Registration Dossier

Administrative data

Description of key information

The read-across supporting substance N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine was investigated for its oral repeated dose toxicity according to the OECD Guideline 422. Stomach, hematopoietic system, and liver were identified as target organs at 125 mg/kg bw.  The NOAEL of 30 mg/kg bw was derived for systemic toxicity and for reproductive toxicity.

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
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Between 16 October 2009 and 08 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Scientific rationale for the read-across: the registration substance is a tertiary amine oxide and the read-across substance the corresponding tertiary amine so that an inter-convertibility of the registraiton substance and the proposed read-across substance can be presumed.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™:HsdRccHan™:WIST strain rat
Sex:
male/female
Details on test animals and environmental conditions:
Test Animals
- Source:
Wistar Han™:HsdRccHan™:WIST strain rats from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.

- Age at study initiation:
Approximately 12 weeks old

- Weight at study initiation:
297 to 342g (male); 184 to 233g (female)
- Fasting period before study:
Not applicable

- Housing:
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

- Diet:
The animals were allowed free access to food. A pelleted diet Rodent 2018C
Teklad Global Certified Diet Harlan UK Ltd, Oxon, UK was used throughout the study period. The diet was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

- Water:
Water intake was measured and recorded daily for each cage group (with the exception of non-recovery (satellite) animals during the mating phase). Individual daily water intakes were measures for females during the gestation and lactation phases of the study

- Acclimation period:
For 12 days

ENVIRONMENTAL CONDITIONS

- Temperature:
21 ± 2 °C

- Humidity:
55 ± 15 %

- Air changes (per hr):
At least fifteen air changes per hour

- Photoperiod (hr dark / hrs light):
12 hours continuous light and 12 hours darkness

IN-LIFE DATES:
20 October 2009 and 15 December 2009 (including recovery phase animals)

Route of administration:
oral: gavage
Vehicle:
other: Arachis oil BP
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services (Harlan Laboratories Ltd. Project Number: 0142-0416). Results from the previous study showed the formulations to be stable for at least twenty days. Formulations were therefore prepared twice monthly during the treatment period and stored at approximately +4ºC in the dark, under nitrogen.
Samples of each test material formulation were taken and analysed for concentration of test material at Harlan Laboratories Ltd., Shardlow, UK Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 26. The results indicate that the prepared formulations were within plus or minus 9% of the nominal concentration.

DIET PREPARATION
- Not applicable

- Rate of preparation of diet (frequency):
Not applicable

- Mixing appropriate amounts with (Type of food):
Not applicable

- Storage temperature of food:
No data

VEHICLE
Arachis oil BP

- Justification for use and choice of vehicle (if other than water):
Not applicable

- Concentration in vehicle:
31.3, 7.5 and 2.5 mg/ml

- Amount of vehicle (if gavage):
4 ml/kg bodyweight

- Lot/batch no. (if required):
Not applicable

- Purity:
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in the formulations was determined by gas chromatography (GC) using an external standard technique. The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.1 mg/ml. Procedural recoveries were performed at each dose level on every analysis occasion.
Standard solutions of test material were prepared in methanol at a nominal concentration of 0.1 mg/ml. The test material formulations were sampled and analysed within five days of preparation. The results indicate that the prepared formulations were within +9% of the nominal concentration.


Duration of treatment / exposure:
The oral administration of the test substance to rats for a period of up to fifty-four consecutive days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
Dose levels of 10, 30 and 125 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
0 mg/kg/day – control: 10 animals per sex.
10 mg/kg/day : 10 animals per sex.
30 mg/kg/day : 10 animals per sex.
125 mg/kg/day : 10 animals per sex.
Recovery (Satellite ) 0 mg/kg/day – control: 5 males only.
Recovery (Satellite ) 125 mg/kg/day : 5 males only.
Control animals:
yes, concurrent vehicle
Details on study design:

