Amides, C18-unsatd., N,N-bis(hydroxyethyl)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Well conducted and comparable to guideline study, no information on GLP status

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mus-Rattus, Brunntal, Germany
- Strain: Wistar rat, MuRa Han 67 SPF
- Age at study initiation: between 6-7 weeks
- Weight at study initiation: 109 (f) - 114 (m) g
- Housing: plastic cages, 3 males and 5 females/cage
- Diet (e.g. ad libitum): ad libitum (Altromin Ratdiet No. 1424 DK, Altromin GmbH, Lage, Germany)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 60-80
- Air changes (per hr): 11
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

Doses were adapted weekly to the body weight; application volume - 5 mL/kg bw

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 d

Frequency of treatment:

Daily once, 5 times/wk

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

70 mg/kg bw/day (actual dose received)

Remarks:

Days 1-28

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Days 1-28

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

Days 1-14

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Remarks:

Days 15-28

No. of animals per sex per dose:

10/sex/dose for main study; 5/sex/dose for 4 month recovery period

Control animals:

yes, concurrent no treatment

Details on study design:

According to standard procedure

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: end of study

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 10 per dose and sex
- Parameters checked: Hematocrit, erythrocytes, leukocytes, hemoglobin, thrombocytes, mean corpuscular volume, white blood cell differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- How many animals: 10 per sex and dose
- Parameters checked: GPT, GOT, AP, glucose, urea, total protein, calcium, phosphate, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: end of study
- Metabolism cages used for collection of urine: No
- Parameters checked: urea, creatinine, sodium, potassium, glucose, calcium, AP

NEUROBEHAVIOURAL EXAMINATION: No

Other: Groups of 5 male and 5 female rats kept for an additional 4 month recovery period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
the following tissues/organs were examined:
adrenal gland
aorta thoracica
brain (cornu ammonis)
coagulating gland with seminal vesicle
epididymis
eye with optic nerve
heart
intestine, large
intestine, small
kidney
liver
lungs
lymph node (cervical)
lymph node (mesenteric)
mucles
oesophagus
ovary
pancreas
prostate
salivary glands (mandibular,
parotid and sublingual gland)
skin
spleen
stomach
testicle
thymus
thyroid (incl. parathyroids)
tongue
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix and oviducts)


HISTOPATHOLOGY: Yes
see gross pathology

Statistics:

't' test used for statistical analysis of all parameters except organ weight (U-test)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

The doses up to 1500 mg/kg body weight/day were tolerated by all animals without lethality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight gain and total increase of body weights did not differ from the control group.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterine, GGT, GOT, GPT and LDH did not show any critical signs. Only slight shifts which were not dose-dependent could be observed. These signs were considered as not substance depending.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative organ weights showed no significant changes in the substance groups compared to the control group, except the organ weight of the liver which is slightly increased for the males of group 4 (750/1500 mg/kg bw) and increased adrenal glands weight in high dose females. This result is considered of no relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The pathological and histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose dependent reversible local findings were restricted to the fore stomach mucosa (important hyperplasia and ulcerations, in some cases also hyper- and parakeratosis of the forestomach of males and females at the high dose. Similar effects but less intense at the medium and low doses).

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The pathological and histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose dependent reversible local findings were restricted to the fore stomach mucosa (important hyperplasia and ulcerations, in some cases also hyper- and parakeratosis of the forestomach of males and females at the high dose. Similar effects but less intense at the medium and low doses).

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No biologically relevant treatment-related effects observed on any of the parameters recorded at any dose, also test animals treated with 1500 mg/kg bw (Days 15-28) showed no adverse effect

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C12-18 and C18-unsatd. DEA, according to 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).

Reference 3

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'

Remarks on result:

other: equivalent to 50 mg/kg bw/day (i.e., 46.18 mg a.i./kg bw/day)

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 (i.e., 46.18 mg a.i./kg bw/day).

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C12 DEA (92.36% active). 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 (i.e., 46.18 mg a.i./kg bw/day) (Gaunt, 1965). Based on the results of the read across study, a similar NOAEL value is expected for the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 June 1992 to 22 September 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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:

A 13-week toxicity study in B6C3F1 mice was conducted to evaluate the effects of repeated dermal exposure to 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:

- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 8 wk
- Housing: Housed individually in polycarbonate cages
-Method of distribution: Animals were distributed randomly into groups of approximately equal initial mean body weights.
- Bedding: Sani-Chip® heat-treated hardwood chips (PJ Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum
- Acclimation period:Time held before studies: Males: 21 d and Females: 22 d
- Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly and rotated every 2 wks
- Animal number per cage: 1
- Cage Filters: Spun-bonded polyester Du Pont 2024 (Snow Filtration, Co., Cincinnati, OH), changed every 2 wks
- Racks: Stainless steel drawer-type (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 wks

ENVIRONMENTAL CONDITIONS
- Temperature: 20.6-25.6°C
- Relative humidity: 39-65 %
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1992-06-02 To: 1992-09-22

