N,N'-ethane-1,2-diylbisoleamide

Endpoint summary

Description of key information

NOAEL (subchronic, oral, rat): 731.8 (m) and 834.5 (f) mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: FDA guidelines for the testing of food additives (Toxicological Principles of the Safety Assessment of Direct Food Additives and Colour Additives used in Food; US Food and Drug Administration Bureau of Foods, 1982)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar (Bor: WISW (SPF; Cpb)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Institute for the Breeding of Laboratory Animals GmbH & Co. KG; Borchen, Germany
- Age at study initiation: approx. 3 weeks
- Weight at study initiation: 40-60 g
- Housing: animals were housed in groups of 5 per sex and dose level in suspended, stainless steel cages, fitted with wire mesh floor and front. The cages of each dose group were allocated at random to the cage racks.
- Diet: powdered cereal-based diet (stock diet), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): at least 40
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): one batch of 7.5 kg of each diet and the respective concentrations of the test substance and the positive control substance (oleic acid) was prepared once a week during the study, except in the last week, in which the remainder of the batch of the previous week was used.
- Mixing appropriate amounts with (Type of food): powdered cereal-based diet (stock diet)
- Storage temperature of food: in a refrigerator

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content of the test substance in the diet preparations was analysed 10 times during the whole study period by High Performance Liquid Chromatography (HPLC) after extraction from the diet as described in a previous report. The concentration of positive control substance (oleic acid) was determined as methylester by Gas Liquid Chromatography (GLC) after petroleum-ether (40:60) extraction of the fat-soluble compounds from the diet for 4 h. Five individual samples from each of the diets prepared at the beginning of the study were used to check the homogeneity of the test substance or the positive control substance in the diet. The stability of the test substance was checked in the last batch prepared, after a storage period of 7 day in a refrigerator and 7 days at room temperature, respectively. Under similar storage conditions, the stability of the positive control substance was examined in one batch produced during the study. The results of the analysis showed that the content of the test substance in the diet was close to the intended levels throughout the study. The content of the positive control substance (oleic acid) in the diet was about 2.5%, which was 0.9% less than the supplemented diet should have (3.4% should be the total level after addition of 2% oleic acid, since the oleic acid content of the unsupplemented control diet was already 1.4%). The analysis of the batch of oleic acid used for supplementation showed that the oleic acid content was only 58%, which explained the too low oleic acid content in the supplemented diet. The test substance and the positive control were found to be stable during the 7-day storage in the refrigerator and at room temperature, respectively. The homogeneity of the test substance at 0.2 and 1% in the diet was satisfactory, whereas the homogeneity of the test substance at 0.05% in the diet was less sufficient due to the possible inaccuracy of determination (concentration was close to the detection limit). The homogeneity of the positive control in the diet was satisfactory.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

500 ppm

Remarks:

corresponding to approx. 35.9 and 41.1 mg/kg bw/day in males and females, respectively (calculated based on the reported average food consumption and body weight (see Table 1 under "Any other information on results incl. tables"))

Dose / conc.:

2 000 ppm

Remarks:

corresponding to approx. 139.9 and 166.9 mg/kg bw/day in males and females, respectively (calculated based on the reported average food consumption and body weight (see Table 1 under "Any other information on results incl. tables"))

Dose / conc.:

10 000 ppm

Remarks:

corresponding to approx. 731.8 and 834.5 mg/kg bw/day in males and females, respectively (calculated based on the reported average food consumption and body weight (see Table 1 under "Any other information on results incl. tables"))

Remarks:

Doses / Concentrations:
0.05, 0.2 and 1.0%
Basis:
nominal in diet

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Positive control:

