but non-adverse (low number of animals affected and low grades). No test item related changes were seen at 100 or 300 mg/kg/day bw.

Miscellaneous changes
- Seminal vesicles
Minimal apoptosis was seen in 1/5 males given 100 mg/kg/day bw and 3/6 animals given 1000 mg/kg/day bw. As this change was minimal, and in the absence of a dose-related trend and other test item-related changes in testes or epididymides, any relationship with the test item was considered to be unlikely.

- Bone marrow
There was a trend towards higher presence of adipose tissue in the bone marrow of females at all dose levels when compared with controls. As this was not seen in males, poorly dose-related and as this was not correlated with any changes in the hematological parameters, any relationship with the test item was excluded.

- Lungs
Mucus was seen in bronchi and/or bronchioli of 3/6 males given 1000 mg/kg/day bw and eosinophilic infiltrate in 3/6 males at this dose-level. Increased alveolar macrophages were seen at a similar incidence and severity between controls and treated animals in both sexes. These changes were consistent with aspiration or regurgitation of the oily vehicle or test item during the technical gavage procedures. Any direct relationship with the test item was excluded.

A careful examination of testes, epididymides, and female genital tract did not show any test item-related effects on these organs. Other histopathological changes were considered to be part of the normal background of changes commonly seen in rats and without any relationship with the test item.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating and fertility data
There were no test item-related effects on mean mating and fertility data. At 1000 mg/kg/day bw, one female was blocked in metestrous/diestrous for 12 days and mated with its male after 13 days, and one female was prematurely sacrificed on day 23 p.c. because of difficulty to deliver as previously mentioned. Both events were considered to be incidental as isolated. A total of five females (three controls and two females treated at 1000 mg/kg/day bw) were sacrificed on day 25 p.c. because of no delivery. They were non-pregnant.

Delivery data
There were no test item-related effects on mean delivery data. Mean pre- and post-implantation loss data were lower than the highest percentages seen in historical control data, thus there were no effects of the test item treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Parental toxicity (non adverse)
Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive performance (mating and fertility)
Key result
Critical effects observed:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
None of the clinical signs were considered to be test item related (comparable incidences in control pups).
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no effects on pup viability.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects of the test item treatment on mean body weight and mean body weight changes in pups. The trend toward higher mean body weights and mean body weight gain at 100 mg/kg/day bw was considered to be incidental (not dose-related).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Both findings are not very often recorded in controls of such studies, although they can appear spontaneously. An effect of the test item treatment was suspected since they were observed with a dose relationship at both the highest dose-levels. However, the effect was considered as of minor toxicological significance as these findings are generally considered as morphological variations (not malformations) and as their incidence was not severe.
Other effects:
no effects observed
Description (incidence and severity):
SEX RATIO
Despite the fact that the pup sex ratio at 1000 mg/kg/day bw was slightly above the historical control range, it was considered to be of no toxicological significance in view of its low amplitude.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Toxic effects on progeny (minor toxicological significance)
Key result
Critical effects observed:
not specified
Key result
Reproductive effects observed:
no
Conclusions:
Under the study conditions, the NOAEL for parental toxicity, the NOEL for reproductive performance (mating and fertility) and the NOAEL for toxic effects on progeny were considered to be 1000 mg/kg/day bw.
Executive summary:

A study was conducted to determine the repeated dose toxicity of the read across substance, C18-unsatd. MIPA, in rats according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. The concentration of the dose formulation was checked in study Weeks 1, 3 and 6. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 p.c. and lactation on Days 1 and 5 p.p. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1 and 5 p.p. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (hematology and blood biochemistry) were carried out on five males and five females. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control- and high-dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low- and intermediate-dose groups and on all macroscopic lesions. The pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/day and in most of the animals at 100 mg/kg bw/day (minor toxicological significance), as well as hypoactivity, loud breathing, piloerection and/or round back observed in a few animals at 300 and 1000 mg/kg bw/day for a few days (limited toxicological significance). There were no relevant effects on mean body weight, mean Functional Observation Battery (FOB), as well as on mean hematology parameters in any group and sex. An effect of the read across substance treatment on mean motor activity data at 300 and/or 1000 mg/kg bw/day was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg bw/day was reduced in the first week of treatment only (23 g/male/day, vs. 27, p<0.001). This effect was considered to be of limited toxicological significance. Mean food consumption in males at 100 and 300 mg/kg bw/day and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg bw/day (up to 1.9 mmol/L, vs.1.2, p<0.01) and considered to be non-adverse in absence of adverse correlates in the study. There were no relevant blood biochemistry findings in females at 100 mg/kg bw/day or in males. At histopathology at 1000 mg/kg bw/day, minimal hepatocellular hypertrophy correlating with higher mean liver weight was seen in the liver of both sexes (about +28% in males and +20% in females compared to controls, p<0.01 generally). In females, mild lymphoid atrophy was seen in the thymus of 2/5 females, correlating with lower mean weight at necropsy (about -22% from controls). At 300 mg/kg/day, only minimal hepatocellular hypertrophy was seen in the liver of a single male correlating with minor higher mean absolute weight in this group. All these microscopic findings were considered to be non-adverse (low number of animals affected and/or minimal to slight grades). There were no histopathological effects at 100 mg/kg bw/day. There were no effects on mean mating, fertility and delivery data in any group. There were no read across substance-related effects on pup viability, clinical signs, body weight and sex ratio. Dilated ureter and renal pelvis were recorded with dose-relationship in a few litters at necropsy at 300 (1 or 2 litters affected out of 10) and 1000 (2/7 litters) mg/kg bw/day, vs. none in the controls. An effect of the read across substance treatment was considered to be of minor toxicological significance. Under the study conditions, the NOAEL for parental toxicity, the NOEL for reproductive performance (mating and fertility) and the NOAEL for toxic effects on progeny were considered to be 1000 mg/kg bw/day (Bentz, 2013).  