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 3 weeks by mixing the test substance by stirring or sonicating with 95% ethanol to give the required concentrations. The test substance formulations were applied on shaved skin of the test animals.
- Storage of dose formulations: The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.
- Stability studies of a 10 mg/mL formulation prepared from lot CH1F980 (not used) were performed by the study laboratory using high-performance liquid chromatography. Stability of the dose formulation was confirmed for at least 28 days when stored in sealed containers, protected from ultraviolet light, at up to room temperature or for 3 hours when stored open to air and light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of the test substance from the beginning, middle, and end of the study were analyzed using HPLC. All the samples from the formulations analyzed during the 13 week study were within 10% of the target concentration.
- Stability of dose formulations: Stability was confirmed for at least 28 days when stored in sealed containers, protected from ultraviolet light, at up to room temperature or for 3 hours when stored open to air and light

Duration of treatment / exposure:

13 wks

Frequency of treatment:

5 exposures/wk

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

800 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Method of animal grouping: Distributed randomly into groups of approximately equal initial mean body weights.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded weekly

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

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

OTHER: Organs weighed were heart, right kidney, liver, lung, right testis, and thymus

Sacrifice and pathology:

SACRIFICE: At the end of the 13-week study animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes.
- Complete histopathology was performed on 0 and 800 mg/kg bw/day mice. In addition to gross lesions and tissue masses, the tissues examined were: 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, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the skin was examined in all mice.
- All major tissues were fixed and preserved in 10% neutral buffered formalin processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination.

Other examinations:

Sperm motility and vaginal cytology:
- Samples were collected for sperm motility or vaginal cytology from all mice in the 0, 200, 400, and 800 mg/kg bw/day dose groups.
- The sperm motility parameters evaluated was: Sperm concentration, sperm motility, sperm count, spermatid heads per testis, and spermatid heads per gram of testis. The left cauda, epididymis, and testis were weighed.
- Vaginal samples were collected for 12 consecutive days prior to the end of the studies for vaginal cytology evaluations. The parameters evaluated were: the length of the estrous cycle and the length of time spent in each stage of the cycle.

Statistics:

Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.

- Analysis of neoplasm and non-neoplastic 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 pair wise 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. Values of P greater than 0.5 are presented as 1-P with the letter N added to indicate a lower incidence or negative trend in neoplasm occurrence relative to the control group (e.g., P=0.99 is presented as P=0.01N).

- Analysis of Continuous Variables: Organ and body weight data were analysed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). 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:

no effects observed

Description (incidence and severity):

Clinical findings included irritation of the skin at the site of application. Irritation occurred in all surviving dosed males and in most females administered 100 mg/kg bw or greater; time of onset was inversely related to dose. Irritation progressed to ulcer in one 800 mg/kg bw male.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Occurred at the site of application in all dosed males and in most of the females administered ≥100 mg/kg bw/day. Irritation progressed to ulcer in one male in the 800 mg/kg bw/day dose group.

Mortality:

no mortality observed

Description (incidence):

All animals except one male mice at 800 mg/kg bw/day survived until the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced final mean body weights and body weight gains of males at 800 and females at ≥400 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Increased absolute and relative heart weights of males at 400 and 800 mg/kg bw/day and of females at ≥200 mg/kg bw/day. Further, the absolute heart weights were increased for females in ≥50 mg/kg bw/day dose groups.
- Increased liver weights in all dosed groups
- Increased kidney weights of males at ≥50 mg/kg bw/day.
- Decreased absolute thymus weight of males at ≥200 mg/kg bw/day and of females at ≥400 mg/kg bw/day. Decreased relative thymus weight was observed with females only the 800 mg/kg bw/day dosed groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Skin: Dose dependent increase in the incidences of lesions (including epidermal hyperplasia, parakeratosis, suppurative epidermal inflammation, chronic active dermal inflammation, sebaceous gland hypertrophy, and ulcer in males and females) were observed.
- Bone marrow myeloid hyperplasia in males and females at 800 mg/kg bw/day and increased hematopoietic cell proliferation of spleen in males at 800 mg/kg bw/day and in females at 400 and 800 mg/kg bw/day were observed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Sperm motility and vaginal cytology parameters of dosed mice were similar to those of the vehicle controls.

Key result

Dose descriptor:

LOAEL

Remarks:

(systemic effects)

Effect level:

ca. 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Altered organ weights (with increased liver weights in all dosed groups) and reduced body weights and body weight gains at 800 mg/kg bw/day in males and ≥400 mg/kg bw/day in females

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Irritation of the skin at the site of application in all dosed males and in most of the females administered ≥100 mg/kg bw/day.

