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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 May 2017 - 11 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Cross-reference
Reason / purpose:
reference to same study
Reference

NOAEL (Reproductive toxicity) = 100 mg/kg /day; OECD 422; N. Barraclough. (2018)

Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The endpoint is concluded based on a single key study with a Klimisch rating of 1. The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band.
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available

In an OECD 422 study, following successful formulation analysis, Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine  was administered via oral garage once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle]), 30, 50, or 100 mg/kg/day test item. The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg.

Observations from the study resulted in test item related findings at all dose levels which were considered non-adverse. The no observed adverse effect level (NOAEL) for male and female systemic toxicity was considered as 100 mg/kg/day.  A no observed effect level (NOEL) was considered as 30 mg/kg/day for the males, although a NOEL for females could not be established, due to the lower thymus weights observed in all dose groups. An NOAEL for development was established to be 100 mg/kg/d as non-adverse effects were seen in bodyweight of the pups in all doses no NOEL could be established.  Reproductive performance and offspring development were not effected adversely. As such, the NOEL and NOAEL for reproductive toxicity was considered as 100 mg/kg/day.

NOAEL (Reproductive toxicity) = 100 mg/kg /day; OECD 422; N. Barraclough. (2018)

Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The endpoint is concluded based on a single key study with a Klimisch rating of 1. The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band.
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available

No adverse effects observed for all measurable parameters on developmental and reproductive toxicity at the maximum dose tested. Since no adverse effects were observed at all dose tested, it can be concluded the test item does not cause reproductive or developmental toxicity.

The substance does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-18
Specific details on test material used for the study:
Clear, light yellow liquid
Storage: 2 to 8 °C, in the dark
Expiry date: 01-Jan-19

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 11 to 12weeks
- Weight at study initiation: Males: 307.4 and 385.4 g; Females: 174.1 and 218.9 g
- Fasting period before study: Not reported
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of five by sex. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels that may have interfered with achieving the objective of the study
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels that may have interfered with achieving the objective of the study.
- Acclimation period: 16 days prior to initiation of dosing (males) or 9 days prior to initiation of smearing (females).

DETAILS OF FOOD AND WATER QUALITY: As described above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): Minimum of 15 hrs
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 15th Feb 2018 To: 13th Apr 2018

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral, by gastric gavage
Vehicle:
corn oil
Details on oral exposure:
Formulations (excluding the control group) were stirred continuously from at least 30 minutes before and throughout dosing.
The test article was administered orally by gavage.
Males were dosed for 42 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]). Females were dosed for up to 57 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, throughout gestation and until LD 13, inclusive, or 25 days post-coitum for females which did not litter. Females were sent to necropsy on LD 14 or 26 days post‑coitum).
Animal R0404 (Group 1 female) and Animals R0705 and R0707 (Group 4 females) were not dosed on GD 22/23 due to signs of parturition.
A dose volume of 5 mL/kg was used. Dose volumes were calculated using the most recent recorded body weight for each animal.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Covance Laboratories Ltd. (2018) Stand-Alone Method Validation and Stability Analysis for One Formulation Used in GLP Studies. Covance Study 8359299. Harrogate (UK): Covance Laboratories Ltd.
Suspensions of 500 mg/mL in corn oil, were found stable for 16 days at room temperature (15 to 25°C) and homogenous (Covance Study 8359299).

Formulations prepared for use in Week 1, Week 3 and Week 6 of dosing were analyzed to determine the achieved concentration. Triplicate samples of 0.5 mL were removed from the test article formulations and control formulations and were analyzed. The mean % target range for the achieved concentration of formulations was 85 to 115% of nominal. Results were within this range. Test article was not detected in Group 1 control samples.
The formulation prepared for use in Week 6 of dosing was also analyzed to determine stability when stored at 2-8 °C. For stability, the change in achieved concentration should be no greater than 15% from the initial time point. Results were within this range and therefore the dose formulations are considered stable for 5 days when stored at 2-8 °C.
The mean % target range for the preparation of the formulations was 85 to 115% of the nominal concentration.

It should be noted that due to test item properties complications arose during chemical analysis. It was therefore not possible to analyse the entire substance within the formulations. Methods to measure N,N,N',N'-tetrabutylmethylenediamine were ineffectual, which is likely due to decomposition of the substance during analysis at increased temperatures during the flame ionisation. Decreasing the temperature and changing the systems still did not allow reliable detection and quantification. It was possible to detect and quantify dibutylamine. Dibutylamine was therefore used as the analyte representative of the whole substance to ensure that the test item was correctly dosed and a range of doses was achieved for delivery to the animal. For further information see the attached report which further explains the issues.
Duration of treatment / exposure:
Males were dosed for 42 consecutive days (2 weeks prior to pairing, during pairing, and approximately 3 weeks post-pairing) and were sent to necropsy on Day 43.