- Dose selection rationale:
Based on Preliminary Fourteen Day Repeated Dose Oral (Gavage) Range-Finder in the Rat (Harlan 0142-0416; see the corresponding endpoint study record)

- Rationale for animal assignment (if not random):
Random

- Rationale for selecting satellite groups:
To determine potential regression of any detected systemic responses elicited by administration of the test material

- Post-exposure recovery period in satellite groups:
Fourteen days

- Section schedule rationale (if not random):
Random
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:

- Yes

- Time schedule:

- Immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, thirty minutes after dosing, and one hour after dosing at weekends and public holidays (except for females during
parturition where applicable). During the treatment-free period, recovery males were observed once daily. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes (see above).
- Time schedule: As above.

NEUROBEHAVIOURAL EXAMINATION:

- Yes

- Functional Observations were performed prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of
functional/behavioural toxicity.

- Functional performance tests (motor activity, forelimb/hindlimb grip strength and sensory reactivity) were also performed on five selected males
during the final week of treatment and five Day 4 post partum females from each dose level.

BODY WEIGHT:

- Yes

- Time schedule for examinations:

- Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating w as evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Bodyweights were
also recorded prior to termination


FOOD CONSUMPTION:

- Yes

- During the maturation period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating
phase. For females showing evidence of mating, food consumption was recorded for the periods covering Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum. Weekly food consumptions were performed weekly for each cage of adults throughout the study period.

- FOOD EFFICIENCY:

- Yes

- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated retrospectively for males throughout the study period, and for females prior to mating.

WATER CONSUMPTION:

- Yes

- Water intake was measured gravimetrically and recorded daily for each cage group (with the exception of non-recovery animals during the mating - phase). Individual daily water intakes were measured for females during the gestation and lactation phases of the study.

OPHTHALMOSCOPIC EXAMINATION:
Not applicable

HAEMATOLOGY AND CLINICAL CHEMISTRY:

- Yes

- Time schedule for collection of blood:

- Haematological and blood chemical investigations were performed on five males and five females selected from each non-recovery test and control group prior to termination (Day 42 for males and Day 4 post partum for females). These investigations were also performed on all recovery
(satellite) males at the end of the treatment-free period (Day 56).

- Blood samples were obtained from the lateral tail vein or by cardiac puncture at termination, if applicable.

- Anaesthetic used for blood collection:
- No

- Animals fasted:
- No

OTHER:

MATING

- Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each
morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal
smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to
their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and
lactation.

PREGNANCY AND PARTURITION

- Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations
were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:

i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

LITTER SIZE

On completion of parturition (Day 0 of post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1.
For each litter the following was recorded:

i) Number of offspring born
ii) Number and sex of offspring alive recorded daily and reported on Day 1 and 4
post partum
iii) Clinical condition of offspring from birth to Day 5 post partum
iv) Individual offspring weights on Day 1 and 4 post partum (litter weights were calculated retrospecively from offsring weights).

PHYSICAL DEVELOPMENT

All live offspring were assessed for surface righting reflex on Day 1 post partum.



















Sacrifice and pathology:
GROSS PATHOLOGY:
- Yes

HISTOPATHOLOGY:
Yes
Other examinations:
MORTALITY DATA
- Yes (no unscheduled deaths ocured during the study)
ORGAN WEIGHTS
- Yes
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Mortality.

No unscheduled deaths were detected.

Clinical Observations.

A higher incidence of increased salivation was detected soon after dosing and up to one hour after dosing for animals of either sex treated with 125 and 30 mg/kg/day, and also for males treated with 10 mg/kg/day when compared to controls. Regression was evident following the cessation of treatment in recovery 125 mg/kg/day males.

Functional Observations.

No treatment-related effects were evident in the weekly behavioural assessments, sensory reactivity, grip strength or motor activity.

Bodyweight.

No adverse effect on bodyweight change was detected for males or for females during the pre-mating and gestation phases. Lower bodyweight gains were evident for females treated with 125 mg/kg/day when compared to controls during the lactation phase of the study. No adverse effects were evident at 30 or 10 mg/kg/day.

