Registration Dossier

Administrative data

Description of key information

Horne (2014) performed a 90-day oral repeated dose toxicity study in rats. The test was performed according to OECD guideline 408 and in compliance with GLP requirements. The No Observed Adverse Effect Level (NOAEL) was considered to be 50 mg/kg/day. The study is scored as reliable (K1) and selected as key study.

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
Study period:
from 2013-03-04 to 2014-03-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: CR:CD (Sprague-Dawley)
Details on species / strain selection:
No further data available
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: between 202.1 and 263.3g. Females: between 150.8 to 205.1
- Fasting period before study: None (but one fasting night during week 13 for blood sampling)
- Housing: The animals were housed in a single, exclusive room, and in groups up to 4 rats of the same sex and dose level par cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45 to 65 %
- Air changes (per hr): 15-20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours nominal

IN-LIFE DATES: From: 22 March 2013 to 24 June 2013
Route of administration:
oral: gavage
Details on route of administration:
No further data available
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was formulated as a solution in purified water following dispensary standard operation procedures and a formulation method develpoped in the laboratory for this study. Formulations were prepared weekly and stored refrigerated (2 to 8°C) in a sealed container, protected
from the light. The formulations were stirred on arrival at the animal room and were stirred continuously before and throughout dosing.

The dose levels of 50, 150 and 500 mg/kg/day were expressed as the active ingredient. The test article was supplied as a 356 mg/mL solution and final concentrations of 5, 15 and 50 mg/mL were formulated by serial dilution.

OTHER:
After the dose formulation was drawn into the syringe, the catheter was washed in potable water, wiped clean and then washed again before dosing the animal. he test article was administered orally by gavage, as it is possible human exposure route. A dose volume of 10 mL/kg was used. Individual dose volumes were based on individual body weight.





Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- SAMPLE COLLECTION:
Samples (3 x 1 mL aliquots from test formulations; 2 x 1 mL aliquots from control formulations) prepared for use on Week 1 and Week 13 were taken for analysis of achieved concentration. Samples were stored at ambient until and during analysis that occurred within 2 hours of the formulations being prepared.

- SAMPLE ANALYSIS:
A set of standard solutions over the approximate concentration range 100 to 600 μg/mL was analysed by HPLC. A six point calibration graph was constructed for the standard solutions. Injections of the lowest calibration standard diluted by factors of 100 and 50 were used to determine a limit of detection (LOD). Formulations were diluted to within the range of the calibration line (approximately100.0 to 600.0 μg/mL) using water.
Duration of treatment / exposure:
The animals were dosed once for at least 13 weeks excluding the day of necropsy.
Frequency of treatment:
The test material was administered daily.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
active ingredient, actual ingested
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
active ingredient, actual ingested
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
active ingredient, actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
5 spare males and females were available to replace any animals due to extremes of body weight, ophthalmoscopy changes or ill health.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The high dose of 500 mg/kg/day was chosen as, based on the background information (28 day toxicity study), it was not expected to reveal overt toxicity and should set a No-Observed-Adverse-Effect Level (NOAEL). The intermediate dose of 150 mg/kg/day was chosen to set an NOAEL if effects were seen at 500 mg/kg/day. The low dose of 50 mg/kg/day was chosen to set a No-Observed-Effect Level (NOEL).

Positive control:
No positive control.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at the beginning and the end of the working day.
- Cage side observations : animals were observed for signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
Each animal was given a detailed physical examination at daily intervals, one hour after dosing only. An individual record was maintained of the clinical condition of each animal. Post dose observations were also performed in relation to the time of dosing to coincide with clinical examinations one hour post dose daily from Day 1 to termination.

BODY WEIGHT: Yes
Individual body weights were recorded twice weekly, before dose, from Dosing Day 1 to Week 4, and at weekly intervals from Week 5 to Week 13 and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The amount of food consumed by each cage of animals was determined twice weekly.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
The amount of water consumed by each cage of animals was determined daily for a three day period (Friday to Monday) in Week 1, 6 and 12.

OPHTHALMOSCOPIC EXAMINATION: Yes
Investigations were performed on all animals pre-treatment and on Groups 1 and 4 in Week 12.