Females were dosed for up to 57 days (2 weeks prior to pairing, during pairing, throughout gestation, and up to LD 13).
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control)
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 3 (Intermediate)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 4 (High)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Method: Oral, by gastric gavage. For each dose group the order of administration was all males first and then all females. Each day of treatment the starting order was alternated between males and females. It was stated in the raw data.
Frequency: Once daily
-F0 males:42 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]).
-F0 females:57 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, throughout gestation and until LD 13, inclusive, or 25 days post-coitum for females which did not litter.
-F1: Potential indirect exposure in utero and through the milk during lactation
Dose levels: The amount of test item to be administered was calculated according to the most recent body weight recorded.
Group 1: 0 mg/kg/day
Group 2: 30 mg/kg/day
Group 3: 50 mg/kg/day
Group 4: 100 mg/kg/day
Administration volume 5 mL/kg
Animal R0404 (Group 1 female) and Animals R0705 and R0707 (Group 4 females) were not dosed on GD 22/23 due to signs of parturition.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- pre-test: Twice
- treatment to lactation: weekly

BODY WEIGHT: Yes
- pre-test: once
- treatment to lactation: on day one then weekly, gestation observation Day 0, 7, 14 & 20 and on lactation day 1, 4,7,13 &14 prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- pre-test: weekly
- treatment to lactation: twice weekly (male) and daily (female) from GD 0 to 20 and LD 1 to 13.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- treatment to lactation: Daily (male) and daily (female) from GD 0 to 20.

SENSORY REACTIVITY & GRIP STRENGTH:
- Week 6 (before dosing)

MOTOR ACTIVITY:
- Week 6 (before dosing)
- Days 7 of lactation

HAEMATOLOGY: Yes
- Week 7 of dosing (Post-Pairing Day 22 for males) or Week 8/9 of dosing (LD 14 for females).

BLOOD CHEMISTRY: Yes
- Week 7 of dosing (Post-Pairing Day 22 for males) or Week 8/9 of dosing (LD 14 for females).

URINALYSIS: Yes
- Week 6 of dosing

IMMUNOLOGY: No
- Not examine

OTHER: Yes (see below);

ESTROUS CYCLE: Yes
- Dry smears - taken for 15 days before pairing (reproductive feamles only).
- Wet smears - taken for 14 days before treatment (all females); females that failed to exhibit 4-5 day cycles were not allocated to the reproductive group.
- daily after pairing until mating (reproductive females only) and on LD 14, prior to necropsy.

THYROID HORMONE ANALYSIS: Yes (T3/4 and TSH)
- Time schedule: Week 7 of dosing (Post-Pairing Day 22 for males) or Week 8/9 of dosing (LD 14 for females).
- All culled pups on PND 4
- Pups (2/sex) sacrificed on PND 13

TERMINAL INVESTIGATIONS: yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Other examinations:
Quality Assessment: five selected animals/sex/group (five males with the lowest identification numbers and the first five littered females/group). Assessments were performed during Week 6 of dosing (Post‑Pairing Day 19) for males and on LD 7 for females.
Quantitative assessment parameters are as follows:
Hind limb foot splay
Fore and/or hind limb grip strength
Statistics:
Statistical analyses (ANOVA) were performed, where appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Transient instances of raised fur were noted for up to four males administered 100 mg/kg/day and one male administered 50 mg/kg/day.
An isolated instance of hunched posture was recorded from GD 5 to GD 7 for one female administered 100 mg/kg/day (Animal R0710). One male administered 100 mg/kg/day was observed with a tilted head and was sent to necropsy on Day 15. One female administered 100 mg/kg/day was observed with protruding eyes on LD 13 and 14. These findings were isolated and, as such, were considered unrelated to test article toxicity.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male administered 100 mg/kg/day (Animal R0306) died on day 15 due to congenital abnormality in the brain.
At 50 mg/kg/day two female R0602 and R0603 found dead on GD 8 and 12, respectively. At necropsy, no macroscopic abnormalities were recorded for Animal R0602 but Animal R0603 had dark lungs, a distended caecum, and an enlarged and dark thymus.
One control female (Animal R04010), one female administered 30 mg/kg/day (Animal R0502) and one female administered 50 mg/kg/day (Animal R0610) did not produce a litter; as such, they were sent to necropsy on GD 26.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Actual body weight loss and lower body weight gains were observed during the first week of dosing for males administered 50 or 100 mg/kg/day (P < 0.01 and P < 0.001 respectively), compared with controls. Overall body weight gain for test article-treated males over the study period was, however, essentially similar to controls.
Lower body weight gains were noted from the 1st week of dosing for females administered 100 mg/kg/day (-17.8 compared with controls) and were still evident for the remainder of the study. Overall body weight gain from the start to end of dosing (LD 13) was significantly lower for females administered 100 mg/kg/day compared with controls (P < 0.001).
No test article-related body weight changes were evident for females administered 30 or 50 mg/kg/day or males administered 30 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower mean food consumption was noted during the first 2 weeks of dosing for males administered 50 or 100 mg/kg/day i.e ‑11 or ‑18% respectively. The mean food consumption over the study duration was approximately -10% compared with controls. Similar effects was also observed in female at these doses, (approximately -11%), compared with controls. The mean overall food consumption during the study, for females administered 100 mg/kg/day was 12% lower than controls.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Alkaline phosphatase activity was significantly elevated in females administered 100 mg/kg/day, compared with controls (P < 0.001); total protein and globulin levels were significantly reduced in this group (P < 0.05), with a non-significant increase in A:G ratio, compared with controls.

