Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
2020-2021
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to read across
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl (C8-18 and C18-unsatd. DEA), EC No. 931-329-6

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies : none
- Available non-GLP studies : none
- Historical human data : none
- (Q)SAR : not applicable
- In vitro methods : not applicable
- Weight of evidence : not sufficient
- Grouping and read-across : not sufficient
- Substance-tailored exposure driven testing [if applicable] : none
- Approaches in addition to above [if applicable] : not applicable
- Other reasons [if applicable] : none

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Proposed adaptations were not considered sufficient to address this endpoint.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Proposed study design: according to OECD Guideline 408
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Species:
rat
Sex:
male/female
Route of administration:
oral: gavage
Remarks on result:
other: Testing planned
Critical effects observed:
not specified
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
From June 1965 to September 1965
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 of IUCLID for details on the category justification.
Principles of method if other than guideline:
Rats were administered dietary levels of the test substance at 0, 0.1, 0.5, 1 and 2% for 90 d. The animals were observed daily for clinical signs, body weights were recorded weekly and haematological, clinical chemistry and urine examinations were carried out at termination. Gross and histopathological examinations were also performed.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Carworth Farm E
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Age at study initiation: Weanlings
- Housing: Five animals per cage
- Diet: Powdered Spillers Small Laboratory Animal Diet, ad libitum
- Water: Water, ad libitum
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 d
Frequency of treatment:
Daily
Remarks:
0, 0.1, 0.5, 1 and 2% in diet (nominal)
No. of animals per sex per dose:
15
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and weekly once thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: Yes
- Time schedule for examinations: Day 0 and weekly once thereafter

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study
- Parameters checked in table [No.2]: Total erythrocyte count, hematocrit, hemoglobin, reticulocyte count, total and differential leucocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study
- Parameters examined: Liver and kidney function tests and levels of serum glutamic-oxaloncetic and glutamic-pyruvic transaminases and of blood urea checked

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the study
- Parameters checked in table [No.3] were examined: Urine was examined for colour, pH, microscopic constituents, protein, reducing substances, bile salts and blood and activity of glutamic-oxaloacetic transaminase. Volume and the specific gravity of the urine excreted were measured during a 6 h period of water deprivation and during 4 h period commencing 16 h after a water load of 25 mL/kg.

OTHER: At autopsy, the absolute and relative organ weight of the brain, heart, liver, kidneys, adrenals and gonads were recorded.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (At autopsy, the gross appearance of the brain, heart, liver, kidneys, adrenals and gonads were recorded)
HISTOPATHOLOGY: Yes (Paraffin wax sections of brain, heart, liver, kidneys, adrenals and gonads together with a wide range of other organs were stained with haematoxylin and eosin for histological examination, smears of femoral marrow were stained by the May Grunwald-Giemsa method)
Other examinations:
Palatability test: Pairs of male rats were allowed access to stock diet and to diet containing either one of the four dietary test levels of test material. The consumption of both diets was recorded for a period of 8 d.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In general rats remained in good health apart from 2 males on 1% test material which were killed on Days 23 and 58 because of weight loss and respiratory distress.
Mortality:
mortality observed, treatment-related
Description (incidence):
In general rats remained in good health apart from 2 males on 1% test material which were killed on Days 23 and 58 because of weight loss and respiratory distress.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight gain was observed from 0.5% onwards.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food intake was reduced at all dietary levels except the lowest level of 0.1%.
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):
Reductions in hemoglobin levels, hematocrit values and red cell counts in females at 1 and 2% levels but these effects were much less pronounced in males.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum levels of glutamic-oxaloacetic transaminases were significantly elevated at 0.5% and above in females, but only at 0.5% level in males.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Proteinurea was comparable in test and control groups. Tests for blood, bile salts and reducing substances were negative in all groups. Level of Glutamic-oxaloacetic transaminase were significantly elevated at dietary levels of 0.5% and above in females but only at the 0.5% level in males. No significant effect was seen in Glutamic-pyruvic transaminase level at all dietary levels.
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 principal organ weight changes were increases in the relative kidney weight in all test groups except at 0.1% in females and at 0.1 and 0.5% in males and increases in relative liver weight in female on the two highest levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant findings were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Examination of the femoral marrow smears showed no deviation from normality. There were no adverse histopathological findings in any organs.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Description (incidence and severity):
OTHER FINDINGS: In the palatability test, exclusive preference was shown to the control diet, virtually no test diet being consumed at any of the dietary levels incorporated. This observation suggests that toxic anorexia was not the cause of reduced food intake.
Key result
Dose descriptor:
NOEL
Effect level:
ca. 0.1 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.5 other: %
Organ:
other: bodyweight
Treatment related:
no

For detailed results tables kindly refer to the attached background materials section of the IUCLID.