Based on the results of the read across study, a similar NOAEL values are expected for the test substance.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A study was conducted to determine the repeated dose toxicity of the read across substance, C18-unsatd. MIPA, in rats according to OECD Guideline 422, in compliance with GLP. Groups of ten male and ten female Sprague-Dawley rats received the read across substance at dose-levels of 0, 100, 300 or 1000 mg/kg bw/day daily by oral (gavage) administration 2 weeks before mating, during mating, gestation and until up to Day 5 p.p. The concentration of the dose formulation was checked in study Weeks 1, 3 and 6. The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating and mating periods (food consumption not during mating), and during gestation on Days 0, 7, 14 and 20 p.c. and lactation on Days 1 and 5 p.p. The animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 5 p.p. The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1 and 5 p.p. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (hematology and blood biochemistry) were carried out on five males and five females. The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 p.p. Final body weights and selected organs weights (adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes and thymus) were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on selected organs from five males and five females in the control- and high-dose groups, on liver, thymus, seminal vesicles and bone marrow from five males and/or five females in the low- and intermediate-dose groups and on all macroscopic lesions. The pups were sacrificed on Day 5 p.p. and submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The read across substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no read across substance in control formulations. There were no read across substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg/day bw and in most of the animals at 100 mg/kg/day bw (minor toxicological significance), as well as hypoactivity, loud breathing, piloerection and/or round back observed in a few animals at 300 and 1000 mg/kg/day bw for a few days (limited toxicological significance). There were no relevant effects on mean body weight, mean Functional Observation Battery (FOB), as well as on mean hematology parameters in any group and sex. An effect of the read across substance treatment on mean motor activity data at 300 and/or 1000 mg/kg/day bw was considered to be equivocal but of limited toxicological significance. In males, mean food consumption at 1000 mg/kg/day bw was reduced in the first week of treatment only (23 g/male/day, vs. 27, p<0.001). This effect was considered to be of limited toxicological significance. Mean food consumption in males at 100 and 300 mg/kg/day bw and in females were not affected. In females, mean cholesterol level was higher than in controls at 300 and 1000 mg/kg/day bw (up to 1.9 mmol/L, vs.1.2, p<0.01) and considered to be non-adverse in absence of adverse correlates in the study. There were no relevant blood biochemistry findings in females at 100 mg/kg/day bw or in males. At histopathology at 1000 mg/kg/day bw, minimal hepatocellular hypertrophy correlating with higher mean liver weight was seen in the liver of both sexes (about +28% in males and +20% in females compared to controls, p<0.01 generally). In females, mild lymphoid atrophy was seen in the thymus of 2/5 females, correlating with lower mean weight at necropsy (about -22% from controls). At 300 mg/kg/day, only minimal hepatocellular hypertrophy was seen in the liver of a single male correlating with minor higher mean absolute weight in this group. All these microscopic findings were considered to be non-adverse (low number of animals affected and/or minimal to slight grades). There were no histopathological effects at 100 mg/kg/day bw. There were no effects on mean mating, fertility and delivery data in any group. There were no read across substance-related effects on pup viability, clinical signs, body weight and sex ratio. Dilated ureter and renal pelvis were recorded with dose-relationship in a few litters at necropsy at 300 (1 or 2 litters affected out of 10) and 1000 (2/7 litters) mg/kg/day bw, vs. none in the controls. An effect of the read across substance treatment was considered to be of minor toxicological significance. Under the study conditions, the NOAEL for parental toxicity, the NOEL for reproductive performance (mating and fertility) and the NOAEL for toxic effects on progeny were considered to be 1000 mg/kg/daybw (Bentz, 2013).

Also, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a reproductive and developmental screening study according to OECD Guideline 421 is planned with C8-18 and C18-unsatd. DEA to provide data on the substance and further support the read across approach proposed for the FAA category members. 

 

In addition, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a testing proposal is submitted for the conduct of an extended one generation reproductive toxicity study (EOGRTS) according to OECD Guideline 443 with C8-18 and C18-unsatd. DEA.

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Official Journal of European Community L 133, May 30, 1988; 87/302/EEC
Deviations:
no
Qualifier:
according to
Guideline:
other: Official Journal of European Community L 180, March 01, 1991; 91/325/EEC
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Wiga D-Sulzfeld
- Age at study initiation: 8-10 wk
- Weight at study initiation: 209 g (mean)
- Housing: Single animal in Makrolon Type M3 cage (Ebeco) with standard softwood bedding
- Diet: Pelleted Altromin Maintenance Diet 1324, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 43-66
- Air changes (per h): 10-15
- Photoperiod (h dark/h light): 12/12
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was suspended in Arachidis oil, DAB 9 such that the required dose per kg body weight was contained in 5 mL.