No effects observed in males.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Higher incidence of decreased activity for males administered 100 or 50 mg/kg/day, compared with controls.
Hind limb foot splay was shorter for males administered 100 mg/kg/day, compared with controls, with statistically significant differences noted for 1 of 2 tests (P < 0.001).

The mean number of rears was reduced during lactation for females administered 100 mg/kg/day, compared with control (LD 1: P < 0.05; LD 7: P < 0.01, LD 13: P < 0.001). Similar effects were also evident following administration of 50 mg/kg/day (LD 1: P < 0.05; LD 7 and 13: P < 0.01), compared with control.

Locomotor activity was unaffected by test article administration.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test article-related decreases in thymus weights were recorded for females in all groups administered the test article; increases in liver weights were recorded for females administered 50 or 100 mg/kg/day, and increases in kidney weights were recorded for males administered 100 mg/kg/day, compared with concurrent controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Kidneys of males administered 100 mg/kg/day, increased severity of hyaline droplets characterized by small, generally round eosinophilic droplets in the proximal tubular epithelium were observed.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Test article-related effects observed were considered not to have represented adverse health effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 4: Summary of Absolute Organ Weights - Terminal Sacrifice

Organ

Dose level mg/kg/day

Male

Female

2M

3M

4M

2F

3F

4F

30

50

100

30

50

100

Thymus

group mean absolute weights 

0.309

0.350

0.320

0.208

0.171

0.159

% total body weight

0.079

0.087

0.086

0.082

0.070

0.063

Liver

group mean absolute weights 

9.584

9.633

9.772

9.603

9.781

10.255

% total body weight

2.467

2.399

2.634

3.781

4.018

4.100

Kidney

group mean absolute weights 

2.410

2.357

2.492

1.714

1.647

1.693

% total body weight

0.620

0.590

0.671

0.676

0.676

0.677

F = Female; M = Male.

Note: Organ weights were not taken from decedent animals.

Table 5. Incidence of Selected Findings; Kidney - Terminal Sacrifice

Tissue and finding

Dose level mg/kg/day

Male

Female

1M

2M

3M

4M

1F

2F

3F

4F

30

 

50

100

30

 

50

100

Kidney

No. examined:

5

5

5

5

5

-

-

5

hyaline droplets

Grade -

-

-

-

-

5

-

-

5

1

5

5

5

1

-

-

-

-

2

-

 

-

4

-

-

-

-

- = Finding not present; 1 = Minimal; 2 = Slight; 3 = Moderate; F = Female; M = Male.

Table 3. summary of mean body weight by day (g)

Group

Male

Female

Predose

Day: 1

Session 1

Pre‑pairing

Day: 1

Session 1

Day: 8

Session 1

Day: 15

Session 1

Pairing

Day: 7

Session 1

Post Pairing

Day: 8

Session 1

Day: 15

Session 1

Day: 21

Session 1

Predose

Day: 8

Session 1

Pre‑pairing

Day: 1

Session 1

Day: 8

Session 1

Day: 15

Session 1

Gestation

Day: 0

Session 1

Day: 7

Session 1

Day: 14

Session 1

Day: 20

Session 1

Lactation

Day: 1

Session 1

Day: 4

Session 1

Day: 7

Session 1

Day: 13

Session 1

Control

333.7

356.3

371.1

382.9

  389.1

403.3

414.1

413.7

180.4

189.4

  195.1

204.3

204.6

  225.3

252.3

309.3

238.8

247.2

254.4

271.0

2

323.5

343.6

355.8

370.4

377.0

389.2

401.4

404.1

185.9

194.1

201.2

210.3

211.1

  227.9

  251.3

310.9

245.6

252.6

261.0

277.0

 

3

331.4

353.0

360.2

377.3

382.3

400.0

  413.0

413.6

185.2

191.1

197.1

205.8

205.0

  224.6

253.2

312.4

238.3

246.4

256.1

272.0

4

323.9

345.3

344.2

355.6

362.9

381.4

  394.4

398.6

191.3

  200.2

204.9

213.6

  209.5

228.4

  253.0

306.0

240.7

244.2

252.1

261.7

Table 5. Summary of mean body weight gain per interval (g)

Group

Male

Female

PA:1‑PA:8

PA:8‑PA:15

PA:15‑PR:7

PR:7‑PP:8

PP:8‑PP:15

PP:15‑PP:21

 

PA:1‑PP:21

PA:1‑PA:8

PA:8‑PA:15

 

GD:0‑GD:7

GD:7‑GD:14

GD:14‑GD:20

GD:20‑LA:1

LA:1‑LA:4

LA:4‑LA:7

LA:7‑LA:13

 

PA:1‑LA:13

Control

14.8

11.8

6.2

14.2

  10.8

-4

57.4

 