Key result

Critical effects observed:

not specified

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

Conclusions:

Under the study conditions, the 13-week LOAEL for both systemic and local effects was considered to be 50 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the repeated dose dermal toxicity in B6C3F1 mice of the test substance, C18-unsatd. DEA, according to design based on OECD Guidance 411, in compliance with GLP. Groups of 10 male and 10 female mice were dermally exposed to 0, 50, 100, 200, 400 or 800 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 13 weeks. Mortality, clinical findings, body weight and histopathology were evaluated at specific time intervals. All male and female mice except one 800 mg/kg bw/day male survived until the end of the study. Final mean body weights and body weight gains of 800 mg/kg bw/day males and females and 400 mg/kg bw/day females were significantly lower than those of the vehicle controls. Clinical findings in dosed mice included irritation of the skin at the site of application. Irritation occurred in all surviving dosed males and in most females administered ≥100 mg/kg bw/day. The heart weights of 400 and 800 mg/kg bw/day males and females and 200 mg/kg bw/day females and the kidney weights of 50, 100, and 400 mg/kg bw/day males were significantly greater than those of the vehicle controls. Relative to the vehicle controls, the liver weights were increased in all dosed groups. A dose-dependent increase in the incidences of non-neoplastic skin lesions included epidermal hyperplasia, parakeratosis, suppurative epidermal inflammation, chronic active dermal inflammation, sebaceous gland hypertrophy and ulcer. Under the study conditions, the 13-week LOAEL for both systemic and local effects was considered to be 50 mg/kg bw/day (NTP, 1999).

Reference 2

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 02 June 1992 to 24 September 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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:

A 13-week toxicity study in F344/N rats was conducted to evaluate the cumulative toxic effects of repeated dermal exposure to 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:

- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 8 wk
- Housing: Housed individually in polycarbonate cages
-Method of distribution: Animals were distributed randomly into groups of approximately equal initial mean body weights.
- Bedding: Sani-Chip® heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum
- Acclimation period: Males: 23 d and Females: 24 d
- Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly and rotated every 2 wks
- Animal number per cage: 1
- Cage Filters: Spun-bonded polyester Du Pont 2024 (Snow Filtration, Co., Cincinnati, OH), changed every 2 wks
- Racks: Stainless steel drawer-type (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 wks

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1-22.8°C
- Relative humidity: 37-65 %
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1992-06-02 To: 1992-09-24 (males); From: 1992-06-02 To: 1992-09-25 (females)

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 3 weeks by mixing (i.e., by stirring or sonicating) the test substance with 95% ethanol to give the required concentrations. The test substance formulations were applied on shaved interscapular skin of the test animals.
- Storage of dose formulations: The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.
- Stability studies of a 10 mg/mL formulation prepared from lot CH1F980 (not used) were performed by the study laboratory using high-performance liquid chromatography. Stability of the dose formulation was confirmed for at least 28 days when stored in sealed containers, protected from ultraviolet light, at up to room temperature or for 3 hours when stored open to air and light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of the test substance from the beginning, middle, and end of the study were analyzed using HPLC. All the samples from the formulations analyzed during the 13 week study were within 10% of the target concentration.

- Stability of dose formulations: Stability was confirmed for at least 28 days when stored in sealed containers, protected from ultraviolet light, at up to room temperature or for 3 hours when stored open to air and light.

Duration of treatment / exposure:

13 wks

Frequency of treatment:

5 exposures/wk

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Method of animal grouping: Distributed randomly into groups of approximately equal initial mean body weights.

Positive control:

Not used in the study

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded weekly

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Days 5 and 19 and all core study rats surviving to the end of the study
- Method: Blood was collected from the retro-orbital sinus of special study rats from each dose group
- Anaesthetic used for blood collection: Yes, carbon dioxide/oxygen mixture
- How many animals: All animals
- Parameters examined.: Hematocrit, hematocrit, hemoglobin, erythrocyte, nucleated erythrocyte, reticulocyte, and platelet counts; mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, leukocyte counts and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Days 5 and 19 (blood was collected from the retro-orbital sinus of special study rats from each dose group) and all core study rats surviving to the end of the study.
- How many animals: All animals
- Parameters examined: Urea nitrogen, creatinine, alanine aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, total protein, albumin, and bile salts

OTHER: Organs weight: Organ weights were determined for heart, right kidney, liver, lung, right testis, and thymus

Sacrifice and pathology:

SACRIFICE: At the end of the 13-week study animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes.

- Complete histopathology was performed on 0 and 400 mg/kg bw/day study rats. In addition to gross lesions and tissue masses, the tissues examined were: 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, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the skin was examined in all rats.
- All major tissues were fixed and preserved in 10% neutral buffered formalin processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination.

Other examinations:

Sperm motility and vaginal cytology:
- Samples were collected for sperm motility or vaginal cytology from all rats in the 0, 100, 200, and 400 mg/kg bw/day groups.
- The sperm motility parameters evaluated was: Sperm concentration, sperm motility, sperm count, spermatid heads per testis, and spermatid heads per gram of testis. The left cauda, epididymis, and testis were weighed.
- Vaginal samples were collected for 12 consecutive days prior to the end of the studies for vaginal cytology evaluations.
- The parameters observed were: Estrous cycle length and relative frequency of estrous stage.

Statistics:

Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.