VEHICLE
Concentration in vehicle: 0, 20, 60 and 200 mg/mL
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Impregnation procedure: Purchased timed pregnant
Duration of treatment / exposure:
From Day 6 up to Day 15 post coitum
Frequency of treatment:
Once daily
Duration of test:
20 d
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of toxicological examinations done before (Report No. 486 = TBD 830034, June 27, 1983) (details not reported)


Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examination: On Day 0, 6, 16 and 20 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All maternal organs, with emphasis on the uterus and uterine contents



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
- Other: Position of fetus in the uterus
Fetal examinations:
External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [approximately half per litter]
- Skeletal examinations: Yes: [approximately half per litter]
- Head examinations: Yes: [approximately half per litter]

(See Table 1 for exact number of fetuses examined)
Statistics:
The following statistical methods were used:
If the variables could be assumed to follow a normal distribution, the Dunnett-Test, based on a pooled variance, was applied for the comparison between the treated groups and the control group.
The Steel-Test was applied when the data could not be assumed to follow a normal distribution.
Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information (Bonferroni-Holm-corrected).

Indices:
- Pre-implantation loss (%) = [(Number of corpora lutea - number of implantations)/number of corpora lutea] X 100
- Post-implantation loss (%) = [(Number of implantations - number of live fetuses)/number of implantations] X 100
- Sex ratio (%) = [(number of males/females)/number of fetuses] X 100
Historical control data:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation and propulsion of the head in all dose groups. Additionally, the highest dose group showed a severe salivation. These symptoms were noted variable in the individual groups during the application period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality at any dose level.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects on body weight gain were observed in the dams.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Placenta and uterus weight: No significant differences between the control and the treatment groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were observed in the survived dams except for one dam at 100 mg/kg/d bw, which showed greenish-brownish fluid in the uterine horn.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
None.
Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation loss was not affected by the treatment. The post-implantation loss and total embryonic deaths were significantly increased in all treatment groups. However, these findings were considered to be incidental (non-treatment-related) because the values in the 100 mg/kg/d bw group were significantly greater than other two higher dose groups and in each group there was one single female with a high incidence of embryonic death.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No treatment-related effects observed on any of the parameters examined
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The weights of live fetuses exhibited no significant differences on a litter and individual basis.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio of the fetuses was not affected by the treatment.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were observed at external examination of fetuses which were considered to be an effect of the treatment. 1 dead fetus in control and 7 dead fetuses (4 out of 7 partly mummified) in 100 mg/kg/d bw group were recorded. One fetus showed a stump tail at 300 mg/kg/d bw and paleness was observed in one fetus at 1000 mg/kg/d bw. These singular findings are normal observations in the animal strain used.
Skeletal malformations:
no effects observed
Description (incidence and severity):
(i) Retardations: No significant finding at 100 mg/kg/d bw. Two sternebrae were non-ossified in 19 and 29 fetuses (statistically significant) at 300 and 1000 mg/kg/d bw, respectively. Statistically significant increase in the number of fetuses with incomplete ossification of skull bones (17 fetuses) and decrease in the number of fetuses with incomplete ossification of 13th rib (0 fetus) was observed at 1000 mg/kg/d bw. The increased "incomplete ossified skull bones" was essentially due to only 2 dams. The other statistically significant differences were considered to be incidental because these retardation effects were not accompanied by weight retardation and were within the normal range of variation for this strain.
(ii) Variations: No variations in any group.
(iii) Malformations: One fetus with stump tail and missing vertebrae coccigycae at 300 mg/kg/d bw (not considered to be treatment-related).
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related abnormalities.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects observed on any of the parameters examined
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 2. Summary of performance of mated females

Treatment dose (mg/kg/d)

0

100

300

1000

No. of mated females

30

30

30

30

No. of pregnant females

30

29

28

29

No. of females

with premature litter

1

0

2

3

No. of mortalities

0

0

0

0

No. of females with live

fetuses at termination

29

29*

26

26

* One dam out of these was not included because the weights of fetuses were not determined

Conclusions:
Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C12-18 and C18-unsatd. DEA, according to OECD Guideline 414, in compliance with GLP. The substance was administered to groups of 30 female rats by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day, once daily from Gestation Days (GD) 6 to 15 inclusive. Control animals were dosed with the vehicle alone (arachis oil, DAB 9). Clinical condition and reaction to treatment were recorded at least once daily. Body weights were reported on GD 0, 6, 16 and 20. All surviving females were sacrificed on GD 20 and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities. No deaths or treatment-related changes in body weight gain and necropsy findings were observed in dams at any dose level. Treatment-related symptoms observed in all groups were salivation and propulsion of the head. The highest dose group showed severe salivation. Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. Pre-implantation loss and mean numbers of resorptions were not affected by treatment. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related. Mean placental and uterus weights were not affected by the treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related visceral and skeletal abnormalities/variations. One foetus at 300 mg/kg bw/day showed a stump tail and missing coccigycae vertebrae. Further, the data for skeletal ossifications showed some deviations in the two highest dose groups. However, all these effects were assessed to be non-treatment-related. Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day (Pitterman, 1994). Based on the results of the read across study, a similar NOAEL values are expected for the test substance.