5.7

9.2

20.8

  26.9

57.0

  ‑70.5

8.4

7.3

16.6

81.5

2

12.2

14.5

6.6

12.2

12.2

2.8

60.5

7.1

9.0

16.8

23.4

59.6

  ‑65.3

7.0

8.4

16.0

84.1

3

7.2+H

17.1

5.0

17.7

13.1

0.5

  60.5

5.9

8.7

19.6

26.9

59.2

‑74.1

8.1

9.8

15.9

79.1

4

‑1.1#H

11.3

5.9

18.5

13.0

4.2

  56.7

4.7

8.7

18.9

24.6

53.0

‑65.3

3.5

7.9

9.6

61.5#r

 

PA ‑ Pre‑pairing,   PR – Pairing, PP ‑ Post Pairing,  LA – Lactation, GD ‑ Gestation    

#r = Wilcoxon rank Sum Test Significant at 0.001 level, +H = Dunnett Exact Homogeneous Test Significant: 0.01 level,   #H = Dunnett Exact Homogeneous Test Significant: 0.001 level

Table 6. Summary of mean food consumption

Group

Male

Female

Pre‑pairing

Post Pairing

Pre‑pairing

Gestation

Lactation

1 ‑ 4

4 ‑ 8

8 ‑ 11

11 ‑ 15

1 ‑ 6

6 ‑ 8

8 ‑ 11

11 ‑ 15

15 ‑ 18

18 ‑ 21

1 ‑ 4

4 ‑ 8

8 ‑ 11

11 ‑ 15

GD:8‑GD:9

GD:9‑GD:10

GD:10‑GD:11

GD:11‑GD:12

GD:12‑GD:13

GD:13‑GD:14

GD:14‑GD:15

GD:15‑GD:16

 

GD:16‑GD:17

GD:17‑GD:18

GD:18‑GD:19

GD:19‑GD:20

LA:1‑LA:2

LA:2‑LA:3

LA:3‑LA:4

LA:4‑LA:5

 

LA:5‑LA:6

LA:6‑LA:7

LA:7‑LA:8

LA:8‑LA:9

LA:9‑LA:10

LA:10‑LA:11

LA:11‑LA:12

LA:12‑LA:13

1

19.2

21.2

18.7

18.0

18.1

17.6

18.7

8.6

17.8

17.8

 

13.5

 14.9

 13.5

13.9

 

12.7

15.7

16.0

14.6

16.2

16.1

16.1

15.7

16.8

16.1

17.5

22.1

17.8

23.3

25.1

19.3

19.1

19.9

       20.2

21.6

28.1

28.1

       32.3

34.6

2

18.7

18.7

17.3

16.9

 17.0

 16.3

 17.2

6.2

16.6

 17.5

15.6

14.2

 12.5

 15.4

 

       14.1

15.0

16.0

14.0

16.2

14.8

15.3

16.6

16.0

16.4

16.8

17.8*H

16.2

19.3

25.1

21.5

19.8

18.2

20.5

21.4

23.6

29.1

       32.3

34.3

3

17.8

17.7

17.3

 15.7

 17.1

16.7

17.6

  5.9

 15.8

17.3

11.2

13.2

11.4

 13.7

14.0

14.6

       14.3

14.9

16.3

16.4

15.2

16.1

15.2

       17.1

17.3

       18.8

15.4

19.4

       18.1

19.4

22.6

20.2

18.9

21.1

22.7

27.2

       32.4

27.8

4

16.6

15.4

15.1

 16.2

17.8

17.3

 

 18.9

  5.7

18.4

18.9

  12.1

12.6

11.6

12.7

11.4

13.8

       13.8

14.3

       15.5

14.7

14.9

15.8

14.7

14.5

16.0

17.4*H

       16.4

20.8

17.3

*H = Dunnett Exact Homogeneous Test Significant:  0.05 level

@ = Number examined reduced due to excluded data

Table 7. Summary of Fertility and Reproductive indices

Treatment Group

Control

30 mg/kg

50 mg/kg

100 mg/kg

Total males

10

10

10

10

Unscheduled Deaths Prior to Cohabitation

 0

 0

 0

 0

Males Cohabitated

10

10

10

10

Unscheduled Deaths Prior to Cohabitation

0

0

0

1

Males mating with at least 1 female

10

10

10

9

Males impregnating at least 1 female

10

9

9

9

Mating Index (%)

100

100

100

90

Fecundity Index (%)

100

90

90

100

Fertility Index (%)

100

90

90

90

Total Females

10

10       

10

10       

Unscheduled Deaths Prior to Cohabitation

0

0

0

0

Females Cohabited

10

10       

10

10       

Unscheduled Deaths During Cohabitation

0

0

0

0

Females Mated

10

10       

10

10       

Pregnant Females

10

9

9

10

      Found Dead

0

0

2

0

Non Pregnant Females

0

1

1

0

Matings Per Day Periods Of Cohabitation – Day 1

 

