Registration Dossier

Administrative data

Description of key information

A NOAEL of 1000 mg/kg bw/d was derived based on absence of treatment-related effects at any of the dose levels in an OECD combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study conducted with the read across substance, Oleamide MIPA.

Further, dermal 90-day and 2-year studies have been conducted by NTP for the read across substance, C8-18 and C18-unsatd. DEA in both rats and mice. The sub-acute dermal studies lead to NOAELs of 50 mg/kg bw/day (based on renal tubule regeneration) in rats and 100 mg/kg bw/day (based on organ-weight changes) in mice respectively. The chronic dermal studies lead to an overall NOAEL of 50 mg/kg bw/day (based on skin irritation at site of application in the 100 mg/kg bw dose (females) and significant increases in the epithelial ulcer of the forestomach) in rats and a LOAEL of 100 mg/kg bw/day (based on body weight changes and skin irritation at site of application in the 200 mg/kg bw/d dose (females) and several non-neoplastic lesions at all dose levels) in mice. The carcinogenic activity seen in the chronic mice study was attributed to the presence of free diethanolamine (DEA) as a residue/contaminant in the test substance. However, recent evidences suggest that DEA should not be classified as a carcinogen, as the hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism for renal/liver tumours are not relevant to humans/primates (Leung et al., 2005)

Overall, considering the available oral and dermal studies with the read across substances, no toxicologically significant critical systemic effects are expected for the test substance, C10-12 and C18-unsatd. DEA. However, considering the likely lower bioavailability potential of the read across substance, Oleamide MIPA (due to longer chain length), the lower NOAEL from the chronic rat dermal studyhas been considered further for hazard/risk assessment as a conservative approach.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
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 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
not specified
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 d before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per h): approximately 12 cycles/h of filtered, non-recycled air
- Photoperiod (h dark / h 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 oral exposure:
Preparation of dosing solutions:
The test substance 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 substance 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 substance 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 substance 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 bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: GC-FID
Test substance 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 d 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
Remarks:
Doses / Concentrations: 100, 300 and 1000 mg/kg bw/day
Basis: actual ingested
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 substance as a suspension in corn oil at 100, 300 or 1000 mg/kg bw/day 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 substance 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 substance-related macroscopic findings.
- Animal assignment: computerized stratification procedure.
Positive control:
Not required
Observations and examinations performed and frequency:
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 post coital. and lactation on Days 1 and 5 post partem. 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 post partem. 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.
Sacrifice and pathology:
The males were sacrificed after at least 5 weeks of treatment and the females on Day 6 post partem. 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.
Statistics:
Body weight, food consumption and reproductive data :Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
Hematology and blood biochemistry: A specific sequence was used for the statistical analyses of hematology and blood biochemistry data.
Organ weight: PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) according to a specific sequence
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 bw/day from the first or second week of treatment and in most of the animals at 100 mg/kg bw/day but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg bw/day but more at 1000 mg/kg bw/day 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.
Mortality:
mortality observed, treatment-related
Description (incidence):
Ptyalism, considered of minor toxicological significance, was observed in all animals at 300 and 1000 mg/kg bw/day from the first or second week of treatment and in most of the animals at 100 mg/kg bw/day but later. Hypoactivity, loud breathing, piloerection and/or round back observed for some days in a few animals at 300 mg/kg bw/day but more at 1000 mg/kg bw/day 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.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See details below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details below
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
See details below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See details below
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Food consumption: In males, mean food consumption at 1000 mg/kg bw/d 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 bw/d was not affected. Mean food consumption in females was considered to be unaffected by the test substance treatment.

Hematology: 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 substance 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 substance treatment was considered to be doubtful (no clear dose-relationship, individual values generally included in historical control data, and no statistical level).

Blood biochemistry: Mean cholesterol level was higher in test substance treated female groups in a dose-related manner, reaching statistical and toxicological significance at 300 and 1000 mg/kg bw/d. 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 substance may be suspected in two males treated at 1000 mg/kg bw/d 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 bw/d 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 substance treatment on mean albumin concentration at 300 mg/kg bw/d in both sexes was considered unlikely as there was no dose-relationship. The increase observed in triglyceride levels at 300 and 1000 mg/kg bw/d 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 bw/d was due to isolated animals per sex only.

Functional Observation Battery (FOB) and motor activity: An effect of the test substance treatment in males and females at 300 and/or 1000 mg/kg bw/d 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 weights: When compared with controls, the mean absolute and relative liver weights were higher in males and females given 1000 mg/kg bw/d and in males given 300 mg/kg bw/d, reaching statistically significant values in both sexes at 1000 mg/kg bw/d (p<0.01 except for the absolute weight in females where it was p<0.05). This correlated histologically with minimal hepatocellular hypertrophy and was considered to be test substance related. The mean absolute and relative thymus weights were lower in females given 1000 mg/kg bw/d, without reaching a statistically significant value. This correlated with minimal to slight atrophy in two females and was considered to be probably test substance related. The mean absolute heart weight was higher in males given 100 mg/kg bw/d. In the absence of a dose-related trend, any relationship with the test substance was considered to be excluded. Other changes in the mean organ weights were considered to be part of the normal variation in rats and without relationship with the test substance.