Food Consumption.

No adverse effects on dietary intake were evident for males or for females during the pre-mating or gestation phases of the study. A slight reduction in dietary intake was evident for females treated with 125 mg/kg/day when compared to controls during lactation.

Water Consumption.

No overt intergroup differences in water intake were detected for males or for females during the pre-mating or gestation phases of the study. A reduction in water intake was evident for females treated with 125 mg/kg/day when compared to controls during lactation.

Reproductive performance.

Mating.

No treatment-related effects were detected in mating performance.

Fertility.

No treatment-related effects were detected in fertility.

Gestation.

No treatment-related effects were detected on gestation length.

Litter responses.

Litter size and Viability.

Lower litter sizes, live birth indices and reduced numbers of viable litters were evident at 125 mg/kg/day when compared to controls. Slightly lower numbers in corpora lutea and implantation sites were evident for females treated with 125 mg/kg/day when compared to controls, and higher post-implantation losses were also evident.

Offspring Growth and Development.

Lower total litter weights were evident at 125 mg/kg/day in comparison to control values. Bodyweights and surface righting assessments were not
affected.

Laboratory Investigations.

Haematology.

Males treated with 125 mg/kg/day showed a reduction in haemoglobin, haematocrit, mean cell haemoglobin, mean cell volume and reticulocyte counts when compared to controls. These findings were considered to be of no toxicological significance.

No treatment-related effects were evident for females treated with 125 mg/kg/day, or for animals of either sex treated with 30 and 10 mg/kg/day.

Blood Chemistry.

No significant effects were detected in the blood chemical parameters investigated.

Pathology.

Organ Weights.

Males treated with 125 mg/kg/day showed slightly higher absolute and bodyweight-relative spleen and liver weights. These findings were considered to be of no toxicological importance.

No treatment-related effects were detected for females treated at 125 mg/kg/day, or for animals of either sex treated with 30 or 10 mg/kg/day.

Necropsy.

Offspring: No treatment-related macroscopic abnormalities were detected for offspring from treated animals when compared to control litters.

Adults: Treatment-related findings were confined to the presence of a thickened non-glandular region of the stomach for one male treated with
125 mg/kg/day.

Histopathology. The following treatment-related changes were observed:

STOMACH: Acanthosis, frequently with associated hyperkeratosis, was seen in the forestomach of all animals of either sex treated with 125 mg/kg/day, and in males treated with 30 mg/kg/day. There was evidence of regression of the condition in recovery 125 mg/kg/day males following an additional fourteen days without treatment.

Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
30 other: mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity.
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
other: Reproductive toxicity
Sex:
female
Basis for effect level:
other: Lower litter sizes due to lower numbers of corpora lutea and implantation sites, and higher post implantation losses were evident at 125 mg/kg/day. A NOEL was therefore considered to be 30 mg/kg/day for reproductive toxicity
Critical effects observed:
not specified

Tab 1a: Body weight development for males [g]

Dose group

Day numbers relative to start date

 

1

8

15

22

29

36

43

50

57

Control

Mean

318.3

329.7

341.2

353.7

363.5

375.7

385.1

403.8

411.0

S.D.

12.8

16.7

20.3

19.8

21.6

26.3

28.2

21.1

22.3

N

15

15

15

15

15

15

15

5

5

10 mg/kg bw/day

Mean

320.9

336.9

348.0

360.7

371.6

379.7

389.6

-

-

S.D.

10.2

13.3

17.9

20.3

21.3

23.2

24.4

-

-

N

10

10

10

10

9

10

10

0

0

30 mg/kg bw

Mean

325.5

340.1

353.9

365.0

376.5

387.3

398.7

-

-

S.D.

9.8

14.7

18.2

19.7

21.1

21.5

22.4

-

-

N

10

10

10

10

10

10

10

0

0

125 mg/kg bw/day

Mean

320.9

330.4

340.7

353.3

361.5

372.3

382.9

395.2

404.8

S.D.