2

2

2

1

Matings Per Day Periods Of Cohabitation – Day 2

3

5

6

4

Matings Per Day Periods Of Cohabitation – Day 3

1

2

2

1

Matings Per Day Periods Of Cohabitation – Day 4

4

1

0

2

Matings Per Day Periods Of Cohabitation – Day 6

0

0

0

1

Matings Per Day Periods Of Cohabitation – Day 7

0

0

0

1

Mating Index %

100

100

100

100

Fecundity Index %

100

90

90

100

Fertility Index %

100

90

90

100

Summary of Mean Parturition and Litter Data

 

Duration of gestation (days)

23.3

23.1

23.1

23.1

Number of implantation sites

11.4

12.1

11.6

10.9

Number of pups born

10.3

10.6

11.1

9.4

Number of pups alive PND 1

10.3

10.6

10.4

9.2

% male pups PND 1

49.1

56.0

52.9

48.4

Number of pups alive PND 4 before culling

10.3

10.6

10.4

9.1

Number of pups culled PND 4

1.4

1.0

1.0

0.2

Number of pups alive PND 4 after culling

8.9

9.6

9.4

8.9

Number of pups alive PND 7

8.9

9.6

8.9

8.9

Number of pups alive PND 13

8.9

9.6

8.9

8.9

Summary of Pup Survival (mean value)

Post-implantation survival index %

93.9

87.4

96.3

86.7

Live birth index %

94.4

100.0

94.9

98.2

Survival index PND 1-4 %

100.0

100.0

100.0

98.6

Survival index PND 4-7 %

100.0

100.0

94.3

100.0

Survival index PND 7-13 %

100.0

100.0

100.0

100.0

Summary of Pup Clinical Observations (Number of litters with sign)

Abdomen; mass:

 

 

1

 

Culled

5

6

4

2

Discoloration: body

 

 

 

1

Front legs; deformed, limited mobility

 

 

1

 

Found dead.

1

 

1

1

Front paws: swollen

1

 

 

 

Front leg; bent: right

1

 

 

 

Hemorrhagic area: neck

 

 

1

 

Hind leg; bent: inwards, left.

1

 

 

 

Hind leg; facing inwards, left

1

 

 

 

Mis-sexed

 

2

1

1

Missing, presumed cannibalized

 

 

3

1

Nose; sore

 

1

 

 

pale

 

 

 

1

Respiration: noisy

 

 

 

1

Small

 

2

2

 

Sores/lesion: mouth and head.

 

 

 

1

Sent to necropsy

1

 

1

2

Thin

1

 

 

1

Tail; bent

1

 

 

 

Tail; damaged

 

 

 

1

 

Unfed

 

 

1

 

Summary of Functional Observational Battery in male

 

Mild vocalization

 

1

2

2

1

Moderate vocalization

1

1

 

 

Mild decreased activity

2

1

2

1

Moderate decreased activity

 

1

 

4

Severe decreased activity

 

 

1

 

Posture (body/head)

tilting, head, severe, right

 

 

 

1

Behavior ‑ other

head shaking, occasional

 

 

 

1

Behavior ‑ other

jaw chomping

 

 

1

1

Table 8. Summary of Functional Observattional Battery in male

Group

Latency sec

Rears

F boli

Ur pools

Pre-Dose

Pre‑pairing

Pairing

Post Pairing

Pre-Dose

Pre‑pairing

Pairing

Post Pairing

Pre-Dose

Pre‑pairing

Pairing

Post Pairing

Pre-Dose

Pre‑pairing

Pairing

Post Pairing

Day 5

Day 5

Day 12

Day 4

Day 5

Day 12

Day 19

Day 5

Day 5

Day 12

Day 4

Day 5

Day 12

Day 19

Day 5

Day 5

Day 12

Day 4

Day 5

Day 12

Day 19

Day 5

Day 5

Day 12

Day 4

Day 5

Day 12

Day 19

1

0.0

3.0

1.0

0.0

1.0

1.0

0.0

5.0

4.0

2.0

3.0

4.0

5.0

1.0

2.0

1.0

1.0

0.0

0.0

1.0

1.0

1.0

0.0

0.0

0.0

0.0

0.0

1.0

2

0.0

3.0

2.0

1.0

1.0

3.0

1.0

5.0

4.0

3.0

3.0

4.0

5.0

7.0

1.0

1.0

0.0

0.0

0.0

0.0

1.0

1.0

0.0

0.0

0.0

0.0

0.0

0.0

3

0.0

5.0

14

2.0

1.0

1.0

1.0

6.0

2.0

1.0

1.0

4.0

3.0

3.0

3.0

2.0

 

0.0

1.0

1.0

1.0

1.0

0.0

0.0

0.0

0.0

0.0

0.0

4

0.0

7.0

5**

3.0

1.0

2.0

1.0

6.0

1.0

1.0

1.0

3.0

2.0

3.0

1.0

1.0

0.0

0.0

0.0

1.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Table 9. Summary of Functional Observational Battery in female

oup

Latency sec

Rears

F Boli

Ur pools

Pre-Dose

Pre‑pairing

Pairing

Gestation

Lactation

Pre-Dose

Pre‑pairing

Pairing

Gestation

Lactation

Pre-Dose

Pre‑pairing

Pairing

Gestation

Lactation

Pre-Dose

Pre‑pairing

Pairing

Gestation

 