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Once at Day 90.
- Animals fasted: Yes, samples were collected after an overnight period without food.
- How many animals: Investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90)
- Parameters examined: Haematology Tests: haemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte count, red cell distribution width, haemoglobin distribution width, total and differential white cell count, platelet count (includes platelet clump assessment - clump count below 100 considered as none detected; clump count over 100 considered as plateletclumps present and confirmed by review of Advia cytogram or blood film examination), platelet crit, mean platelet volume, platelet distribution width. Coagulation Tests: prothrombin time activated partial thromboplastin time. Clinical Chemistry Tests: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, creatinine, urea, glucose.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and at once weekly intervals thereafter. In Week 13, an detailed assessment was done.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity and some open filed test were performed weekly. Functional observationanl battery, open field examinations and locomotor activity were assessed in Week 13.
Sacrifice and pathology:
NECROPSY AND GROSS PATHOLOGY: All animals including the decedent animal were subjected to necropsy. The scheduled necropsies were performed in Week 14 (Day 95), after an overnight period without food. Where possible, they were carried out in replicate order. Each animal was given an overdose of isoflurane anaesthesia. Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal. A full macroscopic examination was performed under the general supervision of a pathologist and all lesions were recorded.

ORGAN WEIGHTS: Animals were weighed immediately before being killed for necropsy. The organs denoted by ‘W’ in the tissue list were dissected free from fat and other contiguous tissue and weighed before fixation (see table below). Left and right organs were weighed together. For 2F 54, the heart was weighed with adhered pericardium.

HISTOPATHOLOGY: The tissues from each animal presented in the following table were preserved in 10% neutral-buffered formalin unless otherwise indicated.
The following tissues were embedded in paraffin wax BP (block stage), sectioned at anominal 5 μm and stained with haematoxylin and eosin:
Group 1 and Group 4: All tissues denoted by ‘E’ in the tissue list above
Group 2 and Group 3: Lungs, liver, spleen, kidney, urinary bladder, (fore)stomach and gross lesions
Decedent: All tissues denoted by ‘E’ in the tissue listed below

MICROSCOPIC OBSERVATIONS
The following tissues were examined microscopically by the Principal Investigator for Histopathology:
Group 1 and Group 4: All tissues denoted by ‘E’ in the tissue list above
Group 2 and Group 3: Lungs, liver, spleen, kidney, urinary bladder, (fore)stomach and gross lesions
Decedent: All tissues denoted by ‘E’ in the tissue listed below

