Amides, C18-unsatd., N-hydroxyethyl

Endpoint summary

Description of key information

A NOAEL of >750 mg/kg bw/day was derived based on no treatment-related effects observed at any of the dose levels in a 28 day oral study conducted on the read across substance amides, C12-18 (even numbered) and C18-unsatd., N-(hydroxyethyl).

Dermal 90 day and 2 year chronic studies on structurally similar amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) have been conducted in both rats and mice. The following dose descriptors were derived:

•       Subchronic dermal rat NOAEL: 50 mg/kg bw/day based on renal tubule regeneration

•       Subchronic dermal mouse NOAEL: 100 mg/kg bw/day organ-weight changes

•       Chronic dermal rat NOAEL: 50 mg/kg bw/day based on skin irritation at site of application in the 100 mg/kg bw dose (females) and significant increases in the epithelial ulcer of the forestomach

•       Chronic dermal mouse LOAEL: 100 mg/kg bw/day based on body weight changes and skin irritation at site of application in the 200 mg/kg bw dose (females) and several non-neoplastic lesions at all dose levels

Since both subchronic and chronic dermal studies are available for structurally similar amides, C8-18 and C18-unsatd., N,N-bis(hydroxyethyl) and since the NOAELs for both were 50 mg/kg bw/day, the NOAEL from the chronic rat study was further used in this safety assessment as the point of departure for deriving the DNEL dermal for long term systemic effects.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across 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:

guideline study

Remarks:

Not GLP. Read across study.

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

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
- Age at study initiation: between 6-7 wk
- Weight at study initiation: 111 (f) - 125 (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): 18-20
- Humidity (%): 60-75
- 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 adpated weekly to the body weight

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily once, 5 times/wk

Remarks:

Doses / Concentrations:
70 mg/kg bw/d (Days 1-28)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
250 mg/kg bw/d (Days 1-28)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
750 mg/kg bw/d (Days 1-14)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1500 mg/kg bw/d (Days 15-28)
Basis:
actual ingested

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 differntial


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, cholestrol


URINALYSIS: Yes
- Time schedule for collection of urine: end of study
- Metabolism cages used for collection of urine: No
- Parameters checked: urea, creatinin, 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 (2)
aorta thoracica
brain (cornu ammonis)
coagulating gland with seminal vesicle
epididymis (2)
eye with optic nerve (2)
heart
intestine, large
intestine, small
kidney (2)
liver
lungs
lymph node (cervical) (1)
lymph node (mesenteric) (1)
mucles
oesophagus
ovary (2)
pancreas
prostate
salivary glands (mandibular,
parotid and sublingual gland)
skin
spleen
stomach
testicle (2)
thymus
thyroid (2) (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

Body weight and weight changes:

no effects observed

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

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

The doses up to 1500 mg/kg bw/d were tolerated by all animals without lethality. The body weight gain and total increase of body weights did not differ from the control group. Absolute and relative organ weights showed no significant changes in the substance groups compared to the control group, accept the organ weight of the adrenal gland which is increased for the females of group 3 (250 mg/kg) and 4 (750/1500 mg/kg bw). This result is considered of no relevance. The pathological, histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose independent reversible local findings were restricted to the fore stomach mucose of the highest group (hyperplastic and cellular changes in the forestomach but not dose-related and also found in controls. Attributed to the use of olive oil as carrier. Effects disappeared during 4 month recovery period). The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterine, GGT, GOT, GPT and LDH did not show any irregular signs. Slight alterations of phosphate in the highest group were noted and regarded as dose/compound related but not critical effect.

Key result

Dose descriptor:

NOAEL

Effect level:

> 750 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

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

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, the systemic NOAEL was considered to be >750 mg/kg bw.