11.7

15.4

17.8

21.0

20.9

22.8

23.8

20.3

23.0

N

15

15

15

15

15

15

15

5

5

Tab 1b: Body weight development for females [g]

Dose group

 

Pre-mating

Gestation

Lactation

 

1

8

15

0

7

14

20

1

4

Control

Mean

207.8

211.6

217.9

218.0

243.2

268.6

333.8

235.1

248.6

S.D.

7.3

3.6

7.3

7.9

10.1

14.7

18.9

11.9

11.4

N

10

10

10

10

10

10

10

10

10

10 mg/kg bw/day

Mean

205.6

207.4

213.7

214.9

240.4

267.7

327.0

247.0

257.1

S.D.

12.7

9.7

14.4

11.2

15.7

19.2

24.8

23.0

21.1

N

10

10

10

9

9

9

9

9

9

30 mg/kg bw

Mean

210.6

212.8

217.7

224.1

246.0

272.9

339.8

243.8

256.6

S.D.

10.2

8.9

12.6

12.7

11.9

10.9

16.7

14.6

13.5

N

10

10

10

10

10

10

10

10

10

125 mg/kg bw/day

Mean

212.3

211.4

218.6

222.0

243.6

269.3

321.9

259.3

260.1

S.D.

9.6

11.7

11.3

14.2

15.2

17.7

24.2

18.1

18.5

N

10

10

10

9

9

9

9

9

9

Tab 2: Hematology for males (N=5) at termination and after recovery:

- data for females not shown as no difference was found for control and treated animals

Dose group

 

Hb

RBC

Hct

MCH

MCV

MCHC

WBC

Neut

Lymph

CT

PLT

APTT

Retics

 

g/dl

10^12/l

%

pg

g/dl

g/dl

10^9/l

10^9/l

10^9/l

sec

10^9/l

sec

%

At termination

Control

Mean

16.60

8.974

48.68

18.48

54.28

34.06

7.56

0.902

6.562

11.06

530.4

16.84

4.80

S.D.

0.41

0.386

1.17

0.42

1.34

0.38

1.29

0.280

1.026

0.89

77.3

0.81

0.71

10 mg/kg bw/day

Mean

16.28

9.158

46.60

17.80

50.88**

34.96

6.66

0.744

5.858

11.72

581.4

16.68

4.56

S.D.

0.27

0.367

1.15

0.82

1.26

1.23

1.61

0.190

1.635

1.41

73.2

1.32

0.47

30 mg/kg bw/day

Mean

16.62

9.176

48.32

18.10

52.60**

34.44

7.98

0.966

6.942

12.36

577.8

16.84

4.68

S.D.

0.77

0.362

2.71

0.19

1.13

0.58

1.73

0.523

1.528

1.99

33.5

1.27

0.40

125 mg/kg bw/day

Mean

15.60**

8.854

45.62*

17.62*

51.52**

34.20

8.06

0.756

7.206

12.86

632.2*

16.62

5.78*

S.D.

0.34

0.240

0.80

0.11

1.26

0.64

1.70

0.336

1.833

2.29

58.4

1.11

1.02

After recovery

Control

Mean

15.66

8.962

47.64

17.48

53.20

32.88

7.22

0.778

6.356

9.12

638.0

13.30

4.58

S.D.

0.39

0.312

1.43

0.51

1.67

0.18

0.75

0.160

0.717

0.41

46.9

1.22

0.40

50 mg/kg bw/day

Mean

15.40

9.168

46.76

16.80*

51.00*

32.94

7.64

1.042

6.512

9.12

690.8

14.66

5.54

S.D.