D13

D6

D13

D5

D0

D7

D14

D20

D1

D7

D13

D13

D6

D13

D5

D0

D7

D14

D20

D1

D7

D13

D13

D6

D13

D5

D0

D7

D14

D20

D1

D7

D13

D13

D6

D13

D5

D0

D7

D14

D20

 

1

0.0

0.0

2.0

-

1.0

1.0

1.0

1.0

0.0

1.0

0.0

8.0

9.0

6.0

-

7.0

8.0

5.0

 

9.0

8.0

9.0

6.0

1.0

0.0

-

1.0

0.0

0.0

0.0

0.0

0.0

0.0

1.0

1.0

0.0

-

0.0

0.0

0.0

0.0

 

2

0.0

0.0

3.0

-

1.0

3.0

2.0

2.0

0.0

0.0

1.0

7.0

5*

5.0

-

3.0

4.0

2*

1.0

7.0

5.0

7.0

4.0

1.0

1.0

-

0.0

0.0

0.0

0.0

0.0

0.0

0.0

1.0

1.0

0.0

-

1.0

0.0

0.0

0.0

 

3

0.0

0.0

2.0

-

0.0

1.0

2.0

2.0

0.0

0.0

1.0

7.0

8.0

5.0

-

3.0

3*

2*

2.0

1.0

4*

3**

2**

0.0

0.0

-

0.0

1.0

0.0

0.0

0.0

0.0

0.0

1.0

1.0

1.0

-

1.0

1.0

0.0

0.0

 

4

0.0

1.0

1.0

3.0

0.0

2.0

4*

4.0

0.0

0.0

0.0

7.0

5*

5.0

4.0

3.0

4.0

2.0

2.0

1.0

4*

3**

2***

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

1.0

0.0

1.0

0.0

0.0

0.0

0.0

 

 

Lactation

 

D1

D7

D13

* P<=0.05

** P<=0.01

*** P<=0.001

 

 

1

1.0

0.0

0.0

 

2

0.0

0.0

0.0

 

3

1.0

1.0

0.0

 

4

0.0

0.0

Table 10. Summary of Hematology

Male

Group

HB g/dL

RBC 10E12/L

PCV %

MCV fL

MCH pg

MCHC g/dL

 

RETA %

RABS 10E9/L

RDW %

HDW g/dL

WBC 10E9/L

N 10E9/L

 

L 10E9/L

M 10E9/L

E 10E9/L

B 10E9/L

LUC. 10E9/L

N% %

L% %

M% %

E% %

B% %

LUC% %

PLT 10E9/L

PCT %

MPV fL

PDW %

PT sec

APTS sec

FIB g/L

1

14.1

8.27

43.9

53.1

17.0

32.0

2.0

163.9

13.3

2.48

4.9

1.15

 

3.46

0.10

0.14

0.0

0.03

24

71

2

3

0

1

753

0.56

7.4

53.2

21.9

17.8

1.45

 

2

14.1

8.38

44.1

52.6

16.9

32.0

2.2

179.6

12.6

2.53

6.4

1.22

 

4.92

0.12

 0.08

0.01

0.04

  19

 

77

2

1

0

1

738

 

0.56

7.6

53.5

PT sec

17.4

1.46

3

14.1

8.43

44.0

52.2

16.7

32.0

2.0

168.4

12.9

2.55

5.6

1.07

 

 4.29

0.13

 0.09

0.01

0.03

20

75

2

2

0

0

725

0.55

7.6

55.2

26.2

18.3

1.45

4

13.8

8.34

43.5

52.2

16.5

31.7

2.2

189.4

13.0

2.52

6.7

1.58

 

4.86

0.17

 0.06

0.01

0.04

23

73

2

1*

0

1

722

 

0.54

7.5

54.3

22.0

17.4

1.44

Female

group

HB g/dL

RBC 10E12/L

PCV %

MCV fL

MCH pg

MCHC g/dL

 

RETA %

RABS 10E9/L

RDW %

HDW g/dL

WBC 10E9/L

N 10E9/L

 

L 10E9/L

M 10E9/L

E 10E9/L

B 10E9/L

LUC. 10E9/L

N% %

L% %

M% %

E% %

B% %

LUC% %

PLT 10E9/L

PCT %

MPV fL

PDW %

PT sec

APTS sec

FIB g/L

1

 14.3

7.51

43.8

58.4

19.1

32.8

 

3.7

276.6

13.6

1.88

4.8

2.04

 

2.51

0.21

0.04

0.0

0.03

42

52

5.0

1.0

0.0

1.0

965

0.68

7.0

47.5

23.8

16.0

1.64

2

 14.4

7.67

44.1

57.5

18.7

32.5

 

3.7

280.4

12.5

1.82

5.1

1.95

2.84

0.18

0.06

0.0

0.04

43

56

4.0

1.0

0.0

1.0

977

0.69

7.1

49.7

23.8

16.3

1.65

3

 14.4

7.73

43.2

55.9

18.6

33.3

4.0

 312.3

14.0

1.96

5.1

2.22

 