- Analysis of neoplasm and non-neoplastic 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 pair wise 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. Values of P greater than 0.5 are presented as 1-P with the letter N added to indicate a lower incidence or negative trend in neoplasm occurrence relative to the control group (e.g., P=0.99 is presented as P=0.01N).

- Analysis of Continuous Variables: Organ and body weight data were analysed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data were analysed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). 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:

no effects observed

Description (incidence and severity):

The only chemical-related clinical finding was irritation of the skin at the site of application in most males administered 100 mg/kg bw or greater and in all females administered 50 mg/kg bw or greater.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

The only chemical-related clinical finding was irritation of the skin at the site of application in most males administered 100 mg/kg bw or greater and in all females administered 50 mg/kg bw or greater.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced final mean body weights and body weight gains of males and/or females at 200 or 400 mg/kg bw/day

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):

Increased segmented neutrophil counts in the 400 mg/kg bw/day male group on day 5 and 19, in the 200 mg/kg bw/day female group on day 19 and at wk 13 and in the 400 mg/kg bw/day female group on day 5 and 19 and at week 13.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant increase in alkaline phosphatase activity on day 19 in males at 200 mg/kg bw/day, at week 13 in females at 200 mg/kg bw/day and at week 13 in males and females at 400 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased kidney weights in females at 200 and 400 mg/kg bw/day; decreased heart, liver, lung and thymus weights in males and females in the 200 and 400 mg/kg bw/day groups were associated with the lower mean body weights of these groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nonneoplastic lesions of the skin related to administration of oleic acid diethanolamine condensate included epidermal hyperplasia, parakeratosis, chronic active inflammation of the dermis, suppurative epidermal inflammation, and sebaceous gland hypertrophy in males and females. The severities of epidermal hyperplasia and sebaceous gland hypertrophy increased with increasing dose in males and females.


Lesions of the skin were also present at the site of application in groups administered 100 mg/kg bw/day; however, the incidences were somewhat less than those observed in the 200 and 400 mg/kg bw/day groups. In addition, the severities of the lesions were increased only slightly in the 200 and 400 mg/kg bw/day groups compared to the severities in the 100 mg/kg bw/day groups. Moreover, it was considered unlikely that these lesions would progress and become life threatening over the period of a 2 -year study. In groups treated with 50 mg/kg bw/day, the incidences of skin lesions diminished considerably and lesion severities were minimal.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no significant differences in sperm motility and vaginal cytology parameters between dosed and vehicle control groups.

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Overall effects (i.e., decreased body weight and body weight gain, increased segmented neutrophil counts, increased alkaline phosphatase concentrations, increased kidney weights in males and/or females at ≥200 mg/kg bw/day)

Key result

Dose descriptor:

NOAEL

Remarks:

(local effects)

Effect level:

ca. 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

not specified

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

Conclusions:

Under the study conditions, the 13-week NOAELs for systemic and local effects can be considered to be 100 and 25 mg/kg bw/day, respectively.

Executive summary:

A study was conducted to evaluate the repeated dose dermal toxicity in F344/N rats of the test substance, C18-unsatd. DEA, according to design based on OECD Guideline 411, in compliance with GLP. Groups of 10 male and 10 female rats were dermally exposed to 0, 25, 50, 100, 200, or 400 mg test substance/kg bw/day in ethanol at a frequency of 5 d/week for a period of 13 weeks. Survival, clinical findings, body weight, hematology, clinical chemistry and histopathology were evaluated at specific time intervals. All animals survived until study termination. The final mean body weights and body weight gains of 200 and 400 mg/kg bw/day males and the mean body weight gain of 400 mg/kg bw/day females were significantly lower than those of the vehicle controls. The only treatment-related clinical finding was irritation of the skin at the site of application in most males administered 100 mg/kg bw/day or greater and in all females administered 50 mg/kg bw/day or greater. Segmented neutrophil counts and alkaline phosphatase concentrations were increased significantly on specific days in the 200 and/or 400 mg/kg bw/day male and female groups. Kidney weights of 200 and 400 mg/kg females were significantly greater than those of the vehicle controls. There was a dose-dependent increase in the incidence of non-neoplastic lesions of the skin at the site of application, including epidermal hyperplasia, parakeratosis, chronic active dermal inflammation, suppurative epidermal inflammation and sebaceous gland hypertrophy in dosed rats. However, these effects were minimal in the 50 mg/kg bw/day dose group. Under the study conditions, the 13-week NOAELs for systemic and local effects can be considered to be 100 and 25 mg/kg bw/day, respectively (NTP, 1999).