Executive summary:

A study was conducted to determine the repeated dose oral toxicity of the read across substance, Amides, C12-18 (even-numbered) and C18-unsatd., N-(hydroxyethyl), to rats according to a method similar or equivalent to OECD Guideline 407. Groups of 10 male and 10 female rats were orally gavaged with the test substance diluted in olive oil 5 d/wk at 0, 70, 250, 750 (Days 1-14) or 1500 (Days 15-28) for 28 d. Clinical signs, bodyweight, hematology, 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. The doses up to 1500 mg/kg bw/d were tolerated by all animals without lethality. The body weight gain and total increase of body weights did not differ from the control group. The pathological, histological evaluation did not show significant compound related gross or microscopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads. Dose-independent reversible local findings were restricted to the fore stomach mucosa of the highest group. The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterol, GGT, GOT, GPT and LDH did not show any irregular signs. Slight alterations of phosphate in the highest group were noted and regarded as dose/compound related but not critical effect. Hence, under the study conditions, the systemic NOAEL was considered to be >750 mg/kg bw (Gloxhuber, 1983).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Read across study

Justification for type of information:

Refer to the section 13 for details on the read across justification.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Principles of method if other than guideline:

A two-year dermal study was conducted in mice to evaluate the carcinogenic potential of the test substance.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
- Source: Taconic Laboratory Animals and Services, (Germantown, NY)
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 13 to 14 days

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

In-life dates: From: Jan. 20, 1993 - To: Jan. 20, 1995

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

The test material formulation was applied to the shaved skin of test animals. No further details provided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed approximately every 2 months using HPLC. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

104 wk

Frequency of treatment:

Five exposures per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

0 mg/mL in ethanol

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

50 mg/mL in ethanol

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

100 mg/mL in ethanol

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Exposure to coconut oil acid diethanolamine condensate in the 14 wk study produced only a minimal toxic response in mice except in the skin at the site of application. The incidences of chronic active inflammation as well as several other skin lesions were significantly increased at doses of 200 mg/kg bw and greater in both male and female mice. The incidences of ulceration were increased in males exposed to 400 and 800 mg/kg bw and in females exposed to 800 mg/kg bw. Therefore, 400 and 800 mg/kg bw were considered inappropriate for a 2-year study. However, ulceration was present in only one 200 mg/kg bw male and no females, and the severities of these lesions in all affected groups were minimal to mild. Below 200 mg/kg bw, the incidences of skin lesions decreased markedly with a minor difference in response between 50 and 100 mg/kg bw. Therefore, 200 mg/kg bw was selected as the high dose and 100 mg/kg bw as the low dose for this 2-yr study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily

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

DERMAL IRRITATION (if dermal study): Yes

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

Sacrifice and pathology:

SACRIFICE: Carbon dioxide asphyxiation
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues
were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.

Other examinations:

-

Statistics:

- Survival Analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Possible dose-related effects on survival were analysed by Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses were two-sided.
- Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.
- Analysis of Continuous Variables: Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of the dose-related trends. Average severity values were analyzed for significance with the Mann-Whitney U test. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across dose levels.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related clinical finding was irritation of the skin at the site of application in males that received 200 mg/kg bw.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

The only treatment-related clinical finding was irritation of the skin at the site of application in males that received 200 mg/kg bw.

Mortality:

mortality observed, treatment-related

Description (incidence):

The survival of dosed males and 100 mg/kg bw females was similar to that of the vehicle controls. Survival of the 200 mg/kg bw group of female mice was reduced compared to the vehicle control group, but the difference was not significant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of dosed males were similar to those of the vehicle controls throughout most of the study; those of 100 and 200 mg/kg bw females were less than those of the vehicle controls from wk 93 and 77, respectively .