Oleic acid was used as positive control at a nominal dietary concentration of 2%, since it was believed to be a degradation product of the test substance. However, the analytical concentration of oleic acid was only determined to be 1.2%.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the rats were observed twice a day on working days and once a day on weekends and holidays for clinical signs of toxicity and mortality.
- Cage side observations involved the inspection of general condition and behaviour of all rats.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: the individual body weights of all rats were determined at the beginning of the study, and then weekly thereafter. All surviving males were weighed on Day 93 and all surviving females were weighed on Day 92 or 94 in order to calculate the correct organ to body weight ratios at the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each group of 5 rats determined and mean weekly diet consumption calculated as g food/rat/week: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: water intake of all groups was measured per cage from Day 72 to Day 79.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: examinations were performed prior to the start of the study (Day -4), and at study termination in Week 13 (on Day 93)
- Dose groups that were examined: all animals of the control and high dose (prior to study); 10 rats per sex of the control, mid, high and positive control group (at study termination)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 42 and 84 (males) and on day 43 and 85 (females) from the tip of the tail
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10 animals/sex/group
- Parameters examined: haemoglobin, packed cell volume, red blood cells, white blood cells, differential white blood cell count, thrombocytes, prothrombin time, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: samples for glucose determination were obtained on Day 45 and 87 from the tip of the tail. Samples for determination of all other parameters were collected on Day 51 for males and on Day 52 for females by orbital puncture as well as at autopsy (on Day 91 for males and on Day 92 for females) from the aorta.
- Anaesthetic used for blood collection: Yes, for collection by orbital puncture and from the aorta (ether)
- Animals fasted: Yes ( deprivation of water 24 h and of food 16h before blood sampling for glucose determination)
- How many animals: 10 rats per sex and group
- Parameters examined: glucose, alkaline phosphatase (ALP), aspartate amino transferase (ASAT), alanine amino transferase (ALAT), gamma-glutamyl transpeptidase (GGT), total protein, albumin, total bilirubin, triglycerides, urea, cholesterol, creatinine, inorganic phosphate, chloride, sodium, potassium, calcium

URINALYSIS: Yes
- Time schedule for collection of urine: freshly voided, 2-h urine samples were collected from 10 male and 10 female unfasted rats per group on Day 44 and 86 for determination of pH, and from fasted rats during the last 16 h of the 24-h deprivation period (24 h water, 16 h food) on Day 45 and 87 from 10 animals per sex and group for determination of the other parameters.
- Animals fasted: Yes (except for pH determination)
- Parameters examined: volume, density, appearance, protein, glucose, occult blood, ketones, bilirubin, urobilinogen, sediment (erythrocytes, leukocytes, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells and worm eggs), pH

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all tissues and organs listed below were examined
HISTOPATHOLOGY: Yes, the following tissues were examined of all rats of the control and top-dose group: adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum and cerebellum), caecum, colon, duodenum, epididymides, eyes, heart, ileum, jejunum, kidneys, liver, lungs with mainstem bronchi, mammary gland (females), mesenteric lymph nodes, oesophagus, ovaries, pancreas, parotid salivary glands, pituitary, prostate, rectum, sciatic nerve, skeletal muscle (thigh), spinal cord (2 levels), spleen, sternum (with bone marrow), stomach (cardia, fundus and pylorus), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid (with parathyroid), trachea, urinary bladder, uterus (with cervix), all gross lesions.
Microscopic examination of kidneys, liver, lungs, mesenteric lymph nodes and all gross lesions observed at autopsy was carried out in all groups.
ORGAN WEIGHTS: Yes, organ weights of the following organs were determined: adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, spleen, testes, thyroid and parathyroid, thymus

Statistics:

Mean values and standard errors of the mean (SEM) were calculated from the parameters examined. Data on organ weights, red blood cell parameters, total white blood cell counts, clinical chemistry values, and volume and density of the urine, were evaluated by analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. Data on body weights were evaluated by analysis of co-variance followed by Dunnett’s multiple comparisons test. Data on food intake, food efficiency and water intake were evaluated by ANOVA followed by the L.S.D.-test. Differential blood counts and the remaining urinary parameters were analysed by the Mann-Whitney U-test. The results of ophthalmoscopy and the histopathological changes were examined by Fisher’s exact probability test. Statistical significance was indicated by *p < 0.05 and **p < 0.01, except for the Mann-Whitney U-test, where significance was indicated at *p < 0.05, **p < 0.02 and ***p < 0.002.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1% in the diet: focal alopecia in 13/20 males compared to 6/20 males in the control group (non-adverse)

Mortality:

mortality observed, treatment-related

Description (incidence):

1% in the diet: focal alopecia in 13/20 males compared to 6/20 males in the control group (non-adverse)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1% in the diet (males): slight decrease in body weights during the last few weeks (non-adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