Reference 3

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 29 April 1993 to 10 May 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A 2-year study was conducted in B6C3F1 mice to evaluate the the chronic dermal toxicity 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:

- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 wks
- Housing: Housed individually in Polycarbonate cages
- Method of distribution: Animals were distributed randomly into groups of approximately equal initial mean body weights.
- Bedding: Sani-Chip® heat-treated hardwood chips (PJ Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum
- Acclimation period:Time held before studies: Males: 11 d and Females: 12 d
- Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly and rotated every 2 wks
- Animal number per cage: 1
- Cage Filters: Spun-bonded polyester Du Pont 2024 (Snow Filtration, Co., Cincinnati, OH), changed every 2 wks
- Racks: Stainless steel drawer-type (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 wks

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1-25.0°C
- Relative humidity: 36-68%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1993-04-29 To: 1995-05-10

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 3 weeks by mixing the test substance by stirring or sonicating with 95% ethanol to give the required concentrations.The test substance formulations were applied on shaved skin of the test animals.
The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of the test substance from the beginning, middle, and end of the studies were analyzed at the study laboratory using HPLC. All the samples from the formulations were analysed every 9 weeks during the 2-year study and were within 10% of the target concentration.
Stability of dose formulations: Stability was confirmed for at least 28 days when stored in sealed containers, protected from ultraviolet light, at up to room temperature or for 3 hours when stored open to air and light.

Duration of treatment / exposure:

2-yr

Frequency of treatment:

5 exposures/wk

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

50/sex/dose; 5 M/F (for 3-month interim evaluation in mice)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose selection is based primarily on the incidences and severities of skin lesions observed at the site of application during the 13-week dermal studies. All groups of mice administered 100 mg/kg bw/day or greater exhibited high incidences of skin lesions at the site of application; thus, 100 mg/kg bw/day or greater were considered inappropriate for the 2-year study.
- The severities of the skin lesions increased with increasing dose in groups administered doses greater than 100 mg/kg bw/day; however, the severities of other lesions generally were increased only slightly between 100 and 800 mg/kg compared to the eightfold increase in dose. Therefore, the skin response appeared to plateau at 100 mg/kg, and higher doses did not produce a proportional increase in response. The incidences of skin lesions in groups administered 50 mg/kg bw/day were slightly less (minimal to mild) than those observed in groups administered 100 mg/kg bw/day. The skin response at the site of application in 50 mg/kg bw/day groups was such that 50 mg/kg bw/day was also considered inappropriate for a 2-year study; however, the slight reduction in incidences and the lower severities observed in the 50 mg/kg bw/day groups compared to those in the 100 mg/kg bw/day groups indicated that 50 mg/kg was below the plateau and at the upper end of a dose range in which skin response at the site of application exhibited a greater dose dependency. Therefore, at doses below 50 mg/kg bw/day, a proportional reduction in incidences and severities of skin lesions at the site of application would be expected. Accordingly, a high dose of 30 mg/kg and a low dose of 15 mg/kg were selected for the 2-year study in mice.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded monthly and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly for 13 wks, approximately monthly thereafter and again at the end of the studies

DERMAL IRRITATION (if dermal study): Yes

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

Sacrifice and pathology:

SACRIFICE: At the end of 3rd month 5 males and females were sacrificed for skin evaluation and at the end of the 2-year study all animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Skin from the site of application was examined from all mice at the 3-month interim evaluation.
- Complete histopathology was performed on all the rats at the end of the study. In addition to gross lesions and tissue masses, the tissues examined were: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum and 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 (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
- All major tissues were fixed and preserved in 10% neutral buffered formalin processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination

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 non-neoplastic 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 pair wise 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. Values of P greater than 0.5 are presented as 1-P with the letter N added to indicate a lower incidence or negative trend in neoplasm occurrence relative to the control group (e.g., P=0.99 is presented as P=0.01N).

Analysis of Continuous Variables: Organ and body weight data were analysed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Irritation of the skin at the site of application in 30 mg/kg bw/day males (vehicle control: 0/55; 15 mg/kg bw/day: 1/55; 30 mg/kg bw/day: 20/55).

Mortality:

no mortality observed

Description (incidence):

Survival of dosed male and female mice was similar to that of the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced mean body weights of 30 mg/kg bw/day females at the beginning of week 76.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Skin lesions: Minimal to mild non-neoplastic lesions of the skin at the site of application were observed in females more than males. The incidences of epidermal hyperplasia, sebaceous gland hyperplasia, and chronic active inflammation of the dermis in all dosed groups were significantly increased relative to the vehicle controls at 3 months and at 2 years. There was increased incidence of hyperkeratosis in dosed males at 3 months and in dosed males and females at 2-years. The increased incidences of parakeratosis in 30 mg/kg bw/day males at the 3-month interim evaluation and at 2-years and ulcer in 30 mg/kg bw/day males and exudate in 30 mg/kg bw/day males and females at 2 years were also attributed to the test substance administration.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

- Skin lesions: No significant treatment related effects were observed. Few neoplastic lesions observed (i.e., one fibrosarcoma in vehicle control female and two fibrosarcomas in females at 15 mg/kg bw/day) were not considered significant as they did not follow a dose dependent pattern.

- Malignant lymphoma: No significant treatment related effects were observed. The increased incidence of the lymphomas observed in females at 30 mg/kg bw/day was found to be similar to the incidences observed in the other dermal studies with ethanol as the vehicle (i.e., comparable to historical control values).