Endpoint:
developmental toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
November 2016 - June 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Remarks:
no major deviations
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
SPF (Specific Pathogen Free) Sprague-Dawley - Crl: OFA (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 L'ARBRESLE Cedex, France
- Age at study initiation: 10 to 11 weeks at the beginning of the treatment period
- Weight at study initiation: Between 224.4 and 326.7 g on the day of randomisation (d5pc) The weight variation of animals used was minimal (+/- 20% of the mean weight). About 10% more animals were ordered to allow selection of animals according to the criterion of body weight and they were used as spare animals in case of unforeseen events happen
- Fasting period before study:
- Housing: Animals were housed individually in cages of standard dimensions with sawdust bedding.
- Diet (e.g. ad libitum): RM3 (E)-SQC SDS/DIETEX feed (quality controlled/radiation sterilised) was available ad libitum except during the fasting experimental period. The certificate of analysis concerning this feed product is included in the report. The criteria for acceptable levels of contaminants in the feed supply were within the limits of the analytical specifications established by the diet manufacturer.
- Water (e.g. ad libitum): Drinking water was available ad libitum in polycarbonate bottles with a stainless steel nipple. A specimen of water is obtained approximately every 6 months and sent to Laboratoire de la Touraine - ZA n°1 du Papillon - Rue de l'Aviation - 37210 Parçay-Meslay - France, for analysis. The certificates of analysis are included in the report. The criteria for acceptable levels of contaminants in the water supplied were within the limits of the analytical specifications.
- Acclimation period:Animals arrived at CERB on day 1 of pregnancy (d1pc). Animals were supplied in several batches for logistical reasons. Each animal had five days in the laboratory animal house where the experiment took place before beginning of dosing. Only animals without any visible sign of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The animals were placed in an air-conditioned (20-24°C) animal house
- Humidity (%): relative humidity between 45% and 65% (except during the cleaning slot). Between 28 Nov at 07.35 p.m. up to 29 Nov at 11.10 a.m., an abnormal decrease of hygrometry was noted in animal room.
- Air changes (per hr): non-recycled filtered air was changed approximately 10 times per hour
- Photoperiod (hrs dark/hrs light): The artificial day/night cycle involved 12 hours light and 12 hours darkness with light on at 7.30 a.m

IN-LIFE DATES: From: 25 November 2016 To: 22 December 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
DIET PREPARATION no information available

VEHICLE
Corn oil will be used (Reference C8267)
- Lot/batch no. (if required): MKBS6944V and MKBW9504V, Expiry dates: 08 Oct 2020 and 06 Oct 2021 respectively
- Amount of vehicle (if gavage): 5 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of test item in formulations were checked during the first and last week of the study. Each concentration level and the vehicle were checked. 1 mL samples of each test item dosing formulation were taken in duplicate by top, middle and bottom sampling. Similar samples from the vehicle were taken, from the middle of the formulation only. Only the middle samples were assayed. Samples were collected in glass ontainers and stored at room temperature. Labels on the containers were marked in waterproof ink with Testing Facility, Study Number, Name of the test item and Concentrations, Sampling Date, Sampling Time and Storage conditions. The sample labels also indicated whether the samples were taken from the top, middle (for vehicle) or bottom of the formulation container. The identity and concentration of the test item in the samples and the absence of the test item in the control formulation was determined by liquid chromatography with UV-detection within the validated stability period available at this moment i.e. one week after sampling Acceptance criteria of the formulation analysis were fixed usually +/- 15% to the nominal concentration. The analytical method is validated within the range 85-115% of the theoretical concentration for formulations between 19.765 mg/mL and 198.120 mg/mL, with a precision better than 10%. The samples in corn oil must be analysed within the stability period (i.e. 30 days at room temperature) and must be within the range 85-115% to meet the acceptance criteria.

Formulation analysis was performed on one formulation prepared during the first and the last week of the study. The concentrations tested were 20 mg/mL, 60 mg/mL and 200 mg/mL. The concentrations found were within the range of acceptance (15% of the intended concentration). The absence of test item was also confirmed in the vehicle samples. Therefore, these data confirmed that the formulations were properly prepared.
Details on mating procedure:
- Impregnation procedure: The breeding establishment will be responsible for mating. Females will be mated at the beginning of the morning. They will be inspected for the presence of a vaginal plug.

- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy (d0pc)
Duration of treatment / exposure:
The test item administered in pregnant rats from day 6 to day 19 of gestation
Frequency of treatment:
N-(2-hydroxypropyl) Oleamide or its vehicle will be administered once a day at approximately the same time (a maximum range of 4 hours between the start and the end of the daily treatment) at each chosen dose level
Duration of test:
21 days (days 0-20 post coitum)
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
20 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Proposed doses are selected in agreement with the Sponsor. The choice is based on previous studies (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test by oral route (gavage) in rats - OECD 422 - Study No. 39644 RSR). Moreover, the highest dose should reveal signs of toxicity and the lowest dose should represent a no-observed-adverse effects level.
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were observed in the home cage before the first dosing and at least once a day during the study except on d7 for 3/20 female of group 2. The time of observation during the treatment period was at 60 min post dose (+/- 30 min). Females showing signs of abortion or of premature delivery during the study, the day on which such findings seen were noted.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the following days:
• on d1 and d5 (day of randomisation)
• daily during treatment (from d6 to d19)
• on d20, day of necropsy, not fasted and not exsanguinated