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Skin: Several nonneoplastic lesions of the skin at the site of application were determined to be chemical related. Incidences of epidermal hyperplasia, sebaceous gland hyperplasia, and hyperkeratosis in all dosed groups of males and females were significantly greater than those in the vehicle control groups. The incidences of ulceration in 200 mg/kg bw males and inflammation and parakeratosis in 200 mg/kg bw females were increased.
- Thyroid Gland: The incidences of follicular cell hyperplasia in all dosed groups of males (vehicle control, 11/50; 100 mg/kg bw, 20/50; 200 mg/kg bw, 23/50) and females (27/50, 36/50, 33/50) were significantly greater than those in the vehicle controls. Follicular cell hyperplasia consisted of focal areas of thyroid gland follicles lined with increased numbers of epithelial cells, which formed papillary projections in some instances.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Liver: Dosed male and female mice had significantly greater incidences of hepatic neoplasms (hepatocellular adenoma, hepatocellular carcinoma, and hepatoblastoma (males) than the vehicle controls. There was a morphologic continuum from adenoma to carcinoma, with less differentiation and typical trabecular formations in the carcinomas. Carcinomas were often a centimeter or more in diameter, whereas adenomas were generally smaller and more discrete. Carcinomas metastasized to the lung in a few males and females. Adenomas, carcinomas, and hepatoblastomas displaced normal liver parenchyma, and none contained normal lobular architecture. Hepatoblastomas were characterized by well-demarcated focal areas composed of bundles of deeply basophilic, spindle-shaped cells.
- Kidney: The incidences of renal tubule adenoma (1/50, 1/50, 7/50) and of renal tubule adenoma or carcinoma (combined) (1/50, 1/50, 9/50) in 200 mg/kg bw males were significantly greater than those in the vehicle controls. Renal tubule hyperplasia, adenoma, and carcinoma formed a morphological continuum. Adenomas were focal, compressive masses approximately five or more tubules in diameter; carcinomas were morphologically similar to adenomas but were larger and often showed cellular debris and/or mineralization. Renal tubule neoplasms were located in the cortex or outer medulla. Focal proliferative masses less than five tubules in diameter were classified as focal hyperplasia.

Key result

Dose descriptor:

LOAEL

Remarks:

(systemic and local effects)

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

dermal irritation

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

other: body weight and see "Organ"

Organ:

kidney

liver

skin

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

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

 

Conclusions:

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

Executive summary:

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

Reference 2

Endpoint:

sub-chronic toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Read across study

Justification for type of information:

Refer to the section 13 for details on the read across justification.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

The test substance was applied daily to the skin in graduated doses to several groups of mice, one dose per group, for a period of 14 wk. During the period of application the animals were observed daily to detect signs of toxicity. Animals which die during the test were necropsied, and at the conclusion of the test the surviving animals were sacrificed and necropsied.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 16 to 17 d

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

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

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

The test material formulations were applied on shaved skin of the test animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of test material were conducted at the study laboratory using HPLC. Dose formulations from the beginning, middle, and end of the studies were analyzed

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

5 exposures/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose group

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once


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


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

Sacrifice and pathology:

SACRIFICE: Yes (Carbon dioxide asphyxiation)
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 800 mg/kg bw mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, gallbladder, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.

Other examinations:

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

Statistics:

Not reported

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only treatment related clinical finding was irritation of the skin at the site of application in males and females administered 800 mg/kg bw.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

The only treatment related clinical finding was irritation of the skin at the site of application in males and females administered 800 mg/kg bw.

Mortality:

no mortality observed

Description (incidence):

All mice survived until the end of the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Final mean body weights and body weight gains of dosed males and females were similar to those of the vehicle controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute and relative liver and right kidney weights of 800 mg/kg bw males and females and the absolute and relative liver weights of 400 mg/kg bw females were significantly greater than those of the vehicle controls. The absolute and relative lung weights of 800 mg/kg bw females were also significantly greater than those of the vehicle controls.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathologic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis, and ulcer. The incidences and severities of these skin lesions generally increased with increasing dose in males and females

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Epididymal spermatozoal concentration was significantly increased in 800 mg/kg bw males. Estrous cycle lengths of dosed females were similar to that of the vehicle controls

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

(local effects)

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: skin inflammation

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: As of 400 mg/kg bw/d, significant increase in relative liver weights (females). At 800 mg/kg bw/d, significant increase in relative kidney (male and females) and lung (females) weights.

Organ:

kidney

liver

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day

System:

other: As of 200 mg/kg bw/d, incidence of chronic active inflammation.

Organ:

skin

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the results of the read across study, the 14-week NOAEL in mice for systemic effects was considered to be 200 mg/kg bw/day and for the local effects 100 mg/kg bw/day.