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

ca. 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight changes at 30 mg/kgt bw/d

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

ca. 15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: incidences of non-neoplastic lesions of the skin

Key result

Critical effects observed:

not specified

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

Conclusions:

Under the study conditions, the 2-yr NOAEL for both local and systemic effects can be considered to be 15 mg/kg bw/day and for local effects, the 2-yr LOAEL can be considered to be 15 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal toxicity in B6C3F1 mice of the test substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female mice were dermally exposed to 0, 15 or 30 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 105 weeks. 5 males and 5 females were considered for the 3 month interim assessment. Survival, clinical findings, body weight and histopathology were evaluated at specific time intervals. Survival of the dosed male and female rats was similar to that of the vehicle control groups. The mean body weights of females (week 76 onwards) were reduced than those of the vehicle control group at 30 mg/kg bw/day. The only significant treatment-related clinical finding was irritation of the skin at the site of application in 30 mg/kg bw/day males. The incidences of epidermal hyperplasia, sebaceous gland hyperplasia and chronic active inflammation of the dermis in all dosed groups were significantly increased relative to the vehicle controls at 3 months and at 2 years. The increased incidences of hyperkeratosis in dosed males at 3 months and in dosed males and females at 2 years, of parakeratosis in 30 mg/kg bw/day males at 3 months and 2 years, and of ulcer in 30 mg/kg bw/day males and exudate in 30 mg/kg bw/day males and females at 2 years were also attributed to the test substance administration. No significant neoplastic lesions or evidence of carcinogenic activity was observed at any tested dose levels in skin and lymph nodes. Under the study conditions, the 2-yr NOAEL for both local and systemic effects can be considered to be 15 mg/kg bw/day and for local effects, the 2-yr LOAEL can be considered to be 15 mg/kg bw/day (NTP, 1999).

Reference 4

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 May 1993 to 16 May 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A 2-year study was conducted in F344/N rats to evaluate the the chronic dermal toxicity 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:

- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 7 wk
- Housing: Housed individually in Polycarbonate cages
- Method of distribution: Animals were distributed randomly into groups of approximately equal initial mean body weights.
- Bedding: Sani-Chip® heat-treated hardwood chips (PJ Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum
- Acclimation period:Time held before studies: Males: 13 d and Females: 14 d
- Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly and rotated every 2 wks
- Animal number per cage: 1
- Cage Filters: Spun-bonded polyester Du Pont 2024 (Snow Filtration, Co., Cincinnati, OH), changed every 2 wks
- Racks: Stainless steel drawer-type (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 wks

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1-23.3°C
- Relative humidity: 31-73%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1993-05-06 To: 1995-05-16

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 3 weeks by mixing the test substance by stirring or sonicating with 95% ethanol to give the required concentrations. The test substance formulations were applied on shaved skin of the test animals.
The dose formulations were stored at room temperature, protected from light, in amber glass bottles for up to 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of the test substance from the beginning, middle, and end of the studies were analyzed at the study laboratory using HPLC. All the samples from the formulations were analysed every 9 weeks during the 2-year study and were within 10% of the target concentration.

Stability of dose formulations: Stability was confirmed for at least 28 days when stored in sealed containers, protected from ultraviolet light, at up to room temperature or for 3 hours when stored open to air and light.

Duration of treatment / exposure:

2-yr

Frequency of treatment:

5 exposures/wk

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose selection was based primarily on the incidences and severities of skin lesions observed at the site of application during the 13-week dermal studies. The doses of 200 and 400 mg/kg bw/day exhibited reduced mean body weight and body weight gain along with high incidences of skin lesions at the site of application; thus were considered inappropriate for a 2-year study. Further, lesions of the skin were also present at the site of application in groups administered 100 mg/kg bw/day; however, the incidence were less than those observed in the 200 and 400 mg/kg bw/day groups. Moreover, it was considered unlikely that these lesions would progress and become life threatening over the period of a 2-year study. Therefore, 100 mg/kg bw/day was selected as the high dose for rats in the 2-year study. In groups treated with 50 mg/kg bw/day, the incidences of skin lesions diminished considerably and lesion severities were minimal. Therefore, 50 mg/kg bw/day was selected as the low dose.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded monthly and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly for 13 weeks, approximately monthly thereafter and again at the end of the studies

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

Sacrifice and pathology:

SACRIFICE: At the end of the 2-year study animals were sacrificed by carbon dioxide asphyxiation.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Complete histopathology was performed on all the rats at the end of the study. In addition to gross lesions and tissue masses, the tissues examined were: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart with aorta, large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum and 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 (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, trachea, urinary bladder, and uterus.
- All major tissues were fixed and preserved in 10% neutral buffered formalin processed and trimmed, embedded in paraffin, sectioned to a thickness of 5 to 6 μm, and stained with hematoxylin and eosin for microscopic examination.

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 non-neoplastic 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 pair wise 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. Values of P greater than 0.5 are presented as 1-P with the letter N added to indicate a lower incidence or negative trend in neoplasm occurrence relative to the control group (e.g., P=0.99 is presented as P=0.01N).