FOOD CONSUMPTION : Yes
- Food consumption was measured and presented daily from d6 to d19

WATER CONSUMPTION : No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20: On day 20 of pregnancy, all surviving animals were killed by subtotal exsanguination following isoflurane inhalation. Females showing signs of abortion or of premature delivery during the study were killed on the day of such findings
- Organs examined: Animals showing signs of abortion or of premature delivery were necropsied as quickly as possible and specimens as required by the study plan obtained whenever practically possible. The main organs cited below were examined macroscopically. All animals were subjected to gross necropsy and their organs (brain, liver, stomach, small intestine (duodenum, jejunum, ileum), large intestine (caecum, colon, rectum), kidneys, spleen, adrenals, lungs, heart) were examined macroscopically. Uterus and ovaries from each female were macroscopically observed and fixed in an appropriate fixative. Gravid uteri were weighed before extraction of foetuses

OTHER:
All organs showing macroscopic signs of pathology and corresponding organs of control groups for comparison were fixed in an appropriate fixative. Remaining tissues were destroyed 6 months after issue of the draft release.
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
- Number and uterine location of foetuses (live and dead): Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: No data
- Skeletal examinations: Yes: [performed in the first instance only on control and high dose groups]
- Number of live or dead foetuses: Yes
- Individual foetal body weight (live and dead): Yes : [all per litter]
- Caudo-cranial measurement of live and dead foetuses: Yes : [all per litter]
- Gross evaluation and weight of placenta of all foetuses: Yes : [all per litter]
- Sexing of all foetuses: Yes : [all per litter]
Statistics:
See "Any other information on materials and methods incl. tables"
The validated computerised system used in this phase was the Xybion Path/Tox System, Version 4.2.2.
Indices:
Pre-implantation loss and post-implantation loss were calculated according to the following formula:
Pre-implantation loss (%):
((Number of corpora lutea - number of implantations) / Number of corpora lutea) x 100
Post-implantation loss (%):
((Number of implantations - number of live foetuses) / Number of implantations) x 100

Foetal or litter incidence was calculated according to the following formula:
Foetal or litter incidence (%):
(Total number of foetus or litter with a particular finding / Total number of foetus per group) x 100
Historical control data:
No data available
Clinical signs:
no effects observed
Description (incidence and severity):
There was an increased salivation in some females treated with test item at 300 mg/kg bw (in 1/20) on d13 and d14 and at 1000 mg/kg bw from d12 up to the end of the study (between 1 to 3/20). There was also chromodacryorrhoea in 1 or 2/20 different animals following the day of observation in females treated with test item at 300 mg/kg bw or at 1000 mg/kg bw. These signs were of low incidence and were not attributed to a toxicological effect of the test item. There was aggressiveness on d5 in 1/20 female treated with test item at 100 mg/kg bw. This sign was only observed on the first day of treatment and was not attributed to the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality and no relevant clinical signs or signs of reaction to treatment were noted in treated females.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no change in body weight gain.
Food efficiency:
no effects observed
Description (incidence and severity):
There was a statistically significant isolated lower food consumption in females treated with test item at 100 mg/kg bw on d10 (-13%) and d18 ( -20%) or in females treated with test item at 1000 mg/kg bw on d6, d11 or d18 (between -14 and -18%). This lower food consumption is transient and attributable to the high variability in the control group. From d5pc, food consumption was lower for Female No. 1602737 treated with test item at 100 mg/kg bw.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no change in uterus weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings in mated females killed on d20pc. At the necropsy, there was a dark liquid in the vulva and vagina, a black abnormal content in stomach and enlarged spleen in Female No. 1602755 treated with test item at 300 mg/kg bw. In Female No. 1602730 treated with test item at 1000 mg/kg bw, there was dark liquid in the vagina, transparent and abnormal area in stomach, adhesion between lungs and thoracic cavity, adhesion between lungs, heart and diaphragm, cloudy liquid in the thoracic cavity and heart with firm area and granular aspect.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Signs of abortion (blood near genital orifice and in cage) were seen in two females, on d19 in one female treated with the intermediate dose (No. 1602755) and on d16 in one female treated with the highest dose (No. 1602730).
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
There was a higher percentage of pre and post implantation loss for Female No. 1602737 treated with test item at 100 mg/kg bw and Female No. 1602766 treated with test item at 300 mg/kg bw. For these females, there was no foetus, only resorptions were observed.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
In Female No. 1602755, there is 16 implantation sites, with 1 early resorption. In Female No. 1602730, there is 17 implantation sites, with 5 early and 3 late resorptions.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
In Female No. 1602755, there is 16 implantation sites with 1 dead foetus. In Female No. 1602730, there is 17 implantation sites, with 9 dead foetus.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
On d20 of pregnancy (d20pc), all mated females were necropsied and all foetuses were examined.
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean body weight per litter and per group of live foetuses were indicated as well as the mean caudal-cranial measurement per litter and per group and the mean weight of the placenta of these foetuses per litter and per group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were isolated macroscopic findings seen in the control or treated groups at the same incidence such as point or area in head, limbs or back.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The test item administered in pregnant rats from day 6 to day 19 of gestation did not induce any relevant changes in foetuses examined at skeletal examination.
Visceral malformations:
no effects observed
Description (incidence and severity):
The test item administered in pregnant rats from day 6 to day 19 of gestation did not induce any relevant changes in foetuses examined at visceral examination.
Details on embryotoxic / teratogenic effects:
There was no change in pre and post-implantation loss, in the number of corpora lutea, in the number of live and abnormal foetuses, in the number of normal and abnormal dead foetuses or in the early and late resorptions. There was no change in caudo-cranial measurement,foetus weight or proportion of male/female foetus. There was a statistically significant lower placenta weight in the group receiving test item at 100 mg/kg bw. This was low in amplitude (-6%) and was not attributed to a toxicological effect of test item.
Key result
Dose descriptor:
NOEL
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 7.8.2/2: Clinical signs – autonomic profile/miscellaneous - Group incidences