Conclusions:
Under the study conditions, the 90 d NOEL in rats was considered to be 0.1% in the diet, equivalent to 50 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C12 DEA. The rats were administered read across substance concentrations of 0, 0.1, 0.5, 1 and 2% in diet for 90 d. The animals were observed daily for clinical signs. Body weight were recorded weekly and haematological, clinical chemistry and urine examinations were carried out at termination. Gross and histopathological examinations were also performed at termination. No adverse effect on the appearance or condition of the animals was observed. Growth retardation was associated with diminished food intake from the dose level of 0.5%. Food refusal was demonstrably due to an effect of the test material on palatability of the diet. Terminal haematological examination revealed a reduction in the haemoglobin level, haematocrit and red cell count at the 1 and 2% dose levels in females but less pronounced effects were seen in males. Serum levels of glutamic-oxaloacetic transaminase were elevated at 0.5% and above in females but only at 0.5 % in males. No untoward effect was observed in the renal function tests. The principal organ weight changes were: increases in the relative kidney weight in all test groups except at 0.1% in females and at 0.1 and 0.5% in males, and increases in the relative liver weight in females on the two highest levels. The types and incidence of histological lesions were comparable in control and test groups. Under the study conditions, the 90 d NOEL in rats was considered to be 0.1% in the diet, equivalent to 50 mg/kg bw/day (Gaunt, 1965).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
2020 - 2021 (planned study)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to read across
Justification for type of information:
Refer to section 13 of IUCLID for details on the category justification.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Species:
rat
Sex:
male/female
Route of administration:
oral: gavage
Remarks on result:
other: Testing planned
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting 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
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 or test system 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 oral 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.
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
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)
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 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.. 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 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.
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/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.
Mortality:
mortality observed, treatment-related
Description (incidence):
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.
Body weight and weight changes:
no effects observed
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.
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):
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.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, the mean absolute and relative liver weights were higher in males and females given 1000 mg/kg/day bw and in males given 300 mg/kg/day bw, reaching statistically significant values in both sexes at 1000 mg/kg/day bw (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 item-related. The mean absolute and relative thymus weights were lower in females given 1000 mg/kg/day bw, without reaching a statistically significant value. This correlated with minimal to slight atrophy in two females and was considered to be probably test item-related. The mean absolute heart weight was higher in males given 100 mg/kg/day bw. In the absence of a dose-related trend, any relationship with the test item 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 item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The thymus was reduced in one female given 1000 mg/kg/day bw correlating in this animal with a small weight (0.1200 g) and histologically with slight atrophy. A relationship with the test item was considered to be likely. Irregular color was seen in the lungs of 3/5 males given 1000 mg/kg/day bw. 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.
Neuropathological findings:
not examined
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.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
See details below.
Other effects:
not examined
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
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, 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 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. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013).   

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
hepatobiliary

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

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 based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
Not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the category justification.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
not specified
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 are observed daily to detect signs of toxicity. Animals which die during the test are 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 or test system 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 (PJ 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 wk
Frequency of treatment:
5 exposures/wk
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Corresponding to 20 mg/mL in ethanol
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Corresponding to 40 mg/mL in ethanol
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Corresponding to 80 mg/mL in ethanol
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Corresponding to 160 mg/mL in ethanol
Dose / conc.:
800 mg/kg bw/day (nominal)
Remarks:
Corresponding to 320 mg/mL in ethanol
No. of animals per sex per dose:
10/sex/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
No information




Positive control:
Not applicable
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:
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 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:
mortality observed, treatment-related
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
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
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
Effect level:
ca. 50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Overall effects
Critical effects observed:
not specified

For detailed tables kindly refer to the attached background materials section of the IUCLID.

Conclusions:
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.
Executive summary:

A study was conducted to evaluate the repeated dose dermal toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to design based on OECD Guideline 411, 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 (NTP, 2001).

Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
Not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the category justification.
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
not specified
Principles of method if other than guideline:
The test substance is 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 are observed daily to detect signs of toxicity. Animals which die during the test are 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 or test system 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 (PJ 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
Details on exposure:
Not reported


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 wk
Frequency of treatment:
5 exposures/wk
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
Corresponding to 30 mg/mL in ethanol
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Corresponding to 61 mg/mL in ethanol
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Corresponding to 121 mg/mL in ethanol
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Corresponding to 243 mg/mL in ethanol
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Corresponding to 485 mg/mL in ethanol
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
Details on study design:
No information




Positive control:
Not applicable
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 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 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:
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):
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:
mortality observed, treatment-related
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
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
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
Effect level:
ca. 50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Overall effects
Critical effects observed:
not specified

For detailed tables kindly refer to the attached background materials section of the IUCLID.