Other: Bone Marrow Smear Evaluation
Bone marrow smears were prepared at necropsy. Tissues were taken into bovine serum albumin. The smears were prepared, air dried and then were fixed in methanol, but not examined.
Other examinations:
No data
Statistics:
Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
The control group (Group 1) was taken as the baseline group with which the treated groups (Groups 2, 3, 4) were compared.
Body weight gains, terminal body weights, functional observational battery, motor activity, haematology and clinical chemistry variables were analysed using one-way analysis of variance (ANOVA), separately for each sex. Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P>0.05), and the group effect from the ANOVA was significant (P≤0.05), pairwise comparisons with the control group were made using Dunnett's test.
Where Levene’s test was significant (P≤0.05), a rank-transformation was applied to the data prior to analysis. No analysis was performed for variables with values above or below the limit of the assay. Some variables were not analysed due to there being too few values for meaningful analysis.
Organ weights were analysed using Analysis of Covariance (ANCOVA), for each sex separately, using the terminal body weight as the covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this assumption was tested. Where there was no evidence of heterogeneity among the slopes (P>0.01), and the group effect from the ANCOVA was significant (P≤0.05), pairwise comparisons with the control group were made using Dunnett's test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Post dosing observations of salivation and staining around the mouth are indicative of the treated animals not liking the taste of the test article and/or being dosed with an irritant material and are considered a secondary action of the test article. Noisy breathing was generally recorded for animals given 500 mg/kg/day but also in a single male at 150 mg/kg/day and a single female at 50 mg/kg/day; this finding being commonly associated with oral administration of irritant materials.
Mortality:
mortality observed, treatment-related
Description (incidence):
A single male dosed at 500 mg/kg/day was found dead during Week 12; although a cause of death could not be established, as the macroscopic findings (large caecum and liver, dark lungs and thymus, a thickened stomach and red fluid in the thoracic cavity) along with the microscopic findings (congestion of the liver, aggregates of foamy alveolar macrophages within the lung (graded as slight, multifocal), squamous cell hyperplasia with hyperkeratosis (both graded as moderate), together with erosion/ulcer (slight, focal) in the (fore)stomach, and minimal transitional cell hyperplasia in the urinary bladder) match the findings in the high dose animals, it is considered that this death was related to the treatment of Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl], N’-oxides.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When compared with control values, body weight gains for males during the first 8 weeks of treatment did not reveal an effect of treatment with Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl], N’-oxides. Over the second half of the study gains were low for males given 150 or 500 mg/kg/day, when compared with control, resulting in an overall slightly low gain in males at 500 mg/kg/day. There was no effect on gains for males given 50 mg/kg/day. Body weight gains for female at 500 mg/kg/day were variable, but high, over the first 5 weeks of treatment resulting in slightly high overall gains. There was no effect on body weight gains for females at 50 or 150 mg/kg/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect on food consumption in any Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl], N’-oxides-treated male group or for females given 50 or 150 mg/kg/day. Food consumption values were high for females given 500 mg/kg/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption values were high in males and females given 500 mg/kg/day. There was no effect seen in groups given 50 or 150 mg/kg/day. The increase in the water consumed could be associated with the oral dosing of an irritant material resulting in the behavioural response to drink. There was no histopathological evidence of any effect on the kidney although transitional cell hyperplasia in the urinary bladder was recorded, suggesting urinary excretion of an irritant material.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular effects after treatment with Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl], N’-oxides-in any group and sex.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant haematological changes (decreased haemoglobin, red blood cell counts, mean cell haemoglobin concentration, haemoglobin distribution width and red cell distribution width and increased reticulocyte counts and percentage, mean cell volume and mean cell haemoglobin) consistent with a decreased red blood cell mass associated with regeneration were seen in all treated males and female groups with the severity of the finding being dose dependant. The haemopoietic response in the spleen is considered to account for the increased weight of this organ.
For haemoglobin levels and red blood cell numbers, females given 500 mg/kg/day showed a change of between 7 and 17% from control values while males showed a change of between10 and 19% meaning the findings are on the border of non adverse and adverse. Males given 150 mg/kg/day showed a change of between 7 and 11% compared to controls with females showing a change between 1 and 5% with these findings being considered non-adverse due to the smaller magnitude of change. The low dose (50 mg/kg/day) percentages were between 0 and 2% for females and 4 and 6% for males and also not considered adverse. A reduced red blood cell mass of more than 30% is considered adverse and a change of less than 10% is considered non-adverse for rodents (Principles of Methods of Toxicology 5th Ed. (A Wallace Hays) 2003; In Chapter 26 (Robert L Hall and Nancy E Everds) p1331-1332.) This finding was not considered to be anaemia as it wasn’t associated with any additional findings such as an increase plasma and urinary bilirubin.