Macroscopic post-mortem examination: The thymus was reduced in one female given 1000 mg/kg bw/d correlating in this animal with a small weight (0.1200 g) and histologically with slight atrophy. A relationship with the test substance was considered to be likely. Irregular color was seen in the lungs of 3/5 males given 1000 mg/kg bw/d. This correlated histologically with presence of mucus and inflammation (chronic) and/or alveolar macrophages. These changes were consistent with aspiration or regurgitation of the oily vehicle or test item during the technical gavage procedures.

Microscopic examination: Test substance 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 bw/d and 4/6 males and 2/5 females given 1000 mg/kg bw/d. This non-adverse change was considered to be test substance 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 bw/d and 2/5 females at 1000 mg/kg bw/d were considered to be fortuitous and without any relationship with the test substance.

Thymus: Minimal or slight lymphoid atrophy was seen in 2/5 females given 1000 mg/kg bw/d, correlating with the lower weight at necropsy. Any relationship with the test substance was considered to be likely but non-adverse (low number of animals affected and low grades). No test substance related changes were seen at 100 or 300 mg/kg bw/d.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Parental or systemic toxicity (non adverse)
Key result
Critical effects observed:
no
Conclusions:
Under study conditions, the NOAEL for parental or systemic toxicity was determined to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to determine the repeated dose toxicity of the read across substance, Oleamide MIPA, according to OECD Guideline 422, in compliance with GLP. Three groups of ten male and ten female Sprague-Dawley rats received the test substance daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 5 post-partum. The test substance was administered as a suspension in the vehicle, corn oil, at dose-levels of 0,100, 300 or 1000 mg/kg bw/day. Another group of ten males and ten females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg bw/day was used. 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 post coital and lactation on Days 1 and 5 post-partum. 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 post-partum. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (haematology 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 post-partum. 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 test substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no test substance in control formulations. There were no test substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/d and in most of the animals at 100 mg/kg bw/d (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 haematology parameters in any group and sex. An effect of the test 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/d, 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/d 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/d 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 bw/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. Based on the results of the study, the study author determined the NOAEL for parental or systemic toxicity to be at 1000 mg/kg bw/day (Bentz, 2013). Based on the results of the read across study, similar NOAEL for systemic toxicity is considered for the test substance, C10-12 and C18-unsatd. DEA.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Guideline compliant study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
Not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Principles of method if other than guideline:
The test substance was applied daily to the skin in graduated doses to several groups of mice, one dose per group, for a period of 14 wk. During the period of application the animals were observed daily to detect signs of toxicity. Animals which die during the test were necropsied, and at the conclusion of the test the surviving animals were sacrificed and necropsied.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 16 to 17 d

Environmental conditions:
- Temperature : 20.6 -22.8 °C
- Humidity : 41-58 %
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

In-life dates: From: Feb. 12, 1992 - To: May 15, 1992

Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
The test material formulations were applied on shaved skin of the test animals.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of test material were conducted at the study laboratory using HPLC. Dose formulations from the beginning, middle, and end of the studies were analyzed
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 exposures/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
800 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/dose group
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once


BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly, and at the end of the studies


OTHER: Organs weighed were heart, right kidney, liver, lung, right testis, and thymus
Sacrifice and pathology:
SACRIFICE: Yes (Carbon dioxide asphyxiation)
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 800 mg/kg bw mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, gallbladder, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.
Other examinations:
Sperm motility and vaginal cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology from mice of 200, 400 and 800 mg/kg bw groups. The following sperm motility parameters were evaluated: spermatid heads per gram of testis, spermatid heads per testis, spermatid count, and epididymal spermatozoal motility and concentration. The left cauda epididymis, epididymis, and testis were weighed. Vaginal samples for cytology evaluations were collected for 12 consecutive days prior to the end of the studies from all female mice. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.
Statistics:
Not reported
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only treatment related clinical finding was irritation of the skin at the site of application in males and females administered 800 mg/kg bw.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
The only treatment related clinical finding was irritation of the skin at the site of application in males and females administered 800 mg/kg bw.
Mortality:
no mortality observed
Description (incidence):
All mice survived until the end of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Final mean body weights and body weight gains of dosed males and females were similar to those of the vehicle controls.



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:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative liver and right kidney weights of 800 mg/kg bw males and females and the absolute and relative liver weights of 400 mg/kg bw females were significantly greater than those of the vehicle controls. The absolute and relative lung weights of 800 mg/kg bw females were also significantly greater than those of the vehicle controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis, and ulcer. The incidences and severities of these skin lesions generally increased with increasing dose in males and females
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Epididymal spermatozoal concentration was significantly increased in 800 mg/kg bw males. Estrous cycle lengths of dosed females were similar to that of the vehicle controls
Key result
Dose descriptor:
NOAEL
Remarks:
(systemic effects)
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
(local effects)
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: skin inflammation
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (actual dose received)
System:
other: As of 400 mg/kg bw/d, significant increase in relative liver weights (females). At 800 mg/kg bw/d, significant increase in relative kidney (male and females) and lung (females) weights.
Organ:
liver
kidney
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day
System:
other: As of 200 mg/kg bw/d, incidence of chronic active inflammation.
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Under the test conditions, the 14-week NOAEL in mice for systemic effects was considered to be 200 mg/kg bw/day and for the local effects 100 mg/kg bw/day.