Conclusions:
Under the study conditions, the 14-week systemic NOAEL in rats was considered to be 50 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the repeated dose toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to design based on OECD Guideline 411, 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 (NTP, 2001).

Endpoint:
chronic toxicity: dermal
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
Not available
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the category justification.
Reason / purpose for cross-reference:
reference to other study
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 or test system 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 (PJ 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 d

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

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

Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Corresponding to 50 mg/mL in ethanol
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Corresponding to 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.
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
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
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.
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.

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.
Key result
Dose descriptor:
LOAEL
Remarks:
(systemic and local effects)
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on body weight changes, thyroid gland follicular cell hyperplasia, hepatic neoplasms, renal tubule adenoma, and several non-neoplastic lesions of the skin at all dose levels.
Critical effects observed:
not specified

For detailed tables kindly refer to the attached background materials section of the IUCLID.

 

Conclusions:
Under the study conditions, the 2-yr LOAEL was considered to be 100 mg/kg bw/day in mouse.
Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal toxicity of the read across substance, C8-18 and C18-unsatd. DEA, in compliance with GLP. The doses studied included 0, 100 and 200 mg/kg bw/day of read across substance (corresponding to 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 read across 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-yr LOAEL was considered to be 100 mg/kg bw/day in mouse (NTP, 2001).

Endpoint:
chronic toxicity: dermal
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:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the section 13 for details on the category justification.
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
A two-year dermal study was conducted in rat to evaluate the carcinogenic potential of the test substance.

GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system 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 (PJ 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. All dose formulations analysed during the 2 year studies were within 10% of the target concentration.
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)
Remarks:
Corresponding to 85 mg/mL in ethanol
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Corresponding to 170 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: 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
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.

Mortality:
mortality observed, treatment-related
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
Description (incidence and severity):
The mean body weights of dosed male and female rats were similar to those of the vehicle controls throughout the study.

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
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Skin: No neoplasms of the skin were attributed to treatment with test material. Incidences of squamous cell papilloma, keratoacanthoma, trichoepithelioma, basal cell adenoma, or carcinoma (combined) were significantly decreased in 100 mg/kg bw/d male rats. Incidences of epidermal hyperplasia, sebaceous gland hyperplasia, parakeratosis, and hyperkeratosis in all dosed groups were significantly greater than those in the vehicle control groups. The severities of these lesions generally increased with increasing dose and ranged from minimal to mild. Females in the 100 mg/kg bw/d group had a significantly greater incidence of ulceration at the site of application than did the vehicle controls.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidney: Incidences of renal tubule hyperplasia in dosed females were significantly greater than those of the vehicle controls, and the incidence of renal tubule adenoma in 50 mg/kg bw/d males was marginally increased. Incidences of chronic nephropathy were similar between vehicle control and dosed groups of male and female rats; however, the severity of nephropathy increased with increasing dose in female rats. The incidences of renal tubule adenoma in all groups of males and of renal tubule carcinoma in 50 mg/kg bw/d females exceeded the historical control ranges. An extended evaluation of the kidney revealed additional renal tubule adenomas in vehicle control and dosed males, and renal tubule adenomas and/or carcinomas in dosed females. When the single and step sections were combined, the incidences of renal tubule hyperplasia in dosed females and of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/d females were significantly greater than those of the controls. In female rats, the combined single and step section evaluations of the kidney revealed a significant dose- related increase in the incidence of renal tubule hyperplasia and two adenomas and two carcinomas in the 50 mg/kg bw/d group but only one neoplasm (an adenoma), in the 100 mg/kg bw group. Renal tubule neoplasms are uncommon in female F344/N rats, and the presence of four neoplasms in the 50 mg/kg bw/d group, combined with the increased incidence of hyperplasia, is suggestive of an association with chemical exposure. However, the absence of an increase in neoplasms in the 100 mg/kg bw/d group in the presence of increased hyperplasia makes the association with chemical exposure uncertain.

Forestomach: The incidences of chronic active inflammation (vehicle control, 1/50; 50 mg/kg bw, 3/50; 100 mg/kg bw, 10/50), epithelial hyperplasia (2/50, 5/50, 13/50), and epithelial ulcer (1/50, 3/50, 11/50) were significantly increased in the forestomach of 100 mg/kg bw females. The severities of these lesions were similar among all groups.

Pancreas: The incidence of pancreatic acinar atrophy in 100 mg/kg male rats was significantly greater than that in the vehicle controls.
Key result
Dose descriptor:
NOAEL
Remarks:
(systemic effects)
Effect level:
ca. 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: pancreatic acinar atrophy and nephropathy at the higher dose
Key result
Dose descriptor:
LOAEL
Remarks:
(local effects)
Effect level:
ca. 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on non-neoplastic lesions of the skin at all doses
Critical effects observed:
not specified

For detailed tables kindly refer to the attached background materials section of the IUCLID.