Clinicalsigns

Time

Vehicle

Test item100mg/kg

Test item300mg/kg

Test item1000mg/kg

Increased salivation

D5

D12

D13

D14

D15

D16

D17

D18

D19

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

1/20

1/20

0/20

0/20

0/20

0/20

0/19

0/20

1/20

0/20

2/20

1/20

1/19

3/19

2/19

2/19

Chromodacryorrhoea

D5

D9

D10

D11

D12

D14

D15

D17

D18

D19

0/20

0/20

0/20

0/20

0/20

0/20

1/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

0/20

1/20

1/20

1/20

1/20

0/20

0/20

1/20

1/20

0/19

0/20

0/20

1/20

0/20

0/20

1/20

2/20

1/19

1/19

1/19

Only times with clinical signs and showing differences with between groups are reported.

Results are expressed as the group incidence of animals showing the sign.

Corn oil

D: Day

P>0.05, when compared with the control group dosed with the vehicle, Fisher's test.

†: mortality occurred, no statistical analysis was performed.

Table 7.8.2/3: Body weight (mean table)

Treatment

 

D5

D6

D7

D8

D9

D10

D11

 

Vehicle

Mean

SEM

% N

260

6

NA

20

264

6

+2

20

267

6

+3

20

272

6

+5

20

277

6

+7

20

284

6

+9

20

288

6

+11

20

Test item

100mg/kg

Mean

SEM

% N P

262

5

NA

20

NS

267

6

+2

20

NS

270

6

+3

20

NS

275

6

+5

20

NS

279

5

+6

20

NS

286

6

+9

20

NS

289

6

+10

20

NS

Test item

300mg/kg

Mean

SEM

% N P

257

5

NA

20

NS

260

5

+1

20

NS

263

4

+2

20

NS

268

4

+4

20

NS

273

4

+6

20

NS

278

5

+8

20

NS

284

4

+11

20

NS

Test item

1000mg/kg

Mean

SEM

% N P

262

5

NA

20

NS

266

5

+2

20

NS

268

4

+2

20

NS

273

4

+4

20

NS

277

4

+6

20

NS

283

5

+8

20

NS

288

4

+10

20

NS

 

Threshold

18

18

18

18

18

18

18

Treatment

 

D12

D13

D14

D15

D16

D17

D18

D19

D20

Vehicle

Mean

SEM

% N

295

6

+13

20

300

6

+15

20

306

6

+18

20

313

6

+20

20

323

6

+24

20

337

6

+30

20

350

6

+35

20

363

6

+40

20

376

6

+45

20

Test item

100mg/kg

Mean

SEM

% N P

294

6

+12

20

NS

301

6

+15

20

NS

306

6

+17

20

NS

314

6

+20

20

NS

324

7

+24

20

NS

337

8

+29

20

NS

348

9

+33

20

NS

357

9

+36

20

NS

368

10

+40

20

NS

Test item

300mg/kg

Mean

SEM

% N P

289

5

+12

20

NS

294

4

+14

20

NS

300

4

+17

20

NS

308

4

+20

20

NS

318

4

+24

20

NS

333

4

+30

20

NS

342

5

+33

20

NS

355

6

+38

19

NS

367

6

+43

19

NS

Test item

1000mg/kg

Mean

SEM

% N P

293

4

+12

20

NS

299

4

+14

20

NS

306

5

+17

20

NS

311

5

+19

20

NS

320

5

+22

20

NS

336

5

+28

19

NS

348

5

+33

19

NS

359

5

+37

19

NS

370

6

+41

19

NS

 

Threshold

18

18

18

18

18

20

20

22

22

Results expressed in g

D: day

Vehicle: Corn oil

NS:P>0.05, when compared to control group

Analysis of variance for repeated measurements with Dunnett's test

NA: not applicable

%: variation expressed in percentage in relation to predose values

Table 7.8.2/4: Absolute weight of uterus (mean values)

Treatment

 

Uterus Weight (g)

Vehicle

Mean

SEM N

71.6

2.5

20

Testitem

100mg/kg

Mean

SEM N

% P

70.4

5.5

20

-2

NS

Testitem

300mg/kg

Mean

SEM N

% P

67.6

4.0

20

-6

NS

Testitem

1000mg/kg

Mean

SEM N

% P

69.6

3.9

20

-3

NS

 