Executive summary:

A 14-week study was conducted by NTP to evaluate the repeated dose toxicity of the read across substance, C8-18 and C18-unsatd. DEA, when administered via the dermal route to B6C3F1 mice. The experiment was carried out in compliance with GLP. Groups of 10 male and 10 female mice were exposed to 0, 50, 100, 200, 400 or 800 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 14 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At the end of the study, samples were collected for sperm motility and vaginal cytology evaluations in the 0, 200, 400 and 800 mg/kg bw/day dose groups. At necropsy, a gross macroscopic examination was conducted. Selected organs were weighed and a complete histopathological evaluation was carried out for 0 and 800 mg/kg bw/day mice. All mice survived until the end of the study. The only treatment related clinical finding was irritation of the skin at the site of application in males and females administered 800 mg/kg bw/day. There were no effects on body weight. Weights of the liver and kidney of 800 mg/kg bw/day males and females, liver of 400 mg/kg bw/day females and lung of 800 mg/kg bw/day females were significantly increased compared to the controls. Epididymal spermatozoa concentration was significantly increased in 800 mg/kg bw/day males. Histopathologic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis and ulcer. The incidences and severities of these skin lesions generally increased with increasing dose in males and females. Under the study conditions, the 14 week NOAEL in mice for systemic effects was considered to be 200 mg/kg bw/day and for the local effects 100 mg/kg bw/day (Irwin, 2001). Based on the results of the read across study, similar NOAELs can be considered for the test substance, C10-12 and C18-unsatd. DEA.

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
Not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Principles of method if other than guideline:
The test substance was applied daily to the skin in graduated doses to several groups of experimental animals, one dose per group, for a period of 14 wk. During the period of application the animals were observed daily to detect signs of toxicity. Animals which die during the test were necropsied, and at the conclusion of the test the surviving animals were sacrificed and necropsied.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 14 to 15 d

Environmental conditions
- Temperature : 22.2 -23.9°C
- Humidity : 38-55 %
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

In-life dates: From: Feb. 10, 1992 - To: May 13, 1992

Type of coverage:
open
Vehicle:
ethanol
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analyses of the dose formulations of test material were conducted at the study laboratory using HPLC. Dose formulations from the beginning, middle, and end of the studies were analysed.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 exposures/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10/sex/dose in the core study, 10/sex/dose for clinical pathology groups
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once


BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly, and at the end of the studies


HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Days 4 and 24 (blood was collected from the retroorbital sinus of clinical pathology study male and
female rats from each dose group)
- Anaesthetic used for blood collection: Yes (carbon dioxide/oxygen mixture)
- How many animals: All animals
- Parameters checked in table [No.?] were examined.: Hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, nucleated erythrocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Days 4 and 24 (blood was collected from the retroorbital sinus of clinical pathology study male and
female rats from each dose group)
- How many animals: All animals
- Parameters checked in table [No.?] were examined: Urea nitrogen, creatinine, total protein, albumin, cholesterol, triglycerides, alanine aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, and total bile acids


OTHER: Organs weighed were heart, right kidney, liver, lung, right testis, and thymus
Sacrifice and pathology:
SACRIFICE: At the end of the 14 wk studies, blood was collected from the retroorbital sinus of all core study rats for hematology and clinical chemistry analyses. Thereafter the test animals were anesthetised with a carbon dioxide/oxygen mixture.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 400 mg/kg bw. In addition to gross lesions and tissue masses, the following tissues were examined: heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the skin (site of application) was examined in all core study animals, and the kidney was examined in core study male and female rats.
Other examinations:
Sperm motility and vaginal cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology from all rats receiving 100, 200 and 400 mg/kg bw of test material. The following sperm motility parameters were evaluated: spermatid heads per gram of testis, spermatid heads per testis, spermatid count, and epididymal spermatozoal motility and concentration. The left cauda epididymis, epididymis, and testis were weighed. Vaginal samples for cytology evaluations were collected for 12 consecutive days prior to the end of the studies from all female rats. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.
Statistics:
Not reported
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings included irritation of the skin at the site of application in 100, 200, and 400 mg/kg bw males and females.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings included irritation of the skin at the site of application in 100, 200, and 400 mg/kg bw males and females.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Final mean body weights and body weight gains of 200 and 400 mg/kg bw males and females were significantly less than those of the vehicle controls.
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:
effects observed, treatment-related
Description (incidence and severity):
At Week 14, a minimal microcytic, normochromic, nonresponsive anemia occurred in the 100 and 200 mg/kg bw females and 400 mg/kg bw males and females. The anemia also occurred in the 400 mg/kg bw males and females on Day 24. Increased segmented neutrophil counts occurred in 400 mg/kg bw males and females at Week 14 and in 400 mg/kg bw females on Day 24.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol concentrations were significantly decreased in 200 and 400 mg/kg bw males and in females administered 100 mg/kg bw or greater; triglyceride concentrations were also decreased in 200 and 400 mg/kg bw males. At week 14, there was a minimal concentration-related increase of serum albumin concentration in all treated groups of females and in 100 mg/kg bw or greater male rats; on Day 24, increased albumin concentration occurred in the 400 mg/kg bw females. There were minimal increases of urea nitrogen concentration that occurred in the 200 and 400 mg/kg bw female rats on Day 24 and at week 14. At week 14, an increase in alanine aminotransferase activity occurred in 50 mg/kg bw or greater male rats. Additionally, alkaline phosphatase activity was increased in 400 mg/kg bw males.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Kidney weights of females administered 50 mg/kg bw or greater were significantly greater than those of the vehicle control group. Left epididymis weights of 200 and 400 mg/kg bw males were significantly less than those of the vehicle controls, but this was most likely secondary to decreased mean body weights in these groups
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathologic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis, and ulcer. The incidences and severities of these skin lesions generally increased with increasing dose in males and females. The incidences of renal tubule regeneration in 100, 200, and 400 mg/kg bw females were significantly greater than the vehicle control incidence, and the severities in 200 and 400 mg/kg bw females were increased.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle lengths of dosed females were similar to those of the vehicle controls
Key result
Dose descriptor:
NOAEL
Remarks:
(systemic and local effects)
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
other: skin, liver, kidney
Organ:
skin
liver
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Under the study conditions, the 14 week systemic NOAEL in rats can be considered to be 50 mg/kg bw/day.
Executive summary:

A 14-week study was conducted by NTP to evaluate the repeated dose toxicity of the read across substance, C8-18 and C18-unsatd. DEA when administered via the dermal route to F344/N rats. The experiment was carried out in compliance with GLP. Groups of 10 male and 10 female rats were exposed to 0, 25, 50, 100, 200 or 400 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 14 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. Blood was collected on Days 4 and 24 for hematology and clinical chemistry analysis. At the end of the study, samples were collected for sperm motility and vaginal cytology evaluations in the 0, 100, 200 or 400 mg/kg bw/day dose groups. At necropsy, a gross macroscopic examination was conducted. Selected organs were weighed and a complete histopathological evaluation was carried out for 0 and 400 mg/kg/day rats. All rats survived until the end of the study. Clinical findings included irritation of the skin at the site of application in 100, 200 and 400 mg/kg bw/day males and females. Final mean bodyweights and bodyweight gains of 200 and 400 mg/kg bw/day males and females were significantly lower than those of the controls. At Week 14, a minimal microcytic, normochromic, non-responsive anaemia occurred in the 100 and 200 mg/kg bw/day females and 400 mg/kg bw/day males and females. The anaemia was also seen in the 400 mg/kg bw/day males and females on Day 24. Increased segmented neutrophil counts occurred in 400 mg/kg bw/day males and females at Week 14 and in 400 mg/kg bw/day females on Day 24. Cholesterol concentrations were significantly decreased in 200 and 400 mg/kg bw/day males and in females administered 100 mg/kg bw/day or greater. Triglyceride concentrations were decreased in 200 and 400 mg/kg bw/day males. Histopathological lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis and ulcer. The incidence and severity of these skin lesions generally increased with increasing dose in males and females. The incidences of renal tubule regeneration in 100, 200 and 400 mg/kg bw/day females were significantly greater than in controls, and the severity in 200 and 400 mg/kg bw/day females was increased. Under the study conditions, the 14 week systemic NOAEL in rats was considered to be 50 mg/kg bw/day (Irwin, 2001). Based on the results of the read across study, similar NOAEL can be considered for the test substance, C10-12 and C18-unsatd. DEA.

Endpoint:
chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
reference to other study
Reason / purpose:
read-across source
Principles of method if other than guideline:
A two-year dermal study was conducted in mice to evaluate the carcinogenic potential of the test substance.

GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
Test animals:
- Source: Taconic Laboratory Animals and Services, (Germantown, NY)
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 13 to 14 days

Environmental conditions:
- Temperature : 20.6 -23.9°C
- Humidity : 30-67%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

In-life dates: From: Jan. 20, 1993 - To: Jan. 20, 1995
Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
The test material formulation was applied to the shaved skin of test animals. No further details provided.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed approximately every 2 months using HPLC. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:
104 wk
Frequency of treatment:
Five exposures per week

Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
0 mg/mL in ethanol
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
50 mg/mL in ethanol
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
100 mg/mL in ethanol
No. of animals per sex per dose:
50/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure to coconut oil acid diethanolamine condensate in the 14 wk study produced only a minimal toxic response in mice except in the skin at the site of application. The incidences of chronic active inflammation as well as several other skin lesions were significantly increased at doses of 200 mg/kg bw and greater in both male and female mice. The incidences of ulceration were increased in males exposed to 400 and 800 mg/kg bw and in females exposed to 800 mg/kg bw. Therefore, 400 and 800 mg/kg bw were considered inappropriate for a 2-year study. However, ulceration was present in only one 200 mg/kg bw male and no females, and the severities of these lesions in all affected groups were minimal to mild. Below 200 mg/kg bw, the incidences of skin lesions decreased markedly with a minor difference in response between 50 and 100 mg/kg bw. Therefore, 200 mg/kg bw was selected as the high dose and 100 mg/kg bw as the low dose for this 2-yr study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded initially, at 4 wk intervals during the study, and at the end of the study