Compound-related changes in the haematology parameters consisted of generally dose-dependent and statistically significant decreases in a number of erythroid parameters (erythrocyte count and haemoglobin concentrations) in compound-treated rats of both genders. These changes were accompanied by evidence for a robust regenerative response, strongly suggesting an appropriate response by the bone marrow. This regenerative response was indicated by one or more of the following in the compound-treated groups in both genders: 1) robust and dose-dependent reticulocyte response; 2) generally dose-dependent increase in the mean corpuscular volume and decreases in the mean corpuscular haemoglobin concentration which is consistent with increased number of reticulocytes. With this convincing evidence of appropriate bone marrow regeneration (increased reticulocyte response) an investigation of the bone marrow smears was not required (References: Provencher Bollinger, A. Cytologic evaluation of bone marrow in rats: Indications, methods, and normal morphology. Veterinary Clinical Pathology 33(2):58-67, 2004; Reagan, W.J., Irizarry-Rovira, A., Poitout-Belissent, F., Provencher Bollinger, A., Ramaiah, S.K., Travlos, G., Walker, D., Bounous, D., Walter, G. (Bone Marrow Working Groups of the ASVCP/STP). Best practice for evaluation of bone marrow in nonclinical toxicity studies. Toxicologic Pathology 39: 435-448, 2011).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry changes revealed low cholesterol and high urea levels in males and female at 500 mg/k/g/day and high inorganic phosphate in males at 500 mg/kg/day. However, the reason for these findings are not clear and in the absence of any histopathological correlate in the liver and kidney, they were considered slight and not adverse they were not taken into consideration when setting the NOAEL.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There was no effect on functional observational battery tests or locomotor activity.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment related organ weight changes included a statistical decrease in organ weight was seen in the thymus of males at 500 mg/kg/day. Liver weights were high in females at 500 mg/kg/day, with spleen weights high in females at 150 mg/kg/day and males and females at 500 mg/kg/day. Adrenal gland weights were high in males given 500 mg/kg/day compared to concurrent controls. Increased adrenal and decreased thymus weights are considered to be an effect of the low terminal body weight recorded in the males and the high liver weight likely to be a consequence of the high body weight gain seen in the females and as there is no histopathological correlate, these changes are considered not to be of toxicological significance.
There was no effect on organ weights for animals given 50 mg/kg/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
On macroscopic examination, findings related to treatment with Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl],N’-oxides were present in the stomach and caecum of animals treated with 500 mg/kg/day: the (fore)stomach of 5/10 males and 2/10 females of the 500 mg/kg/day
treated group showed macroscopic abnormalities consisting of thickening and in some of those 7 animals also a change in colour (recorded as abnormal and pale), and a distention was noted in the caecum of 5/10 males treated with this dose level. These findings comprised: thickening and/or an abnormal/pale colour of the (fore)stomach, and a large or distended caecum.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
On microscopic examination, findings related to treatment with Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl],N’-oxides were observed in the (fore)stomach, lung, spleen, liver and urinary bladder.
In the stomach, changes observed comprised squamous cell hyperplasia, erosions/ulcers within the affected epithelium, some acute inflammatory changes and submucosal oedema. These changes were noted in a single male treated with 150 mg/kg/day and in males and females treated with 500 mg/kg/day.
In the spleen an increase in severity of (brownish) pigment was observed in males and females from the 50 mg/kg/day treated group onwards: The pigment is believed to be haemosiderin. The incidence of the pigment in the spleen at 50 mg/kg bw/day was 7/10 (slight) and 3/10 (moderate) in males and 1/10 (slight), 7/10 (moderate) and 2/10 (severe) in males. At 150 mg/kg bw/day, 1/10 (minimal), 6/10 (slight) and 3/10 (moderate) in males and, in females, 8/10 (moderate) and 2/10 (severe). At 500 mg/kg bw/day, 5/10 (slight) and 5/10 (moderate) in males and, in females, 9/10 (moderate) and 1/10 (severe). This pigment appeared even in the controls at a moderate grade in females (7/10) and slight in males (8/10).
The changes observed in the urinary bladder were all mild and comprised diffuse transitional cell hyperplasia and/or increased epithelial folding. The transitional epithelium of the urinary bladder showed an increased epithelial folding in a 2/10 males treated with 150 mg/kg/day and in 5/10 males and 7/10 females of the 500 mg/kg/day treated group, while a low grade diffuse transitional cell hyperplasia was observed in few males and one female treated with 500 mg/kg/day. These findings are considered to be due to the irritating properties of the test substance. The epithelium of the urinary bladder is closely related to the squamous epithelium in the (fore)stomach and it is considered that the changes in both organs are related.
An increase in the presence of aggregates of (slight foamy) alveolar macrophages was noted in the lung of the 500 mg/kg/day treated animals, with the significance of these findings unclear.
A treatment-related increase in the incidence of pigmented Kupffer’s cells was observed in the liver of females of the 500 mg/kg/day group only. No microscopic findings were observed in the liver and in the caecum to support the liver weight changes observed in females dosed at 500 mg/kg/day, and the gross changes observed in the caecum of animals dosed at 500 mg/kg/day.