Threshold

14.0

%: variation expressed in percentage in relation to control values

Vehicle: Corn oil

NS:P>0.05, when compared with control group

Analysis of variance with Dunnett's test if P <0.05

Table 7.8.2/5: Macroscopic observations - Group incidences

Organs

Observations

vehicle

Test item100mg/kg

Test item300mg/kg

Test item1000mg/kg

Placenta

Dark

Total animals involved

0/256

0

0/259

0

14/243

14

0/246

0

Head

Punctate or point

Area

Total animals involved

1/256

2/256

3

0/259

2/259

2

1/243

1/243

2

1/246

2/246

3

Limbs

Punctate or point

Area

Total animals involved

1/256

2/256

3

2/259

3/259

5

1/243

5/243

6

1/246

4/246

5

Abdomen

Punctate or point

Total animals involved

1/256

1

0/259

0

0/243

0

0/246

0

Tail

Area

Twisted

Total animals involved

1/256

0/256

1

0/259

0/259

0

0/243

1/243

1

0/246

0/246

0

Back

Punctate or point

Area

Total animals involved

14/256

0/256

14

10/259

5/259

15

11/243

0/243

11

11/246

2/246

13

Table 7.8.2/6: Caudo-cranial measurements and weights of live foetuses, weights of the placenta (mean values)

Treatment

 

Caudo-cranial

measurement(mm)

Foetus

weight(g)

Placenta

weight(g)

Vehicle

Mean

SEM N

35.2

0.2

256

3.77

0.03

256

0.567

0.006

255

Test item

100mg/kg

Mean

SEM N

% P

34.8

0.1

259

-1

NS

3.69

0.02

259

-2

NS

0.533

0.005

259

-6

??

Test item

300mg/kg

Mean

SEM N

% P

34.7

0.3

243

-1

NS

3.70

0.04

243

-2

NS

0.557

0.006

242

-2

NS

Test item

1000mg/kg

Mean

SEM N

% P

35.2

0.1

246

0

NS

3.78

0.02

246

0

NS

0.569

0.005

246

0

NS

 

Threshold

0.6

0.09

0.018

%: variation expressed in percentage in relation to control values

Vehicle: Corn oil

NS:P>0.05, ??:P<0.01, when compared with control group

Analysis of variance with Dunnett's test if P <0.05

Conclusions:
Under the study conditions, the NOAEL for embryo-foetal developmental toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C18-unsatd. MIPA according to OECD Guideline 414, in compliance with GLP. The substance diluted in corn oil was administered by gavage to groups of mated female Sprague-Dawley rats (20 mated females/dose) at the dose levels of 0, 100, 300, 1000 mg /kg bw/ day from days 6 to 19 after mating. The aim of the study was to investigate any possible adverse effects on the pregnant female rat and on the developing embryo and foetus following daily administration to pregnant rats by the oral route from implantation throughout organogenesis and until late gestation. Morbidity/mortality checks were performed twice daily. Clinical observations were performed before the first dosing and daily. Body weight was recorded on d5 then daily from d6 to d20. Food consumption was measured daily from day 6 to day 19. On day 20 of pregnancy (d20pc), all mated females were necropsied and all foetuses were examined. Foetuses were examined macroscopically and the following data were noted: number of live or dead foetuses, individual foetal body weight (live and dead), caudo-cranial measurement of live and dead foetuses, gross evaluation and weight of placenta of all foetuses, external morphological examination of these foetuses, sexing of all foetuses. The clinical signs (increased salivation and chromodacryorrhoea) observed were at low incidence and were not attributed to a toxicological effect of the read across substance. There was no change in pre and post-implantation loss, in the number of corpora lutea, in the number of live and abnormal foetuses, in the number of normal and abnormal dead foetuses or in the early and late resorptions. There was no change in caudo-cranial measurement, foetus weight or proportion of male/female foetus. There was a statistically significant lower placenta weight in the group receiving read across substance at 100 mg/kg bw. This was low in amplitude and was not attributed to a toxicological effect of read across substance. The read across substance administered in pregnant rats from day 6 to day 19 of gestation did not induce any relevant changes in foetuses examined at skeletal and visceral examination. Based on the above results, it could be concluded that the read across substance had no harmful effects on the prenatal development of the rat offspring at doses used in the present study. Under the study conditions, the NOAEL for embryo-foetal developmental toxicity was considered to be 1000 mg/kg bw/day (Mortier, 2018). Based on the results of the read across study, a similar NOAEL value is expected for the test substance.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Sufficient data available.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C12-18 and C18-unsatd. DEA, according to OECD Guideline 414, in compliance with GLP. The substance was administered to groups of 30 female rats by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day, once daily from Gestation Days (GD) 6 to 15 inclusive. Control animals were dosed with the vehicle alone (arachis oil, DAB 9). Clinical condition and reaction to treatment were recorded at least once daily. Body weights were reported on GD 0, 6, 16 and 20. All surviving females were sacrificed on GD 20 and the foetuses were removed by caesarean section. At necropsy, the females were examined macroscopically. Live foetuses were weighed, sexed and examined for visceral and skeletal abnormalities. No deaths or treatment-related changes in body weight gain and necropsy findings were observed in dams at any dose level. Treatment-related symptoms observed in all groups were salivation and propulsion of the head. The highest dose group showed severe salivation. Apart from the control (1 dead foetus) and the 100 mg/kg bw/day groups (7 dead foetuses), all females had viable foetuses. Pre-implantation loss and mean numbers of resorptions were not affected by treatment. The data for post-implantation loss, embryonic deaths and total foetuses showed some deviations, which were considered to be non-treatment-related. Mean placental and uterus weights were not affected by the treatment. Foetal sex ratio was comparable in all groups. No treatment-related foetal abnormalities were found at necropsy. The examined foetuses showed no treatment-related visceral and skeletal abnormalities/variations. One foetus at 300 mg/kg bw/day showed a stump tail and missing coccigycae vertebrae. Further, the data for skeletal ossifications showed some deviations in the two highest dose groups. However, all these effects were assessed to be non-treatment-related. Under the study conditions, the NOAELs for parental toxicity and developmental toxicity were considered to be 1000 mg/kg bw/day (Pitterman, 1994).