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially, weekly during Week 1 through 13, at 4 wk intervals thereafter, and at the end of the studies


Sacrifice and pathology:
SACRIFICE: Carbon dioxide asphyxiation
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues
were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.
Other examinations:
-
Statistics:
- Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Possible dose-related effects on survival were analysed by Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses were two-sided.
- Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.
- Analysis of Continuous Variables: Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of the dose-related trends. Average severity values were analyzed for significance with the Mann-Whitney U test. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across dose levels.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related clinical finding was irritation of the skin at the site of application in males that received 200 mg/kg bw.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related clinical finding was irritation of the skin at the site of application in males that received 200 mg/kg bw.
Mortality:
mortality observed, treatment-related
Description (incidence):
The survival of dosed males and 100 mg/kg bw females was similar to that of the vehicle controls. Survival of the 200 mg/kg bw group of female mice was reduced compared to the vehicle control group, but the difference was not significant.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of dosed males were similar to those of the vehicle controls throughout most of the study; those of 100 and 200 mg/kg bw females were less than those of the vehicle controls from wk 93 and 77, respectively .
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
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Skin: Several nonneoplastic lesions of the skin at the site of application were determined to be chemical related. Incidences of epidermal hyperplasia, sebaceous gland hyperplasia, and hyperkeratosis in all dosed groups of males and females were significantly greater than those in the vehicle control groups. The incidences of ulceration in 200 mg/kg bw males and inflammation and parakeratosis in 200 mg/kg bw females were increased.
- Thyroid gland: The incidences of follicular cell hyperplasia in all dosed groups of males (vehicle control, 11/50; 100 mg/kg bw, 20/50; 200 mg/kg bw, 23/50) and females (27/50, 36/50, 33/50) were significantly greater than those in the vehicle controls. Follicular cell hyperplasia consisted of focal areas of thyroid gland follicles lined with increased numbers of epithelial cells, which formed papillary projections in some instances.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Liver: Dosed male and female mice had significantly greater incidences of hepatic neoplasms (hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma (males) than the vehicle controls. There was a morphologic continuum from adenoma to carcinoma, with less differentiation and typical trabecular formations in the carcinomas. Carcinomas were often a centimeter or more in diameter, whereas adenomas were generally smaller and more discrete. Carcinomas metastasized to the lung in a few males and females. Adenomas, carcinomas, and hepatoblastomas displaced normal liver parenchyma, and none contained normal lobular architecture. Hepatoblastomas were characterized by well-demarcated focal areas composed of bundles of deeply basophilic, spindle-shaped cells.
- Kidney: The incidences of renal tubule adenoma (1/50, 1/50, 7/50) and of renal tubule adenoma or carcinoma (combined) (1/50, 1/50, 9/50) in 200 mg/kg bw males were significantly greater than those in the vehicle controls. Renal tubule hyperplasia, adenoma, and carcinoma formed a morphological continuum. Adenomas were focal, compressive masses approximately five or more tubules in diameter; carcinomas were morphologically similar to adenomas but were larger and often showed cellular debris and/or mineralization. Renal tubule neoplasms were located in the cortex or outer medulla. Focal proliferative masses less than five tubules in diameter were classified as focal hyperplasia.
Key result
Dose descriptor:
LOAEL
Remarks:
(systemic and local effects)
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
dermal irritation
body weight and weight gain
histopathology: non-neoplastic
histopathology: neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
other: body weight and see "Organ"
Organ:
skin
liver
kidney
thyroid gland
Treatment related:
yes
Dose response relationship:
yes

All dose formulations analysed during the 2 year studies were within 10% of the target concentration.

 

Conclusions:
Under the study conditions, the 2-year LOAEL was considered to be 100 mg/kg bw/day in mouse. There was clear evidence of carcinogenic activity in male B6C3F1 mice based on increased incidences of hepatic and renal tubule neoplasms and in female B6C3F1 mice based on increased incidences of hepatic neoplasms. These increases were associated with the the concentration of free diethanolamine present as a contaminant in the diethanolamine condensate. However, recent evidences suggest that DEA should not be classified as a carcinogen, as the hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism for renal/liver tumours are not relevant to humans/primates.
Executive summary:

A 2-year study was conducted by NTP to evaluate the long-term repeated dose toxicity of read across substance, C8-18 and C18-unsatd. DEA, when administered via the dermal route in B6C3F1 mice. The experiment was carried out in compliance with GLP. The doses studied included 0, 100 and 200 mg/kg bw/day of test substance (0, 50, or 100 mg/mL in ethanol). Fifty male/female test animals were used in each group. Five exposures per week were administered for 104 to 105 weeks. The animals were observed twice daily, and body weights and clinical findings were recorded periodically. All animals were necropsied and complete histopathology was performed. Survival of dosed male and female mice was generally similar to that of the vehicle controls. The mean bodyweights of the 100 mg/kg bw/day females from Week 93 and of the 200 mg/kg bw/day females from Week 77 were lower than those of the vehicle controls. The only clinical finding attributed to treatment was an irritation of the skin at the site of application in males administered 200 mg/kg bw/day. The incidences of hepatic neoplasms (hepatocellularadenoma, hepatocellular carcinoma and hepatoblastoma) were significantly increased in male and/or female mice. The number of eosinophilic foci in dosed groups of male mice was higher than in the vehicle controls. The occurrences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) were significantly increased in 200 mg/kg bw/day males. Several non-neoplastic lesions of the skin at the site of application were considered treatment-related. Incidences of epidermal hyperplasia, sebaceous gland hyperplasia and hyperkeratosis were greater in all dosed groups than in the vehicle controls and the number of thyroid gland follicular cell hyperplasia in all dosed groups was also significantly greater than those in the vehicle control groups. There was clear evidence of carcinogenic activity in male B6C3F1 mice based on increased incidences of hepatic and renal tubule neoplasms and in female B6C3F1 mice based on increased incidences of hepatic neoplasms. These increases were associated with the concentration of free diethanolamine present as a contaminant in the test substance. However, recent evidences suggest that DEA should not be classified as a carcinogen, as the hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism for renal/liver tumours are not relevant to humans/primates (Leung, 2005). Under the study conditions, the 2-year LOAEL was considered to be 100 mg/kg bw/day in mouse (Irwin, 2001). Based on the results of the read across study, similar LOAEL can be considered for the test substance, C10-12 and C18-unsatd. DEA.

Endpoint:
chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
reference to other study
Reason / purpose:
read-across source
Principles of method if other than guideline:
Groups of 50 male and 50 female rats were exposed to 0, 50 or 100 mg/kg bw/d of the substance in ethanol by dermal application, 5 times per week, for 104 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At necropsy, a gross macroscopic examination and complete histopathology were carried out.

GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Source: Taconic Laboratory Animals and Services, (Germantown, NY)
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 11 to 12 d

Environmental conditions:
- Temperature : 20.0 -23.9 °C
- Humidity : 33-70 %
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

In-life dates: From: Feb. 1, 1993 - To: Jan. 31, 1995
Type of coverage:
open
Vehicle:
ethanol
Details on exposure:
The test substance formulation was applied to the shaved skin of test animals. No further details provided.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulations were analysed approximately every 2 months using HPLC. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.
Duration of treatment / exposure:
104 wk
Frequency of treatment:
Five exposures per week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
50/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Doses of 200 or 400 mg/kg bw/d in the 14 wk study were associated with reduced mean body weights, mild anemia, and significantly increased incidences and severities of lesions of the skin at the site of application. Therefore, these doses were considered inappropriate for a 2-year study. At 100 mg/kg bw/d, the incidences of skin lesions, especially ulceration, were less than at 200 mg/kg bw/d, and in general, the severities were minimal to mild. Therefore, 100 mg/kg bw/d was selected as the high dose for this 2-yr study.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded initially, at 4 wk intervals during the study, and at the end of the study


DERMAL IRRITATION (if dermal study): Yes


BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially, weekly during Week 1 through 13, at 4 wk intervals thereafter, and at the end of the studies


Sacrifice and pathology:
SACRIFICE: Carbon dioxide asphyxiation
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues
were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (and
epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus
Statistics:
Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Possible dose-related effects on survival were analysed by Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses were two-sided.
Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.
Analysis of Continuous Variables: Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of the dose-related trends. Average severity values were analyzed for significance with the Mann-Whitney U test. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across dose levels.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw/d females.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw/d females. Non-neoplastic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, parakeratosis and hyperkeratosis, and the incidences and severities of these lesions increased with increasing dose.
Mortality:
no mortality observed
Description (incidence):
Survival rates of dosed male and female rats were similar to those of the vehicle controls.
Body weight and weight changes:
no effects observed
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
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- The severity of nephropathy increased with increasing dose in female rats.
- The incidences of chronic active inflammation, epithelial hyperplasia and epithelial ulcer of the forestomach increased with dose in female rats and the increases were significant in the 100 mg/kg bw/day group.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/day females. Under the study conditions, there was no evidence of carcinogenic activity of the test substance in male rats at any dose. There was an equivocal evidence of carcinogenic activity in female rats based on a marginal increase in the incidences of renal tubule neoplasms. However, the absence of an increase in neoplasms in the 100 mg/kg bw/day group in the presence of increased hyperplasia makes the association with treatment uncertain.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
systemic and local effects
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
dermal irritation
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
other: skin irritation at site of application in the 100 mg/kg bw dose group (females) and significant increases in the epithelial ulcer of the forestomach
Organ:
skin
stomach
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

All dose formulations analysed during the 2 year studies were within 10% of the target concentration.