0.66

0.376

2.35

0.14

0.80

0.34

0.36

0.283

0.432

0.16

90.8

0.72

0.91

Tab 3: Organ weight for males and females [g]:

-data shown only for organs with statistical differences for control and treated animals

 

males

females

 

At treatment termination

After recovery of 14 days

At treatment termination

Dose group[g]

Dose group[g]

Dose group[g]

Control

10

30

125

0

125

Control

10

30

125

Terminal body weight

Mean

380.8

389.6

398.7

382.5

411.0

404.8

250.0

257.0

255.4

259.2

S.D.

31.5

24.4

22.4

25.2

22.3

23.0

8.1

24.5

12.8

18.5

N

10

10

10

10

5

5

10

9

10

9

Kidneys

Mean

1.99

2.18*

2.17*

2.15*

2.24

2.24

1.43

1.42

1.52

1.53

S.D.

0.20

0.19

0.13

0.16

0.13

0.18

0.10

0.08

0.08

0.11

N

10

10

10

10

5

5

10

9

10

9

Liver

Mean

11.97

12.34

12.38

13.40**

12.45

12.34

10.59

10.19

10.40

11.43

S.D.

1.20

0.87

0.90

1.13

1.79

0.56

1.26

1.02

0.86

1.13

N

10

10

10

10

5

5

10

9

10

9

Spleen

Mean

0.64

0.63

0.68

0.73*

0.66

0.71*

0.62

0.63

0.63

0.63

S.D.

0.06

0.09

0.09

0.11

0.07

0.03

0.06

0.09

0.09

0.09

N

10

10

10

10

5

5

10

9

10

9

Thyroid

Mean

0.012

0.014

0.017*

0.016*

0.023

0.020

0.014

0.015

0.015

0.017

S.D.

0.004

0.003

0.005

0.004

0.014

0.005

0.003

0.006

0.004

0.007

N

10

10

10

10

5

5

10

9

10

9

The

Conclusions:
The read-across supporting substance N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine was investigated for its oral repeated dose toxicity according to the OECD Guideline 422. Stomach, hematopoietic system, and liver were identified as target organs at 125 mg/kg bw. The NOAEL of 30 mg/kg bw was derived for systemic toxicity and for reproductive toxicity.
Executive summary:

The repeated dose toxicity of the registration substance is derived by use of data on the read-across supporting substance N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine.

N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine was investigated for its oral repeated dose toxicity according to the OECD Guideline 422. The rats were treated at doses of 0, 10, 30 and 125 mg/kg bw for up to forty five days. The irritant effect such as salivation and histopathologcal alteration in the gastric tract was evident at 125 mg/kg bw. Further, a possible effect on the hematopoietic system could be derived based on the minimal changes in hematology parameters and the slightly increased spleen weights in males. Also the liver weight was minimally increased for males that was considered as adaptive responce. Based on the histopathological changes observed in the stomach the NOAEL of 30 mg/kg bw was derived for the systemic toxicity.

At dose of 125 mg/kg bw lower litter size was evident. The NOAEL of 30 mg/kg bw was derived for the reproductive toxicity.

Similar toxicity pattern is expected for the registration substance. Gastric tract, hematopoietic system and liver are likely to serve as target organs at doses of 125 mg/kg bw and higher. Taking account that the chemical structure and the molecular size of the registration substance is closely similar to those of the read-across supporting substance, the NOAEL of 30 mg/kg bw is considered to be also valid for the registration substance.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One key study on read-across supporting substance and three supporting studies on read-across supporting substance or on read-across substance; consistent results obtained for all studies; all avaiable studies are of high quality

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

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for the proposed read-across approach

The registration substance is the oxidation product of the introduced read-across supporting substance, N,N-Bis(2-hyydroxyethyl)-C12-18(even numbered)alkyl-1-amine.The underlying scientific rationale for the proposed read-across is based on:

(a) The source chemical (and accordingly the read-across supporting substance) is a tertiary amine and the target chemical (and accordingly the registration substance) the corresponding tertiary amine oxide, whereby an inter-convertibility within these two chemicals in biological systems can be presumed so that the subsequent metabolism pathway should be identical for both chemicals.