2.62

0.18

0.06

0.0

0.03

34

52

4.0

1.0

0.0

1.0

915

0.64

7.0

53.4

25.0

17.1

1.69

4

 14.4

7.48

43.6

58.3

19.3

33.1

3.6

266.0

14.0

1.97

6.1

2.45

3.25

0.27

0.08

0.0

0.04

40

54

5.0

2.0

0.0

1.0

981

0.68

6.9

49.3

22.9

16.1

1.62

Key to abbreviations

ode

Parameter         

Method of determination

HB

Haemoglobin

Flow Cytometry

RBC

Red Blood Cells

Flow Cytometry

PCV

Packed Cell Volume

Flow Cytometry Calculation

MCV

Mean Cell Volume

Flow Cytometry

MCH

Mean Cell Haemoglobin

Flow Cytometry Calculation

MCHC

Mean Cell Haemoglobin Concentration

Flow Cytometry Calculation

RETA

Reticulocytes %

Flow Cytometry Calculation

RABS

Absolute reticulocytes

Flow Cytometry

RDW

Red Cell Distribution Width

Flow Cytometry

HDW

Haemoglobin Distribution Width

Colorimetry

WBC

White Blood Cells

Flow Cytometry

N

Neutrophils

Calculation

L

Lymphocytes

Calculation

M

Monocytes

Calculation

E

Eosinophils

Calculation

B

Basophils

Calculation

LUC

Large Unstained Cells

Calculation

N%

Neutrophils %

Flow Cytometry

L%

Lymphocytes %

Flow Cytometry

M%

Monocytes %

Flow Cytometry

E%

Eosinophils %

Flow Cytometry

B%

Basophils %

Flow Cytometry

LUC%

Large Unstained Cells %

Flow Cytometry

PLT

Platelets

Flow Cytometry

PCT

Platelet Crit

Flow Cytometry Calculation

MPV

Mean Platelet Volume

Flow Cytometry

PDW

Platelet Distribution Width

Flow Cytometry

PT

Tox Prothrombin time

Turbidometry

APTS

Toxicology activated partial thromboplastin time - Synthasil

Turbidometry

FIB

Fibrinogen

Turbidometry

Table 11. Summary of Clinical Chemistry

Male

group

AST IU/L

ALT IU/L

HALP IU/L

CHOL mmol/L        

T.BI umol/L

TP g/L

ALB g/L

GLOB g/L

A\G RATIO

NA mmol/L

K mmol/L

CL mmol/L

 

CAL mmol/L

P mmol/L

HCRE umol/L

UREA mmol/L

GLUC mmol/L

ISBA umol/L

 

1

 14.3

74

60

83        

1.5

<1.7

59

40

  19

  2.2

 143

3.9

102

2.56

1.7

32

  7.9

9.5

2

 14.4

67

59

83

1.4

<1.7

58

40

  18

  2.3

 142

3.9

101

2.55

1.7

30

 8.5

9.4

3

 14.4

65

44

83

1.4

<1.7

58

40

  18

  2.3

 141

4.0

100*

2.54

1.8

31

8.0

9.3

4

 14.4

78

50

79

1.6

<1.7

58

39

  17

  2.4

 142

4.28

100*

2.57

1.8

32

9.1

 10.4

Female

group

AST IU/L

ALT IU/L

HALP IU/L

CHOL mmol/L        

T.BI umol/L

TP g/L

ALB g/L

GLOB g/L

A\G RATIO

NA mmol/L

K mmol/L

CL mmol/L

 

CAL mmol/L

P mmol/L

HCRE umol/L

UREA mmol/L

GLUC mmol/L

ISBA umol/L

 

1

130

64

65        

2.1

<1.7

59

35

  23

1.5

138

4.0

98

2.67

2.6

31

  9.5

8.2

 36.86

2

113

68

71

1.9

<1.7

57

36

 21

1.7

138

3.8

97

2.75

2.6

30

 10.6

8.8

 57.92

 

3

103

69

70

1.9

<1.8

57

36

  21

1.7

137

4.2

97

2.68

2.6

27

10.6

9.5

85.90

4

122

<82

111***

2.0

<1.7

55*

34

  20*

1.8

138

4.4

97

2.73

2.4

30

11.6

9.5

84.74

Key to abbreviations

Code

Parameter

Method of determination

AST

Aspartate Aminotransferase

Optimised UV method using alpha ketoglutarate without pyridoxal phosphate activation

ALT

Alanine Aminotransferase

Optimised UV method using L-Alanine and alpha‑oxoglutarate as primary substrates

HALP

Alkaline Phosphatase

Colorimetric method using p‑nitrophenyl phosphate as substrate standardised to IFCC

CHOL

Total Cholesterol

Enzymatic method using cholesterol oxidase/esterase

T.BI

Total Bilirubin

A colorimetric method. Indirect bilirubin is liberated by detergent: Total bilirubin is coupled with a diazonium compound to give corresponding azobilirubin.