Conclusions:
Under the study conditions, the 2-year NOAEL was considered to be 50 mg/kg bw/day in rat.
Executive summary:

A 2-year study was conducted by NTP to evaluate the long-term repeated dose toxicity of the test substance when administered via the dermal route to F344/N rats. The experiment was conducted in compliance with GLP. Groups of 50 male and 50 female rats were exposed to 0, 50 or 100 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 104 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At necropsy, a gross macroscopic examination and complete histopathology were carried out. The survival rates of treated male and female rats were similar to those of controls. There were no significant differences in bodyweight throughout the groups. The only treatment-related clinical finding was irritation of the skin at the site of application in 100 mg/kg bw/day females. Non-neoplastic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, parakeratosis and hyperkeratosis, and the incidences and severities of these lesions increased with increasing dose. There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/day females. The severity of nephropathy increased with increasing dose in female rats. The incidences of chronic active inflammation, epithelial hyperplasia and epithelial ulcer of the forestomach increased with dose in female rats and the increases were significant in the 100 mg/kg bw/day group. Under the study conditions, there was no evidence of carcinogenic activity of the test substance in male rats at any dose. There was an equivocal evidence of carcinogenic activity in female rats based on a marginal increase in the incidences of renal tubule neoplasms. However, the absence of an increase in neoplasms in the 100 mg/kg bw/day group in the presence of increased hyperplasia makes the association with treatment uncertain. Based on the results of the read across study, the 2 year NOAEL was considered to be 50 mg/kg bw/day in rat (Irwin, 2001).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Well conducted NTP studies.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Quality of whole database:
Assessed qualitatively

Additional information

Oral:

A study was conducted to determine the repeated dose toxicity of the read across substance, Oleamide MIPA, according to OECD Guideline 422, in compliance with GLP. Three groups of ten male and ten female Sprague-Dawley rats received the test substance daily by oral (gavage) administration before mating, through mating and, for the females, through gestation until Day 5 post-partum. The test substance was administered as a suspension in the vehicle, corn oil, at dose-levels of 0,100, 300 or 1000 mg/kg bw/day. Another group of ten males and ten females received the vehicle alone, under the same experimental conditions and acted as a control group. A constant dosage-volume of 5 mL/kg bw/day was used. 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 post coital and lactation on Days 1 and 5 post-partum. 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 post-partum. At the end of the treatment period, Functional Observation Battery, motor activity and laboratory investigations (haematology 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 post-partum. 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 test substance concentrations checked during the study were within an acceptable range of variations when compared to the nominal values (± 15%). There was no test substance in control formulations. There were no test substance-related deaths. Clinical signs consisted of ptyalism in all animals at 300 and 1000 mg/kg bw/d and in most of the animals at 100 mg/kg bw/d (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 haematology parameters in any group and sex. An effect of the test 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/d, 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/d 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/d 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 bw/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. Based on the results of the study, the study author determined the NOAEL for parental or systemic toxicity to be at 1000 mg/kg bw/day (XXXXXX, 2013). Based on the results of the read across study, similar NOAEL for systemic toxicity is considered for the test substance, C10-12 and C18-unsatd. DEA.

Dermal

14 week studies (mouse and rat):

Study 1: A 14-week study was conducted by NTP to evaluate the repeated dose toxicity of the read across substance, C8-18 and C18-unsatd. DEA, when administered via the dermal route to B6C3F1 mice. The experiment was carried out in compliance with GLP. Groups of 10 male and 10 female mice were exposed to 0, 50, 100, 200, 400 or 800 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 14 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At the end of the study, samples were collected for sperm motility and vaginal cytology evaluations in the 0, 200, 400 and 800 mg/kg bw/day dose groups. At necropsy, a gross macroscopic examination was conducted. Selected organs were weighed and a complete histopathological evaluation was carried out for 0 and 800 mg/kg bw/day mice. All mice survived until the end of the study. The only treatment related clinical finding was irritation of the skin at the site of application in males and females administered 800 mg/kg bw/day. There were no effects on body weight. Weights of the liver and kidney of 800 mg/kg bw/day males and females, liver of 400 mg/kg bw/day females and lung of 800 mg/kg bw/day females were significantly increased compared to the controls. Epididymal spermatozoa concentration was significantly increased in 800 mg/kg bw/day males. Histopathologic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis and ulcer. The incidences and severities of these skin lesions generally increased with increasing dose in males and females. Under the study conditions, the 14 week NOAEL in mice for systemic effects was considered to be 200 mg/kg bw/day and for the local effects 100 mg/kg bw/day (Irwin, 2001).

Study 2: A 14-week study was conducted by NTP to evaluate the repeated dose toxicity of the read across substance, C8-18 and C18-unsatd. DEA, when administered via the dermal route to F344/N rats. The experiment was carried out in compliance with GLP. Groups of 10 male and 10 female rats were exposed to 0, 25, 50, 100, 200 or 400 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 14 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. Blood was collected on Days 4 and 24 for hematology and clinical chemistry analysis. At the end of the study, samples were collected for sperm motility and vaginal cytology evaluations in the 0, 100, 200 or 400 mg/kg bw/day dose groups. At necropsy, a gross macroscopic examination was conducted. Selected organs were weighed and a complete histopathological evaluation was carried out for 0 and 400 mg/kg/day rats. All rats survived until the end of the study. Clinical findings included irritation of the skin at the site of application in 100, 200 and 400 mg/kg bw/day males and females. Final mean bodyweights and bodyweight gains of 200 and 400 mg/kg bw/day males and females were significantly lower than those of the controls. At Week 14, a minimal microcytic, normochromic, non-responsive anaemia occurred in the 100 and 200 mg/kg bw/day females and 400 mg/kg bw/day males and females. The anaemia was also seen in the 400 mg/kg bw/day males and females on Day 24. Increased segmented neutrophil counts occurred in 400 mg/kg bw/day males and females at Week 14 and in 400 mg/kg bw/day females on Day 24. Cholesterol concentrations were significantly decreased in 200 and 400 mg/kg bw/day males and in females administered 100 mg/kg bw/day or greater. Triglyceride concentrations were decreased in 200 and 400 mg/kg bw/day males. Histopathological lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis and ulcer. The incidence and severity of these skin lesions generally increased with increasing dose in males and females. The incidences of renal tubule regeneration in 100, 200 and 400 mg/kg bw/day females were significantly greater than in controls, and the severity in 200 and 400 mg/kg bw/day females was increased. Under the study conditions, the 14 week systemic NOAEL in rats was considered to be 50 mg/kg bw/day (Irwin, 2001).