Executive summary:

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

Reference 3

Endpoint:

sub-chronic toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Read across study

Justification for type of information:

Refer to the section 13 for details on the read across justification.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

The test substance was applied daily to the skin in graduated doses to several groups of experimental animals, one dose per group, for a period of 14 wk. During the period of application the animals were observed daily to detect signs of toxicity. Animals which die during the test were necropsied, and at the conclusion of the test the surviving animals were sacrificed and necropsied.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 14 to 15 d

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

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

Type of coverage:

open

Vehicle:

ethanol

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of test material were conducted at the study laboratory using HPLC. Dose formulations from the beginning, middle, and end of the studies were analysed.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

5 exposures/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose in the core study, 10/sex/dose for clinical pathology groups

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly once


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


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


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


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

Sacrifice and pathology:

SACRIFICE: At the end of the 14 wk studies, blood was collected from the retroorbital sinus of all core study rats for hematology and clinical chemistry analyses. Thereafter the test animals were anesthetised with a carbon dioxide/oxygen mixture.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 400 mg/kg bw. In addition to gross lesions and tissue masses, the following tissues were examined: heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the skin (site of application) was examined in all core study animals, and the kidney was examined in core study male and female rats.

Other examinations:

Sperm motility and vaginal cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology from all rats receiving 100, 200 and 400 mg/kg bw of test material. The following sperm motility parameters were evaluated: spermatid heads per gram of testis, spermatid heads per testis, spermatid count, and epididymal spermatozoal motility and concentration. The left cauda epididymis, epididymis, and testis were weighed. Vaginal samples for cytology evaluations were collected for 12 consecutive days prior to the end of the studies from all female rats. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.

Statistics:

Not reported

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings included irritation of the skin at the site of application in 100, 200, and 400 mg/kg bw males and females.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Clinical findings included irritation of the skin at the site of application in 100, 200, and 400 mg/kg bw males and females.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weights and body weight gains of 200 and 400 mg/kg bw males and females were significantly less than those of the vehicle controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At Week 14, a minimal microcytic, normochromic, nonresponsive anemia occurred in the 100 and 200 mg/kg bw females and 400 mg/kg bw males and females. The anemia also occurred in the 400 mg/kg bw males and females on Day 24. Increased segmented neutrophil counts occurred in 400 mg/kg bw males and females at Week 14 and in 400 mg/kg bw females on Day 24.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Cholesterol concentrations were significantly decreased in 200 and 400 mg/kg bw males and in females administered 100 mg/kg bw or greater; triglyceride concentrations were also decreased in 200 and 400 mg/kg bw males. At week 14, there was a minimal concentration-related increase of serum albumin concentration in all treated groups of females and in 100 mg/kg bw or greater male rats; on Day 24, increased albumin concentration occurred in the 400 mg/kg bw females. There were minimal increases of urea nitrogen concentration that occurred in the 200 and 400 mg/kg bw female rats on Day 24 and at week 14. At week 14, an increase in alanine aminotransferase activity occurred in 50 mg/kg bw or greater male rats. Additionally, alkaline phosphatase activity was increased in 400 mg/kg bw males.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Kidney weights of females administered 50 mg/kg bw or greater were significantly greater than those of the vehicle control group. Left epididymis weights of 200 and 400 mg/kg bw males were significantly less than those of the vehicle controls, but this was most likely secondary to decreased mean body weights in these groups

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathologic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, chronic active inflammation, parakeratosis, and ulcer. The incidences and severities of these skin lesions generally increased with increasing dose in males and females. The incidences of renal tubule regeneration in 100, 200, and 400 mg/kg bw females were significantly greater than the vehicle control incidence, and the severities in 200 and 400 mg/kg bw females were increased.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle lengths of dosed females were similar to those of the vehicle controls

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic and local effects)

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: Liver, kidney, skin

Organ:

kidney

liver

skin

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on the results of the read across study, the 14-week systemic NOAEL in rats can be considered to be 50 mg/kg bw/day.