The kidney was not considered target tissue in this 13-week study.

No treatment-related histopathological changes were noted in any of the reproductive organs investigated in this study.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
The analytical results indicate that the prepared formulations were within ±10 % of the nominal concentration. Test article was
not detected in the control samples.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Toxicologically significant changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg/day and in the (fore)stomach at 150 mg/kg/day.
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
other: gastrointestinal, urinary, and hematopoietic system
Organ:
bladder
blood
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Group mean weekly bodyweights and standard deviation (Tables 1 and 2)

 

Table 1: Males

 

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

BODYWEIGHT(g)AT DAY

 

1*

1

5

8

12

15

19

0 (Control)

10

mean

178.2

235.2

266.8

285.0

309.4

328.3

350.0

sd

17.19

21.43

23.44

23.98

23.48

25.29

23.95

50

10

mean

173.9

232.3

263.4

279.0

310.0

330.4

351.6

sd

10.54

14.79

14.95

16.12

17.67

17.14

17.10

150

10

mean

174.1

228.9

260.3

277.9

306.2

326.8

347.6

sd

15.99

20.47

21.91

22.67

25.52

25.02

26.17

500

10

mean

174.7

228.3

258.6

271.7

300.5

317.6

341.8

sd

11.48

14.42

16.46

16.08

17.90

18.58

20.90

 

 

BODYWEIGHT(g)AT DAY

 

22

26

29

36

43

50

57

0 (Control)

10

mean

362.7

385.6

393.8

419.8

446.9

466.9

478.1

sd

25.26

22.61

23.68

21.86

24.66

21.08

22.65

50

10

mean

365.9

384.4

397.1

423.1

453.1

470.1

489.0

sd

17.46

22.20

22.52

25.11

22.78

23.40

23.87

150

10

mean

363.3

380.6

390.5

414.0

435.5

453.8

468.3

sd

27.76

26.08

26.43

29.08

29.29

30.87

32.52

500

10

mean

354.9

370.8

374.8

400.9

416.4

432.6

451.5

sd

20.97

24.25

20.95

23.74

23.49

30.58

29.86

 

 

BODYWEIGHT(g)AT DAY

 

64

71

78

85

92

 

 

0 (Control)

10

mean

489.8

501.4

515.3

524.1

525.7

 

 

sd

22.28

21.88

19.83

22.67

23.02

 

 

50

10

mean

500.1

511.9

524.4

537.4

536.8

 

 

sd

24.06

23.06

26.48

26.02

22.61

 

 

150

10

mean

476.0

488.3

494.3

498.2

496.4

 

 

sd

32.71

35.66

39.79

37.58

37.75

 

 

500

10/9**

mean

448.5

462.6

469.0

492.8

485.2

 

 

sd

29.83

30.73

32.93

28.50

32.90

 

 

*= predose
** =1 animal died during week 12

 

Table 2: Females

 

Dose Level of Active Ingredient

mg/kg/day

Number of Animals

BODYWEIGHT(g)AT DAY

 

1*

1

5

8

12

15

19

0 (Control)

10

mean

150.3

173.4

185.1

196.2

202.8

212.1

221.1

sd

11.29

12.82

14.90

15.99

11.45

13.46

16.18

50

10

mean

151.0

174.1

187.4

196.5

209.4

220.3

228.1

sd

16.62

15.06

14.62

18.80

17.76

18.66

20.37

150

10

mean

145.7

168.8

182.9

192.1

204.7

212.4

222.3

sd

6.09

9.19

11.51

12.27

14.98

14.05

17.28

500

10

mean

155.7

178.0

192.9

203.8

218.4

229.1

241.7

sd

12.27

17.10

19.81

16.87

15.64

17.38

19.74

 

 

BODYWEIGHT(g)AT DAY

 

22

26

29

36

43

50

57

0 (Control)

10

mean

227.1

236.9

236.4

248.9

260.6

266.9

266.8

sd

18.21

21.28

22.20

20.77

21.10

26.61

24.48

50

10

mean

236.0

245.4

243.5

253.5

268.1

272.1

272.5

sd

20.87

22.13

17.02

19.65

21.69

25.13

21.60

150

10

mean

231.4

239.1

239.7

253.0

263.5

267.4

268.7

sd

18.64

19.45

20.70

21.58

20.78

24.03

22.78

500

10

mean

249.4

255.6

259.1

272.0

278.3

280.1

290.1

sd

21.50

21.46

21.25

21.83

24.74

25.75

26.28

 

 

BODYWEIGHT(g)AT DAY

 

64

71

78

85

92

 

 

0 (Control)

10

mean

273.7

276.2

277.8

283.8

280.8

 

 

sd

23.29

23.65

27.71

25.13

25.36

 

 

50

10

mean

280.1

284.3

286.8

289.1

291.7

 

 

sd

23.89

25.79

26.22

23.38

25.99

 

 

150

10

mean

278.0

281.0

287.8

289.5

286.9

 

 

sd

25.43

26.25

27.18

26.32

27.12

 

 

500

10

mean

293.5

297.0

302.0

306.5

305.0

 

 

sd

25.46

25.57

26.86

29.51

25.43

 

 

For other tables, see 'Attachments" to this endpoint study record.