(b) For the verification purpose of the proposed read-across appraoch, the target chemical was investigated for its reproduction toxicity according to the OECD 421, because the reduction of live fetuses under given exposure conditions were predictable based on the available data on the source chemical. The outcome of the OECD 421 on the target chemical confirmed the similarity of the toxicity profiles for target- and the source chemicals, which serves here as a crucial bridging evidence.

Further detailed justification is provided separately in Chapter 13.

Overall assessment

The repeated dose toxicity of the registration substance is derived from one key-study and one supporting study on the read-across supporting substance, N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine and two supporting studies on the registration substance.

a) N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine was investigated for its oral repeated dose toxicity according to the OECD Guideline 422. The rats were treated at doses of 0, 10, 30 and 125 mg/kg bw for up to forty five days. The irritant effect such as salivation and histopathologcal alteration in the gastric tract was evident at 125 mg/kg bw. Further, the treatment related effect on the hematopoietic system was found based on the minimal changes in hematology parameters and the slightly increased spleen weights in males. Also the liver weight was minimally increased for males, which was considered as adaptive responce. Based on the histopathological changes observed in the stomach the NOAEL of 30 mg/kg bw was derived for the systemic toxicity.

At dose of 125 mg/kg bw lower litter size was evident. The NOAEL of 30 mg/kg bw was derived for the reproductive toxicity.

b) N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine was investigated for its repeated dose toxicity by oral application to rats for up to 14 days at doses of 0, 75, 150 and 250 mg/kg bw. The study was performed for the purpose of dose-range finding for the main study (OECD 422). The investigation parameters were limited to clinical signs, body weight development, food consumption and gross pathology. Severe toxicity such as mortality and emaciation occurred at the dose of 250 mg/kg bw/day. Tissue damages in the gastro-intestinal tract were evident in dose-dependent manner. Further, liver enlargement was found at dose of 150 mg/kg bw and 75 mg/kg bw.

c) The registration substance was investigated for its repeated dose toxicity by the oral application to rats for up to 7 days at doses of 62.5, 250 and 1000 mg/kg bw/day.The study was performed for the purpose of dose-range finding for the main study (OECD 421).The investigation parameters were limited to clinical signs, body weight development, food consumption and macroscopic examination upon gross pathology. Rats treated with 1000 mg/kg bw died after 2-3 treatments and damages in the gastro-intestinal tract were evident upon gross necropsy. At doses of 250 and 62.5 mg/kg bw, body weight decrease and reduced food consumption were found in dose dependent manner and at 250 mg/kg bw the thickened/rough surface non glandular mucosa of the stomach was macroscopically observed.

d) The registration substance was investigated for its oral repeated dose toxicity according to the OECD Guideline 421. The rats were treated at doses of 0, 12.5, 50 and 200 mg/kg bw for up to forty five days. Based on the clinical signs and reduced body weight gain at 200 mg/kg bw/ day the NOAEL of 50 mg/kg bw was derived for the systemic toxicity.

At dose of 200 mg/kg bw/ day lower litter size was evident. The NOAEL of 50 mg/kg bw was derived for the reproductive toxicity.

Based on the available data, the repeated dose toxicity of the registration substance can be reasonably derived. After subacute oral exposure, adverse effects on gastric tract can be expected at dose of 125 mg/kg bw and higher. Adverse effects on hematopoietic system and liver may occur additionally upon chronic exposure. Taking account that the comparable dose-range-finding studies on the read-across supporting substance and the registration substance exhibited similar effects at comparable doses (i.e. tissue lesion in stomach and body weight decrease, the dose of 250 mg/kg bw as the MTD after up to 14 days exposure), comparable potencies can be derived for both substances. Accordingly, the NOAEL of 30 mg/kg bw after subacute exposure for the read-across supporting substance is considered to be also valid for the registration substance.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Scientifically well-performed and well-reported study; recently performed Guideline study; GLP study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

Justification for classification or non-classification