0.2% in the diet: decrease in food intake (m, f), non-adverse

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

0.2% and 1% in the diet: decrease in ALP activity in Weeks 8 and 13 (m, f); decreased plasma triglyceride levels in Week 8 and 13 (f); 1% in the diet: decreased plasma triglyceride levels in Week 8 (m) (non-adverse)

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

1% in the diet: increase in urinary pH in Week 13 (m) and Week 7 (f), non-adverse

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

0.2 and 1%: slight decrease in absolute and relative heart weight (f, non-adverse)

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Focal alopecia was observed at a higher incidence (in 13/20 males) in the high dose group (1% in the diet) during the whole study period compared to the control group (6/20 males). The same observation was also made in females of the positive control group, which all (20/20) showed focal alopecia during the study, while only 8/20 females of the control group exhibited these clinical signs. No other notable differences in appearance and behaviour were observed between treated and control animals. One incidental death occurred in one male of the control group due to anaesthesia during blood sampling. No further mortalities were observed during the study.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights were comparable among the groups throughout the 13-week study period (see Table 1 "Under: Any other information on results incl. tables"). A slight decrease in body weight was observed in rats of the high dose group compared to controls during the last few weeks of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In males treated of the mid dose group (0.2% in the diet) and the positive control group, food intake was decreased during the first 8 weeks of the study, but there was no dose-response relationship and no statistically significant difference in the last 5 weeks of the study compared to controls. Furthermore, the decrease in food consumption was not accompanied by significant changes in the growth rates of these animals. Relatively low food intake values were also observed in females treated with 0.2 and 1% in the diet (see Table 1 "Under Any other information on results incl. tables") and in females of the positive control group. The reduction in food consumption was rather associated with a higher energetic value of the diet supplemented with the test substance or oleic acid, respectively. These assumption was further supported by the relatively high food conversion efficiency in the positive control group.

FOOD EFFICIENCY
Food conversion efficiency did not reveal any effect in treated animals compared to controls. In males of the positive control group, a tendency towards increased values for food conversion efficiency was observed.

WATER CONSUMPTION
Water intake recorded from Days 72 to 79 did not reveal any outstanding differences among the groups. In females of the high dose group (1% in the diet) and positive control group, mean water intake was only slightly above those of the other groups.

OPHTHALMOSCOPIC EXAMINATION
In Week 13, no significant differences were observed at ophthalmoscopic examination between treated and control groups. The ocular changes observed were common findings in rats of this strain and age and thus of no toxicological relevance.

HAEMATOLOGY
No statistically significant changes were observed in red blood cell parameters of samples collected in Weeks 7 and 13, respectively. Differential white blood cell counts only showed a shift in the lymphocyte/neutrophil ratio in males at 0.05% in the diet, and a decrease in the percentage of eosinophils in females at 1% in the diet. However, these changes were not dose-related or consistently present, and thus considered incidental and not toxicologically relevant.

CLINICAL CHEMISTRY
In males and females of the mid (0.2% in the diet) and high dose group (1% in the diet), plasma ALP activities were statistically significantly decreased in Weeks 8 and 13. In contrast, plasma ALP activities were significantly increased in females of the positive control group at both time points. Although rather increases than decreases in the activity of plasma ALP are considered to be of toxicological significance, the authors suggested that the decrease in plasma ALP activity might indicate interference with the intestinal absorption of fat. Plasma triglyceride concentration was decreased in males treated with 1% in the diet and in females treated with 0.2 and 1% in the diet in Week 8. A similar tendency was observed towards the end of the study, but no statistical significance was reached compared to controls. Plasma GPT activity was increased in females receiving 1% in the diet during Week 8, but this finding was not confirmed at the end of the study. Other changes in clinical chemistry parameters observed at 0.05% in the diet, i.e. decreased plasma GPT activity in Week 8, and an increased plasma cholesterol concentration at the end of the study, were considered to be incidental and not toxicologically relevant.

URINALYSIS
At 1% in the diet, urinary pH was significantly increased in males in Week 13 and females in Week 7 of the study. However, these changes were slight and not consistently present. The changes in urinary density in Week 7 were inconsistent, and thus not considered to be of toxicological relevance. No further treatment-related changes in urinary parameters were observed.