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

A study was conducted to evaluate the long-term repeated dose dermal toxicity of the read across substance, C8-18 and C18-unsatd. DEA, in compliance with GLP. 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 read across 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. Under the study conditions, the 2-yr NOAEL was considered to be 50 mg/kg bw/day in rat (NTP, 2001).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral (28 day)

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, C12-18 and C18-unsatd. DEA, according to a design based on OECD Guideline 407. Groups of 10 male and 10 female Wistar rats were orally gavaged with the substance diluted in olive oil, 5 d/week for 28 d at doses of 0, 70, 250, 750 (Days 1-14) and 1500 (Days 15-28) mg/kg bw/d. Clinical signs, bodyweight, haematology, clinical chemistry, urinalysis, gross and microscopic pathology were recorded. Additional groups of 5 male and 5 female rats were kept for a 4 month recovery period. No treatment-related adverse effects were observed at any of the doses. Changes in the forestomach at some doses including controls were attributed to the use of olive oil and found to be reversible after end of exposure. Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day (Potokar, 1983).

Oral (Combined repeated dose and reproductive/developmental screen)

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C18-unsatd. MIPA, 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 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. Under the study conditions, the NOAEL for parent systemic toxicity was considered to be 1000 mg/kg bw/day (Bentz, 2013). 

Also, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a combined repeated dose toxicity testing/reproductive and developmental screening according to OECD Guideline 422 is planned with C16-18 and C18-unsatd. DEA in order to further support the read across approach proposed for the FAA category members.

Oral (90 day)

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C12 DEA. The rats were administered read across substance concentrations of 0, 0.1, 0.5, 1 and 2% in diet for 90 d. The animals were observed daily for clinical signs. Body weight were recorded weekly and haematological, clinical chemistry and urine examinations were carried out at termination. Gross and histopathological examinations were also performed at termination. No adverse effect on the appearance or condition of the animals was observed. Growth retardation was associated with diminished food intake from the dose level of 0.5%. Food refusal was demonstrably due to an effect of the test material on palatability of the diet. Terminal haematological examination revealed a reduction in the haemoglobin level, haematocrit and red cell count at the 1 and 2% dose levels in females but less pronounced effects were seen in males. Serum levels of glutamic-oxaloacetic transaminase were elevated at 0.5% and above in females but only at 0.5 % in males. No untoward effect was observed in the renal function tests. The principal organ weight changes were: increases in the relative kidney weight in all test groups except at 0.1% in females and at 0.1 and 0.5% in males, and increases in the relative liver weight in females on the two highest levels. The types and incidence of histological lesions were comparable in control and test groups. Under the study conditions, the 90 d NOEL in rats was considered to be 0.1% in the diet, equivalent to 50 mg/kg bw/day (Gaunt, 1965).

Furthermore, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a testing proposal is submitted for the conduct of repeated dose toxicity study according to OECD Guideline 408 with the read across substance, C8-18 and C18-unsatd. DEA.

Dermal (14 week)  

A study was conducted to evaluate the repeated dose dermal toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to a design based on OECD Guideline 411, in compliance with GLP. Groups of 10 male and 10 female B6C3F1mice 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 (NTP, 2001).  

A study was conducted to evaluate the repeated dose toxicity of the read across substance, C8-18 and C18-unsatd. DEA, according to a design based on OECD Guideline 411, in compliance with GLP. Groups of 10 male and 10 female Fischer 344 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 (NTP, 2001). 

Dermal (2 year) 

A study was conducted to evaluate the long-term repeated dose dermal toxicity to B6C3F1 mice of the read across substance, C8-18 and C18-unsatd. DEA, in compliance with GLP. The doses studied included 0, 100 and 200 mg/kg bw/day of read across substance (corresponding to 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 (hepatocellular adenoma, 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 read across 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-yr LOAEL was considered to be 100 mg/kg bw/day in mouse (NTP, 2001).  

A study was conducted to evaluate the long-term repeated dose dermal toxicity to Fischer 344 rats of the read across substance, C8-18 and C18-unsatd. DEA, 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 read across 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. Under the study conditions, the 2-yr NOAEL was considered to be 50 mg/kg bw/day in rat (NTP, 2001).

Additional considerations

 

Based on an in-depth analysis of the available information (see read-across justificationin 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 conductedwith 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 (2cnd 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 suggestsignificant differences in type and strength of effects between theDEA, MEA and MIPAsubcategories, 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

Based on the results of oral and dermal repeated dose toxicity studies in rodents with the substance itself or the read across substances C18-unsatd. MIPA, C12 DEA or C8-18 and C18-unsatd. DEA, the test substance does not require classification for this endpoint according to CLP (EC 1272/2008) criteria.