2 year studies (mouse and rat):

Study 1: A 2 -year study was conducted by NTP to evaluate the long-term repeated dose toxicity of read across substance, C8-18 and C18-unsatd. DEA, when administered via the dermal route in B6C3F1 mice. The experiment was carried out in compliance with GLP. The doses studied included 0, 100 and 200 mg/kg bw/day of test substance (0, 50, or 100 mg/mL in ethanol). Fifty male/female test animals were used in each group. Five exposures per week were administered for 104 to 105 weeks. The animals were observed twice daily, and body weights and clinical findings were recorded periodically. All animals were necropsied and complete histopathology was performed. Survival of dosed male and female mice was generally similar to that of the vehicle controls. The mean bodyweights of the 100 mg/kg bw/day females from Week 93 and of the 200 mg/kg bw/day females from Week 77 were lower than those of the vehicle controls. The only clinical finding attributed to treatment was an irritation of the skin at the site of application in males administered 200 mg/kg bw/day. The incidences of hepatic neoplasms (hepatocellularadenoma, hepatocellular carcinoma and hepatoblastoma) were significantly increased in male and/or female mice. The number of eosinophilic foci in dosed groups of male mice was higher than in the vehicle controls. The occurrences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) were significantly increased in 200 mg/kg bw/day males. Several non-neoplastic lesions of the skin at the site of application were considered treatment-related. Incidences of epidermal hyperplasia, sebaceous gland hyperplasia and hyperkeratosis were greater in all dosed groups than in the vehicle controls and the number of thyroid gland follicular cell hyperplasia in all dosed groups was also significantly greater than those in the vehicle control groups. There was clear evidence of carcinogenic activity in male B6C3F1 mice based on increased incidences of hepatic and renal tubule neoplasms and in female B6C3F1 mice based on increased incidences of hepatic neoplasms. These increases were associated with the concentration of free diethanolamine present as a contaminant in the test substance. However, recent evidences suggest that DEA should not be classified as a carcinogen, as the hepatic tumours seen in mice and the proposed mode of non-genotoxic mechanism for renal/liver tumours are not relevant to humans/primates. Under the study conditions, the 2 year LOAEL was considered to be 100 mg/kg bw/day in mouse (Irwin, 2001).

Study 2: A 2-year study was conducted by NTP to evaluate the long-term repeated dose toxicity of the read across substance, C8-18 and C18-unsatd. DEA, when administered via the dermal route to F344/N rats. The experiment was conducted in compliance with GLP. Groups of 50 male and 50 female rats were exposed to 0, 50 or 100 mg/kg bw/day of the substance in ethanol by dermal application, 5 times per week, for 104 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At necropsy, a gross macroscopic examination and complete histopathology were carried out. The survival rates of treated male and female rats were similar to those of controls. There were no significant differences in bodyweight throughout the groups. The only treatment-related clinical finding was irritation of the skin at the site of application in 100 mg/kg bw/day females. Non-neoplastic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, parakeratosis and hyperkeratosis, and the incidences and severities of these lesions increased with increasing dose. There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/day females. The severity of nephropathy increased with increasing dose in female rats. The incidences of chronic active inflammation, epithelial hyperplasia and epithelial ulcer of the forestomach increased with dose in female rats and the increases were significant in the 100 mg/kg bw/day group. Under the study conditions, there was no evidence of carcinogenic activity of the test substance in male rats at any dose. There was an equivocal evidence of carcinogenic activity in female rats based on a marginal increase in the incidences of renal tubule neoplasms. However, the absence of an increase in neoplasms in the 100 mg/kg bw/day group in the presence of increased hyperplasia makes the association with treatment uncertain. Based on the results of the read across study, the 2 year NOAEL was considered to be 50 mg/kg bw/day in rat (Irwin, 2001).

Overall, based on the results of the read across dermal studes, similar NOAELs can be considered for the test substance, C10-12 and C18-unsatd. DEA.

Justification for classification or non-classification

Based on the results of the oral and dermal read across studies, the test substance, C10-12 and C18-unsatd. DEA is concluded not to warrant classification for repeated dose toxicity/STOT RE, according to the EU CLP criteria (Regulation 1272/2008/EC).

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