ORGAN WEIGHTS
No dose-related changes in absolute and relative organ weights were observed. However, in a few organs (brain, kidney, spleen and heart), statistically significant changes were noted, which were not accompanied by any histopathological abnormalities. These involved a slight decrease in absolute and relative brain weight in females of the mid (0.2% in the diet) and high (1% in the diet) dose group as well as the positive control group. Furthermore, absolute organ weight of the kidneys was increased in females receiving 1% in the diet. Increases in the relative weight of hearts in males (0.05% dose group) and relative weights of the spleens in females (0.2% dose group) were not confirmed in the high dose group (1% in the diet), and were therefore considered to be incidental findings.

GROSS PATHOLOGY
One male of the control group, which died during ether-anaesthesia at orbital puncture, showed a thrombus in the heart. All other findings were considered incidental and of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
In all dose groups treated with the test substance, the degree of sinus histiocytosis in mesenteric lymph nodes was somewhat increased, but without showing a dose-response relationship. Moreover, the total incidence of sinus histiocytosis was not altered in any of the groups, therefore, this change was not considered to be treatment-related. Minimal to slight accumulations of “foamy” macrophages were observed in the mesenteric lymph nodes of rats of all groups (incl. positive control), but in naturally occurring quantities. All other abnormalities found in the organs were considered to be common findings in rats of this strain and age and only occurred incidentally.

Key result

Dose descriptor:

NOAEL

Effect level:

1 other: % in the diet

Based on:

test mat.

Remarks:

corresponding to dose levels of approx. 731.8 (males) and 834.5 (females) mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level.

Dose descriptor:

LOEL

Effect level:

0.2 other: % in the diet

Based on:

test mat.

Remarks:

corresponding to dose levels of approx. 139.9 (males) and 166.9 (females) mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: food consumption: decrease (non-adverse); clinical chemistry: decrease in plasma ALP activity (m, f) and triglyceride levels (f) (non-adverse); organ weights (f): slight decrease in absolute and relative heart weight (non-adverse)

Dose descriptor:

NOEL

Effect level:

0.05 other: % in the diet

Based on:

test mat.

Remarks:

corresponding to dose levels of approx. 35.9 (males) and 41.1 (females) mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

no

BODY WEIGHT, FOOD CONSUMPTION AND COMPOUND INTAKE

Table 1. Mean body weight, food consumption and compound intake over the whole study period

Concentration in diet	Mean food consumption [g/rat/day]*	Mean body weight [g]	Mean daily compound intake [mg/kg bw/day]
MALES
Control (0 mg/kg)	17.84	243.5	0
0.05% (500 mg/kg)	17.36	241.7	35.9
0.2% (2000 mg/kg)	16.56	236.7	139.9
1.0% (10000 mg/kg)	17.11	233.8	731.8
FEMALES
Control (0 mg/kg)	13.44	158.0	0
0.05% (500 mg/kg)	13.00	158.0	41.1
0.2% (2000 mg/kg)	12.86	154.1	166.9
1.0% (10000 mg/kg)	12.36	154.1	834.5

*calculated from the reported weekly mean food consumption in g/rat/week

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

731.8 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

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:

sub-chronic toxicity: inhalation

Data waiving:

other justification

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

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are reliable data available on the repeated dose toxicity via the oral route of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis, thus fulfilling the standard information requirements set out in Annex IX, 8.6, of Regulation (EC) No 1907/2006. Furthermore, supporting information is available on the chronic oral toxicity from a structurally related substance according to Annex XI, 1.5, of Regulation (EC) No 1907/2006.

A detailed justification for the grouping of chemicals and read-across is provided in Sections 7.1 and 13.