Executive summary:

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

Reference 4

Endpoint:

chronic toxicity: dermal

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Read across study

Justification for type of information:

Refer to the section 13 for details on the read across justification.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Groups of 50 male and 50 female rats were exposed to 0, 50 or 100 mg/kg bw/d of the substance in ethanol by dermal application, 5 times per week, for 104 weeks. Mortality, clinical signs and bodyweight were recorded throughout the study. At necropsy, a gross macroscopic examination and complete histopathology were carried out.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Taconic Laboratory Animals and Services, (Germantown, NY)
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate cages (Lab Products, Inc., Maywood, NJ)
- Bedding: Sani-Chip heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ)
- Diet : NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water : Tap water (Columbus municipal supply), ad libitum
- Acclimation period: 11 to 12 d

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

In-life dates: From: Feb. 1, 1993 - To: Jan. 31, 1995

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

The test substance formulation was applied to the shaved skin of test animals. No further details provided.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulations were analysed approximately every 2 months using HPLC. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

104 wk

Frequency of treatment:

Five exposures per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Doses of 200 or 400 mg/kg bw/d in the 14 wk study were associated with reduced mean body weights, mild anemia, and significantly increased incidences and severities of lesions of the skin at the site of application. Therefore, these doses were considered inappropriate for a 2-year study. At 100 mg/kg bw/d, the incidences of skin lesions, especially ulceration, were less than at 200 mg/kg bw/d, and in general, the severities were minimal to mild. Therefore, 100 mg/kg bw/d was selected as the high dose for this 2-yr study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observed twice daily


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


DERMAL IRRITATION (if dermal study): Yes


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

Sacrifice and pathology:

SACRIFICE: Carbon dioxide asphyxiation
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues
were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (and
epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus

Statistics:

Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Possible dose-related effects on survival were analysed by Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses were two-sided.
Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.
Analysis of Continuous Variables: Organ and body weight data, which historically have approximately normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of the dose-related trends. Average severity values were analyzed for significance with the Mann-Whitney U test. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across dose levels.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw/d females.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

The only clinical finding attributed to dosing was irritation of the skin at the site of application in 100 mg/kg bw/d females. Non-neoplastic lesions of the skin at the site of application included epidermal hyperplasia, sebaceous gland hyperplasia, parakeratosis and hyperkeratosis, and the incidences and severities of these lesions increased with increasing dose.

Mortality:

no mortality observed

Description (incidence):

Survival rates of dosed male and female rats were similar to those of the vehicle controls.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- The severity of nephropathy increased with increasing dose in female rats.
- The incidences of chronic active inflammation, epithelial hyperplasia and epithelial ulcer of the forestomach increased with dose in female rats and the increases were significant in the 100 mg/kg bw/day group.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were marginal increases in the incidences of renal tubule adenoma or carcinoma (combined) in 50 mg/kg bw/day females. Under the study conditions, there was no evidence of carcinogenic activity of the test substance in male rats at any dose. There was an equivocal evidence of carcinogenic activity in female rats based on a marginal increase in the incidences of renal tubule neoplasms. However, the absence of an increase in neoplasms in the 100 mg/kg bw/day group in the presence of increased hyperplasia makes the association with treatment uncertain.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

systemic and local effects

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

dermal irritation

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: skin irritation at site of application in the 100 mg/kg bw dose group (females) and significant increases in the epithelial ulcer of the forestomach

Organ:

kidney

skin

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

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

Conclusions:

Basd on the results of the read across study, the 2-year NOAEL was considered to be 50 mg/kg bw/day in rat.

Executive summary:

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

Endpoint conclusion

Endpoint conclusion:

no study available

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, rat

A study was conducted to determine the repeated dose oral toxicity of the read across substance, amides, C12-18 (even-numbered) and C18-unsatd., N-(hydroxyethyl), to rats according to a method similar or equivalent to OECD Guideline 407. Groups of 10 male and 10 female rats were orally gavaged with the test substance diluted in olive oil 5 d/wk at 0, 70, 250, 750 (Days 1-14) or 1500 (Days 15-28) for 28 d. Clinical signs, bodyweight, hematology, 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. The doses up to 1500 mg/kg bw/d were tolerated by all animals without lethality. The body weight gain and total increase of body weights did not differ from the control group. The pathological, histological evaluation did not show significant compound related gross or microscopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads. Dose-independent reversible local findings were restricted to the fore stomach mucosa of the highest group. The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterol, GGT, GOT, GPT and LDH did not show any irregular signs. Slight alterations of phosphate in the highest group were noted and regarded as dose/compound related but not critical effect. Hence, under the study conditions, the systemic NOAEL was considered to be >750 mg/kg bw (Gloxhuber, 1983).

Dermal

14 weeks, mouse and rat

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

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

2 years, mouse and rat

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

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