 

 

 

 

 

Conclusions:
Daily oral administration of Amides, C12-C18 (even numbered), N-[3- dimethylamino) propyl], N’-oxides, to Sprague-Dawley rats for 13 weeks (test performed according to OECD 408) at dose levels of 50, 150 and 500 mg/kg/day, resulted in toxicologically significant effects at dose levels of 500 mg/kg/day and 150 mg/kg/day. The changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg/day and the changes in the (fore)stomach at 150 mg/kg/day were considered adverse and toxicologically significant. No adverse effect was observed at 50 mg/kg/day. Therefore the No Observed Adverse Effect Level is set at 50 mg/kg/day. With the LOAEL set at 150 mg/kg/day, the substance is not to be classified as STOT RE toxicant according to CLP Regulation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

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Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

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Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
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:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
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:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

Two reliable studies are available for this endpoint: a 28-day oral repeated dose toxicity study and a 90-day oral repeated dose toxicity study.

Jones et al. (2000) performed a 28-day repeated dose toxicity study according to OECD 407 and EU method B.7, and in compliance with GLP. In this supporting study, Sprague-Dawley rats (5 females and 5 males per dose) were exposed daily by oral gavage with the test substance at 18.4 mg/kg/day (15 mg/kg/day of active ingredient), 184 mg/kg/day (150 mg/kg/day of active ingredient), 1227 mg/kg/day (1000 mg/kg/day of active ingredient). A control group was dosed with the vehicle only (distilled water).

Clinical signs, functional observations, bodyweight gain and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to a gross necropsy examination. Histopathological evaluation of selected tissues was performed in all control animals (0 mg/kg/day) and animals at the highest dose group (1000 mg/kg/day). The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed.

Clinical signs of toxicity, functional incidents as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption and reduced bodyweight gain were observed at dose of 1000 mg/kg/day.

In the blood chemistry, elevations in plasma aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and bilirubin was noted at the dose of 1000 mg/kg/day; ASAT and ALAT were also elevated for 150 mg/kg/day animals. Haematological changes as haemolytic anaemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time were observed at doses of 1000 and 150 mg/kg/day.

A statistically significant increase in spleen weight was observed in males and females treated with 1000 mg/kg/day. The effect extended to the 150 mg/kg/day dose group although statistical significance was not always achieved.

Treatment-related changes in liver and spleen were detected at 1000 mg/kg/day and 150 mg/kg/day. Histopathological changes were also observed in kidneys, urinary bladder and stomach of animals treated with 1000 mg/kg/day. No treatment-related changes were reported in the ovaries, prostate, seminal vesicles, testes and uterus of control animals or animals at 1000 mg/kg/day.

Based on the above-mentioned results, the No Observed Effect Level (NOEL) was considered to be 15 mg/kg/day.

Horne (2014) performed a 90-day repeated oral dose toxicity study in rats with Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl], N’-oxides. The test was performed according to OECD guideline 408 and in compliance with GLP. In this study Sprague-Dawley rats (10 females and 10 males per dose) were exposed daily by oral gavage with the active ingredient at 50 mg/kg/day, 150 mg/kg/day and 500 mg/kg/day of active ingredient). These doses were selected based on the results of the 28 -day repeated oral dose toxicity study described here above. A control group was dosed with the vehicle only (distilled water).

Clinical signs (health monitoring, clinical examinations and post-dose observations), bodyweight, food and water consumption were monitored during the study. All animals were subjected to a functional observational battery weekly and at the Week 13 before sacrifice. Blood samples were collected after an overnight period without food from all the animals at the end of the study and haematology, coagulation and clinical chemistry parameters were evaluated for all animals. All animals were subjected to a gross necropsy examination. Selected organs were weighed. Histopathological evaluation of selected tissues was performed (including reproductive organs) in all control animals (0 mg/kg/day) and animals at the highest dose group (500 mg/kg/day). The lungs, liver, kidney, urinary bladder, (fore)stomach, spleen and other gross lesions observed at the macroscopic evaluation were also processed for animals of the 50 and 150 mg/kg/day dose group.

Only one animal was found dead: one male dosed at 500 mg/kg bw/day. the animal was found dead during Week 12 and this death was considered to be treatment-related.