Analysis of Continuous Variables: Organ and body weight data were analysed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate irritation of the skin at the site of application in dosed males and females; vehicle control, 0/50; 50 mg/kg bw/day, 17/50; 100 mg/kg bw/day, 32/50; females: 3/50, 46/50, 50/50.

Mortality:

no mortality observed

Description (incidence):

Survival of dosed male and female rats was similar to that of the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly reduced mean body weights of males and females at 100 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Skin lesions: Minimal to moderate non-neoplastic lesions of the skin at the site of application were observed. The major alterations included (thickening of the epidermis, sebaceous gland and epidermal hyperplasia, hyperkeratosis, parakeratosis, chronic active dermal inflammation and ulcer) which were significantly increased in dosed males and females relative to the vehicle control.

- Forestomach lesions: No significant treatment related effects were observed. The increased incidence of hyperkeratosis and ulceration in 50 mg/kg bw/day males were not considered to be treatment related as these effects were not observed in females.

- Testis: No significant treatment related effects were observed.

- Thyroid gland lesions: No significant treatment related effects were observed.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

- Skin lesions: No significant treatment related effects were observed. Few observed skin neoplasms (one subcutaneous fibroma in one vehicle control male and one subcutaneous fibrosarcoma in each of the 50 and 100 mg/kg bw/day male groups) were not considered to be significant as the incidences did not follow a pattern indicative of an association with the test substance.

- Testis: No significant treatment related effects were observed. Increased interstitial cell adenoma in males at 100 mg/kg bw/day was not considered significant as similar incidences were reported in vehicle controls in historical NTP dermal studies.

- Thyroid gland: No significant treatment related effects were observed. Marginal increase in the incidence of follicular cell adenoma or carcinoma observed in males at 50 mg/kg bw/day was not considered dose related and no follicular cell hyperplasias were observed .

Other effects:

no effects observed

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: body weight changes at the LOAEL

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: irritation and non-neoplastic lesion of the skin at both the tested doses

Key result

Critical effects observed:

not specified

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

Conclusions:

Under the study conditions, the 2-yr NOAEL for systemic effects can be considered to be 50 mg/kg bw/day and the 2-yr LOAEL for local effects can be considered at the lowest dose of 50 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal toxicity in F344 rats of the test substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female rats were dermally exposed to 0, 50 or 100 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 104 weeks. Survival, clinical findings, body weight and histopathology of different organs were assessed at specific time intervals. Survival of the dosed male and female rats was similar to that of the vehicle control groups. The mean body weights of males and females (week 24 onwards) were reduced than those of the vehicle control group at 100 mg/kg bw/day. A dose dependent increase in irritation (mild to moderate) and non-neoplastic lesions (minimal to moderate) of the skin were observed at the site of application in all animals. The non-neoplastic lesions included epidermal hyperplasia, sebaceous gland hyperplasia, hyperkeratosis, parakeratosis, chronic active dermal inflammation and ulcer. No significant neoplastic lesions or evidence of carcinogenic activity was observed at any tested dose levels in skin, testis and thyroid gland. Under the study conditions, the 2-yr NOAEL for systemic effects can be considered to be 50 mg/kg bw/day and the 2-yr LOAEL for local effects can be considered at the lowest dose of 50 mg/kg bw/day (NTP, 1999).

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 days)

A study was conducted to evaluate the repeated dose oral toxicity of the read across 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 the read across substance, 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 days)

A study was conducted to evaluate the repeated dose dermal toxicity in B6C3F1 mice of the test substance, C18-unsatd. DEA, according to design based on OECD Guidance 411, in compliance with GLP. Groups of 10 male and 10 female mice were dermally exposed to 0, 50, 100, 200, 400 or 800 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 13 weeks. Mortality, clinical findings, body weight and histopathology were evaluated at specific time intervals. All male and female mice except one 800 mg/kg bw/day male survived until the end of the study. Final mean body weights and body weight gains of 800 mg/kg bw/day males and females and 400 mg/kg bw/day females were significantly lower than those of the vehicle controls. Clinical findings in dosed mice included irritation of the skin at the site of application. Irritation occurred in all surviving dosed males and in most females administered ≥100 mg/kg bw/day. The heart weights of 400 and 800 mg/kg bw/day males and females and 200 mg/kg bw/day females and the kidney weights of 50, 100, and 400 mg/kg bw/day males were significantly greater than those of the vehicle controls. Relative to the vehicle controls, the liver weights were increased in all dosed groups. A dose-dependent increase in the incidences of non-neoplastic skin lesions included epidermal hyperplasia, parakeratosis, suppurative epidermal inflammation, chronic active dermal inflammation, sebaceous gland hypertrophy and ulcer. Under the study conditions, the 13-week LOAEL for both systemic and local effects was considered to be 50 mg/kg bw/day (NTP, 1999).

