Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across based on grouping of substances (category approach)
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:
Rational for reliability: 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. Rational for read-across: The registration substance and the read-across supporting substance belong to the category "Primary Fatty Amine Ethoxylates"

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : Bis (2-hydroxyethyl) coco alkylamine
Description : pale brown viscous liquid
Batch number : S-001016
Date received : 08 July 2009
Storage conditions : approximately 4ºC in the dark, under nitrogen
Expiry date :26 June 2017

Test animals

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)

Administration / exposure

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.

See attached Appendix 26 - Chemical Analysis of Test Material Formulations, Methods and Results
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
Doses / concentrations
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

- 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

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:

- Yes see attached tables and appendices

- 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 see attached tables and appendices

- 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 see attached tables and appendices

- 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

- For parameters checked see attached Tables.

FOOD CONSUMPTION:

- Yes see attached tables and appendices

- 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 see attached tables

- 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 see attached tables

- 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 see attached tables and appendices

- 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.

- see attached tables and appendices


















Sacrifice and pathology:
GROSS PATHOLOGY:
- Yes see attached Table 21 and 22 and Appendices 246 to 249 for males and 250 to 253 for females

HISTOPATHOLOGY:
Yes see attatched Table 23 and Appendix 24
Other examinations:
MORTALITY DATA
- Yes (no unscheduled deaths ocured during the study)
ORGAN WEIGHTS
- Yes see attached Tables and Appendices

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Tab 4: Summary report of effects on reproduction/development

 

Dose group

 

Control

10

mg/kg bw/day

30

mg/kg bw/day

125

mg/kg bw/day

No. of females paired

10

10

10

10

No. of females mated

10

10

10

10

No. of females pregnant

10

9

10

10

No. of dams with living pubs

10

9

10

9

Body weight on GD20 [g]

334

327

340

322

Body weight on PN4 [g]

249

257

257

260

Corpora lutea

19.4

16.7

18.2

15.2

Implantation sites

15.6

12.4

14.3

  12.3*

No. of pubs alive at birth

14.3

11.9

13.6

       8.8***

No. of pubs alive at PN4

14.0

11.8

12.5

       7.7***

Applicant's summary and conclusion

Conclusions:
The repeated dose toxicity of the registration substance is derived based on the read-across approach.
The supporting substance CAS 71786 -60 -2 was tested for its repeated dose toxicity according to OECD 422 Test Guideline and the NOAEL of 30 mg/kg/day was obtained. The major effect found at 30 and 125 mg/kg/day was changes in forestomach and in stomach, indicating the local irritation as the mode of action.

Executive summary:

The repeated dose toxicity of the registration substance is derived based on the read-across approach.

Bis (2-hydroxyethyl) coco alkylamine CAS 71786 -60 -2, was tested for its repeated dose toxicity according to OECD 422 Test Guideline. The rats were treated at doses of 0, 10, 30 and 125 mg/kg/day for up to forty five days. The irritant effect such as salivation and histopathological alteration in the gastric tract was evident at 125 mg/kg/day. 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 at 125 mg/kg/day the NOAEL of 30 mg/kg/day was derived for the systemic toxicity.

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