A study was conducted to evaluate the prenatal developmental toxicity of the read across substance, C18-unsatd. MIPA according to OECD Guideline 414, in compliance with GLP. The substance diluted in corn oil was administered by gavage to groups of mated female Sprague-Dawley rats (20 mated females/dose) at the dose levels of 0, 100, 300, 1000 mg /kg bw/ day from days 6 to 19 after mating. The aim of the study was to investigate any possible adverse effects on the pregnant female rat and on the developing embryo and foetus following daily administration to pregnant rats by the oral route from implantation throughout organogenesis and until late gestation. Morbidity/mortality checks were performed twice daily. Clinical observations were performed before the first dosing and daily. Body weight was recorded on d5 then daily from d6 to d20. Food consumption was measured daily from day 6 to day 19. On day 20 of pregnancy (d20pc), all mated females were necropsied and all foetuses were examined. Foetuses were examined macroscopically and the following data were noted: number of live or dead foetuses, individual foetal body weight (live and dead), caudo-cranial measurement of live and dead foetuses, gross evaluation and weight of placenta of all foetuses, external morphological examination of these foetuses, sexing of all foetuses. The clinical signs (increased salivation and chromodacryorrhoea) observed were at low incidence and were not attributed to a toxicological effect of the read across substance. There was no change in pre and post-implantation loss, in the number of corpora lutea, in the number of live and abnormal foetuses, in the number of normal and abnormal dead foetuses or in the early and late resorptions. There was no change in caudo-cranial measurement, foetus weight or proportion of male/female foetus. There was a statistically significant lower placenta weight in the group receiving read across substance at 100 mg/kg bw. This was low in amplitude and was not attributed to a toxicological effect of read across substance. The read across substance administered in pregnant rats from day 6 to day 19 of gestation did not induce any relevant changes in foetuses examined at skeletal and visceral examination. Based on the above results, it could be concluded that the read across substance had no harmful effects on the prenatal development of the rat offspring at doses used in the present study. Under the study conditions, the NOAEL for embryo-foetal developmental toxicity was considered to be 1000 mg/kg bw/day (Mortier, 2018).

In addition, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), testing proposals are submitted for the conduct of prenatal developmental toxicity studies in rat and rabbit according to OECD Guideline 414 with C8-18 and C18-unsatd. DEA.

Additional considerations 

Based on an in-depth analysis of the available information (see read-across justification in Section 13 of the IUCLID dataset), it is the FAA consortium’s hypothesis that a read-across within and across MEA, DEA and MIPA derived alkanolamides is scientifically plausible and justified. While there is at this point no evidence putting the read-across hypothesis in question, the FAA consortium recognizes some limitations, predominantly related to the quality of individual endpoint studies (including quality of the test substance characterisations) and existing higher Tier endpoint data gaps. After discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), the FAA Consortium agrees to the need to strengthen the toxicology data-based link between and within the DEA, MEA and MIPA subcategories.

In view of this, the FAA Consortium decided to proceed with a 3-Tier testing strategy. In Tier 1, a series of bridging studies according to OECD TG 422 will be conducted with a representative short- and a long chain substance of each subcategory (i.e., DEA, MEA, MIPA).

The objective of Tier 2 will be to generate a complete set of higher toxicology data for a selected >1000 tpa substance (i.e., OECD TG 408/443/414 (rats)/414 (2nd species). The testing in Tier 2 will be conducted with C8-18 and C18-unsatd. DEA, the substance that is generally perceived to be the most critical from a reproductive toxicity point of view based on the classification of DEA.

Tier 1 and 2 proposed studies have been included in the present dossier update. Should these suggest significant differences in type and strength of effects between the DEA, MEA and MIPA subcategories, therefore not supporting the current read across justification, further testing may be initiated. The Consortium’s strategy is detailed in a document entitled ‘ECHA-DIAP -FAA testing strategy summary – 24Sep20’ attached in Section 13 of the IUCLID dataset.

Justification for classification or non-classification

The available data suggests that the test substance is not a reproductive toxicant with regard to fertility or developmental effects. However, the registered compositions indicate that some of the test substances may contain ≥3% free DEA. This constituent has now been self-classified by its REACH registrants as Repr. 2 -H361: Suspected of damaging fertility or the unborn child(https://echa.europa.eu/registration-dossier/-/registered-dossier/15770/2/1/?documentUUID=b015c4bf-61f0-4b7b-b858-cfe6f686f088) according to CLP (EC 1272/2008) criteria.Therefore, based on the mixture classification rules, the test substance warrants no classification for reproductive or developmental toxicity when its free DEA content is <3% but classification as Repr. 2 -H361: Suspected of damaging fertility or the unborn child when the free DEA content is ≥3%