Oral

Subchronic

The subchronic oral toxicity of the target substance in male and female Wistar rats was investigated in a study similar to OECD guideline 408 and according to the FDA guidelines for the testing of food additives (Rutten and Til, 1988). Twenty animals per sex and group received the substance for a period of 90 days at dietary concentrations of 0.05, 0.2 and 1%, which corresponded to dose levels of approx. 35.9, 139.9 and 731.8 mg/kg bw/day in males and 41.1, 166.9 and 834.5 mg/kg bw/day in females. Two further groups of each 20 animals per sex received the plain diet or oleic acid at dietary concentrations of 1.2% and served as negative or positive controls. Oleic acid was used as positive control since it was believed to be a degradation product of the test substance. No treatment-related mortalities occurred during the whole study period. Clinical signs involved the increased incidence of focal alopecia in animals receiving 1% of the substance and 1.2% of oleic acid (positive control) via the diet compared to controls. At ophthalmoscopic examination, no treatment-related changes were observed in the test groups compared to controls. No changes in body weights were observed during the study, except for a slight, non-adverse decrease in body weight in males during the last few weeks of the study. During the first 8 weeks of the study, food consumption was slightly decreased in males treated with 0.2% of the test substance in the diet and in males of the positive control group. However, there was no dose-dependent decrease and no statistically significant decrease during the last 5 weeks of the study. Water consumption was not affected in treated animals compared to controls. Haematology did not reveal any treatment-related changes in the animals. At clinical chemistry, a decrease in plasma ALP activity was observed in females treated with 0.05 and 0.2% of the test substance. In contrast, increased plasma ALP activities were observed in animals of the positive control group. Plasma triglyceride concentration was decreased in males treated with 1% in the diet and in females treated with 0.2 and 1% in the diet in Week 8. A similar tendency was observed towards the end of the study, but no statistical significance was reached compared to controls. Other changes in clinical chemistry parameters observed at 0.05% in the diet (decreased plasma GPT activity, increased plasma cholesterol concentration) were considered to be incidental and not toxicologically relevant. At 1% in the diet, urinary pH was significantly increased in males in Week 13 and females in Week 7 of the study, but these changes were slight and not consistently present and thus of no toxicological relevance. The absolute and relative organ weights were not adversely affected by treatment. In a few organs (brain, kidney, spleen and heart), statistically significant changes in the weights were noted. However, these changes were not accompanied by any gross pathology or histopathological abnormalities. Based on the results of the study, the NOEL for rats was considered to be at 0.05% in the diet, corresponding to dose levels of 35.9 mg/kg bw/day in males and 41.1 mg/kg bw/day in females, respectively; the NOAEL was considered to be at 1% in the diet, corresponding to dose levels of 731.8 mg/kg bw/day for males and 834.5 mg/kg bw/day for females.

 

Chronic

No study investigating the chronic toxicity of the target substance via the oral route is available. However, there exists one reliable study from a structurally related substance (source substance), which is used for read-across based on the analogue approach.

In a 2-year feeding study rats were exposed to doses of 5000, 20000 and 50000 ppm via the feed, which were equivalent to 250, 1000 and 2500 mg/kg bw/d (Weigand, 1963). The doses were converted assuming an animal weight of 400 g and a daily food consumption of 20 g/d, according to the recommendations of the WHO or the Toxicologist's pocket Handbook, CRC press, 2000. In the study, mortality, clinical signs, body weights, haematology and urinalysis parameters were assessed, and all survivors of the 104-week exposure period and all animals deceased before scheduled sacrifice were subjected to macroscopic and histopathological examination of heart, lung, liver, kidney and spleen. Despite the unusual high dose for chronic exposure, no adverse effects related to treatment could be detected after 104 weeks of exposure. Frequency and characteristics of findings, including the sporadic occurrence of tumours in the experimental groups, did not differ significantly from those observed in the control group.

Based on the results of the chronic study with Amides, C16-C18 (even), N,N'-ethylenebis, the NOAEL for rats was considered to be ≥ 2500 mg/kg bw/day for this substance. This assumption is considered rather conservative, since the actual NOAEL of this study might range between 5000-7500 mg/kg bw/d, based on the authors information that daily food consumption equalled about 10-15% of the animal’s body weight.

Due to the strong structural relationship with this substance, no toxicity is expected to occur after chronic exposure to Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis, either.

However, the hazard assessment for Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis was done based on the more conservative NOAEL for males derived from the subchronic study for the substance itself.

Justification for classification or non-classification

The available data on the repeated dose toxicity via the oral route of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis and a substance structurally related to Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis according to the criteria laid down in Regulation (EC) No 1907/2006, Annex XI, 1.5 do not meet the criteria for classification according to Regulation (EC) No 1272/2008.