Findings for animals at 500 mg/kg/day included salivation, staining around the mouth and noisy breathing. This is indicative of irritant test materials with the animals not liking the taste of the formulation. Body weight gains were low and, as there was no effect on food consumption; this was considered non-treatment related toxicity. Water consumption values were high for males and females which again was associated with the oral dosing of an irritant material. The changes in the haematology parameters were consistent with a decrease in red cell mass showing regeneration and the magnitude of change (between 7% and 19%) was considered borderline adverse as a reduced red blood cell mass of more than 30% is considered adverse and a change of less than 10% is considered non-adverse for rodents. This finding was not considered to be anaemia as it was not associated with any additional findings such an increase of plasma bilirubin.

Microscopic examination revealed adverse findings in the (fore)stomach (squamous cell hyperplasia) and urinary bladder (transitional cell hyperplasia) which was considered a result of the irritating properties of the test article. The epithelium of the urinary bladder is closely related to the squamous epithelium in the (fore)stomach and it is considered that the changes in both organs are related.

Findings for animals at 150 mg/kg/day included low body weight gains and as there was no effect on food consumption, this was considered non-specific toxicity. The changes in the red blood cell mass were not considered adverse at this dose level as the magnitude of change was less than 11%. Forestomach squamous cell hyperplasia was noted in a single male at 150 mg/kg/day and as this finding matched those recorded at 500 mg/kg/day this was considered adverse. The only treatment-related change seen in males at 50 mg/kg/day were statistically relevant minor changes in the red blood cell parameters, indicating that the change in red blood cell mass was affecting these animals but as the magnitude of change was much lower than animals treated at 500 mg/kg/day, these findings were considered treatment-related but not adverse in nature.

In conclusion, daily oral administration of Amides, C12-C18 (even numbered), N-[3- dimethylamino) propyl],N’-oxides, to rats for 13 weeks at dose levels of up to 500 mg/kg/day, resulted in toxicologically significant effects at dose levels of 500 mg/kg/day and 150 mg/kg/day. The changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg/day and the changes in the (fore)stomach at 150 mg/kg/day were considered adverse and toxicologically significant. No adverse effect was observed at 50 mg/kg/day. Therefore the NOAEL is set at 50 mg/kg/day.

No treatment-related histopathological changes were noted in any of the reproductive organs investigated in this study.

Although the 28-day repeated oral dose toxicity study yields to the lowest NOEL/NOAEL value, this study is considered less sensitive compared to the 90-day repeated oral dose toxicity study as the duration of exposure is shorter and less animals are used per group. In addition, the spacing between doses in the 28-day study of 10-fold (compared to 2-3 fold in the 90-day study) also increases the uncertainty about the derived NO(A)EL. Therefore, the oral 90-day NOAEL value is considered more reliable than the 28-day NOEL value and thus the 90-day repeated oral dose toxicity study is selected as key study for this endpoint. The study is scored as reliable (K1).

Repeated dose toxicity: dermal

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure .

Repeated dose toxicity: inhalation

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure .

Justification for classification or non-classification

The administration of the test material to rats in a 90-day repeated oral (gavage) dose toxicity study performed according to OECD 408 guideline resulted in toxicologically significant effects at dose levels of 500 and 150 mg/kg/day (changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg/day and changes in the (fore)stomach at 150 mg/kg bw/day). No adverse effects were observed at 50 mg/kg/day and this dose was considered to be the NOAEL value.

According to the CLP Regulation (Regulation (EC) No 1272/2008, a substance is classified (or not) based on the dose level at which significant/serious adverse effects are observed. This level should be compared to the corresponding Guidance Values (GVs) in the EU CLP guideline. The lowest dose at which significant/severe effects are observed in the 90-day repeated dose study is 150 mg/kg bw/day but the NOAEL is 50 mg/kg bw/day, that means below the GV for a 90-day study (100 mg/kg bw/day; Table 3.9.3 in the Regulation). Therefore, the first dose at which significant/serious adverse effects are observed (Effective Dose, ED) should be derived by interpolation, as indicated in the ECHA CLP guidance document on classification, labelling and packaging (CLP) of substances and mixtures (Version 4.0 November 2013). On the basis of the observed results and the interpolation, the ED is estimated to be 126,66 mg/kg bw/day (> GV for a 90-day study 100; Table 3.9.3 in the Regulation). Thus, and according to the criteria of the CLP Regulation, the substance does not need to be classified for STOT RE. In addition, the changes observed in the stomach can be considered as an adaptive response to the repeated oral gavage with a corrosive substance.