A study was conducted to evaluate the repeated dose dermal toxicity in F344/N rats of the test substance, C18-unsatd. DEA, according to design based on OECD Guideline 411, in compliance with GLP. Groups of 10 male and 10 female rats were dermally exposed to 0, 25, 50, 100, 200, or 400 mg test substance/kg bw/day in ethanol at a frequency of 5 d/week for a period of 13 weeks. Survival, clinical findings, body weight, hematology, clinical chemistry and histopathology were evaluated at specific time intervals. All animals survived until study termination. The final mean body weights and body weight gains of 200 and 400 mg/kg bw/day males and the mean body weight gain of 400 mg/kg bw/day females were significantly lower than those of the vehicle controls. The only treatment-related clinical finding was irritation of the skin at the site of application in most males administered 100 mg/kg bw/day or greater and in all females administered 50 mg/kg bw/day or greater. Segmented neutrophil counts and alkaline phosphatase concentrations were increased significantly on specific days in the 200 and/or 400 mg/kg bw/day male and female groups. Kidney weights of 200 and 400 mg/kg females were significantly greater than those of the vehicle controls. There was a dose-dependent increase in the incidence of non-neoplastic lesions of the skin at the site of application, including epidermal hyperplasia, parakeratosis, chronic active dermal inflammation, suppurative epidermal inflammation and sebaceous gland hypertrophy in dosed rats. However, these effects were minimal in the 50 mg/kg bw/day dose group. Under the study conditions, the 13-week NOAELs for systemic and local effects can be considered to be 100 and 25 mg/kg bw/day, respectively (NTP, 1999).

Dermal (2 years)

A study was conducted to evaluate the long-term repeated dose dermal toxicity in B6C3F1 mice of the test substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female mice were dermally exposed to 0, 15 or 30 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 105 weeks. 5 males and 5 females were considered for the 3 month interim assessment. Survival, clinical findings, body weight and histopathology were evaluated at specific time intervals. Survival of the dosed male and female rats was similar to that of the vehicle control groups. The mean body weights of females (week 76 onwards) were reduced than those of the vehicle control group at 30 mg/kg bw/day. The only significant treatment-related clinical finding was irritation of the skin at the site of application in 30 mg/kg bw/day males. The incidences of epidermal hyperplasia, sebaceous gland hyperplasia and chronic active inflammation of the dermis in all dosed groups were significantly increased relative to the vehicle controls at 3 months and at 2 years. The increased incidences of hyperkeratosis in dosed males at 3 months and in dosed males and females at 2 years, of parakeratosis in 30 mg/kg bw/day males at 3 months and 2 years, and of ulcer in 30 mg/kg bw/day males and exudate in 30 mg/kg bw/day males and females at 2 years were also attributed to the test substance administration. No significant neoplastic lesions or evidence of carcinogenic activity was observed at any tested dose levels in skin and lymph nodes. Under the study conditions, the 2-yr NOAEL for both local and systemic effects can be considered to be 15 mg/kg bw/day and for local effects, the 2-yr LOAEL can be considered to be 15 mg/kg bw/day (NTP, 1999).

A study was conducted to evaluate the long-term repeated dose dermal toxicity in F344 rats of the test substance, C18-unsatd. DEA, in compliance with GLP. Groups of 50 male and 50 female rats were dermally exposed to 0, 50 or 100 mg/kg bw/day in ethanol at a frequency of 5 d/week for a period of 104 weeks. Survival, clinical findings, body weight and histopathology of different organs were assessed at specific time intervals. Survival of the dosed male and female rats was similar to that of the vehicle control groups. The mean body weights of males and females (week 24 onwards) were reduced than those of the vehicle control group at 100 mg/kg bw/day. A dose dependent increase in irritation (mild to moderate) and non-neoplastic lesions (minimal to moderate) of the skin were observed at the site of application in all animals. The non-neoplastic lesions included epidermal hyperplasia, sebaceous gland hyperplasia, hyperkeratosis, parakeratosis, chronic active dermal inflammation and ulcer. No significant neoplastic lesions or evidence of carcinogenic activity was observed at any tested dose levels in skin, testis and thyroid gland. Under the study conditions, the 2-yr NOAEL for systemic effects can be considered to be 50 mg/kg bw/day and the 2-yr LOAEL for local effects can be considered at the lowest dose of 50 mg/kg bw/day (NTP, 1999).

Additional considerations 

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

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

The objective of Tier 2 will be to generate a complete set of higher toxicology data for a selected >1000 tpa substance (i.e., OECD TG 408/443/414 (rats)/414 (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 suggest significant differences in type and strength of effects between the DEA, MEA and MIPA subcategories, therefore not supporting the current read across justification, further testing may be initiated. The Consortium’s strategy is detailed in a document entitled ‘ECHA-DIAP -FAA testing strategy summary – 24Sep20’ attached in Section 13 of the IUCLID dataset.

Justification for classification or non-classification

Based on the results of oral and dermal repeated dose toxicity studies in rodents with various read-across substances, the test substance does not warrant classification for this endpoint according to CLP (EC 1272/2008) criteria.