TP

Total Protein

Colorimetric method using Biuret reagent

ALB

Albumin

Bromocresol Green method

GLOB

Globulin

globulin = total protein - albumin

A\G

Albumin/Globulin Ratio

Calculation

NA

Sodium

Ion‑selective electrode

K

Potassium

Ion‑selective electrode

CL

Chloride

Ion‑selective electrode

CAL

Calcium Gen 2

Photometric using 5-nitro-5'-methyl-BAPTA (NM-BAPTA)

P

Inorganic Phosphate

UV Assay utilising ammonium molybdate

HCRE

Enzymatic Creatinine

Enzymatic Colorimetric method utilising 4-aminophenazone

UREA

Urea

UV method using a coupled urease procedure

GLUC

Glucose

UV method using a coupled hexokinase procedure

ISBA

Serum Bile Acids - ILab 650

Spectrophotometric/Enzymic with Bile Acids being converted to 3-keto steroids with the production of Thio-NADH

NB: More tables containing raw data are attached in background material.

Applicant's summary and conclusion

Conclusions:
Once daily oral gavage administration of 30, 50, or 100 mg/kg/day Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) resulted in test article related findings at all dose levels which were not considered adverse in nature, therefore, the no observed adverse effect level (NOAEL) for male and female systemic toxicity was considered as 100 mg/kg/day.
Executive summary:

In an OECD 422 study, following successful formulation analysis, Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine  was administered via oral garage once a day to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation). Four groups of 10 male and 10 female sexually mature Crl:WI(Han) rats were administered 0 (control article [vehicle]), 30, 50, or 100 mg/kg/day test item. The control article (vehicle) was corn oil, and formulations were administered at a dose volume of 5 mL/kg.

Assessment of toxicity in the adults was based on clinical observations, body weights, food consumption, functional and behavioral assessments, estrous cycles, mating, fertility and pregnancy indices, and offspring parameters. For pups, clinical observations, litter size, sex, and body weights were recorded. Ano-genital distance was recorded on Postnatal Day (PND) 4, and nipple retention was recorded for male pups on PND 13. One pup/sex/litter/dose group was selected to have thyroid weights recorded and were retained in fixative.

Micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow were not induced following test article administration at any dose level.

Complete necropsy was performed on all animals (except PND 4 pups and selected PND 13 pups), and any macroscopic abnormalities were noted. Blood samples were also collected for clinical pathology  and thyroid hormone assessment. Femur from five males and five females from each dose group were also processed for micronucleus testing.

No postdose observations were evident during the first 3 days of dosing, however, transient instances of raised fur were noted for up to four males administered 100 mg/kg/day and one male administered 50 mg/kg/day.

No test item related deaths occurred but three mortality was observed; one male  in control group, a female in the 50 mg/kg/day and a male at 100 mg/kg/day .

Food consumption was also lower during the first 2 weeks of dosing in both sex at 50 or 100 mg/kg/day treatment groups as such actual body weight loss and lower body weight gains were observed during the first week of dosing. Overall body weight gain for treated males over the study period was essentially similar to that of controls but the overall female body weight gain was lower from the start of dosing to LD 13. No adverse effect on food consumption was evident following administration of 30 mg/kg/day in both sexes. No overt differences in water consumption were noted for treated animals, compared with controls.

Following 100 mg/kg/day in males, initial body weight loss, lower body weight gains and reduced food consumption were observed. Furthermore, raised hair and shorter hind limb foot splay, with reduced activity and reduced rearing observed during the weekly behavioral assessments.  However, locomotor activities were unaffected and in the absence of any overt pathology observation, these findings were not considered as an indication of neurotoxicity. The group mean kidney weights (absolute and body weight-relative) were higher correlating with presence of hyaline droplets; this is a common response in the male rat to xenobiotics and represents accumulations of α2u globulin, a naturally occurring male rat protein. Chemicals that bind to α2u globulin form a complex that is more resistant to catabolism and will result in accumulations of hyaline droplets. This male rat-specific finding is of little relevance to risk assessment in humans.

For females administered 100 mg/kg/day; body weight gain was reduced over the duration of the study, mean absolute liver weights and body weight relative weight ratios were higher with elevated alkaline phosphatase activity, total protein and albumin:globulin (A:G) ratios. Furthermore, lower total protein and globulin levels were observed. In the absence of any overt clinical or pathological findings, these observations were considered to represent adaptive responses to administration of a xenobiotics.  In addition to the above, rearing was reduced but locomotive activities were not affected.

Following 50 mg/kg/day administration Initial body weight losses, reduced body weight gains, and lower food consumptions were observed in both sexes. For females, initial reduction in lower food consumption was evident  with reduced rearing during lactation. Finally, mean thymus weights (absolute and body weight relative) were lower for females administered at all dose levels without any correlating haematological or microscopic abnormalities.  In the absence of any associated decline in physical health, clinical pathology or microscopic changes to indicate an adverse effect, these findings were not considered treatment related.

Reproductive performance was unaffected; the test article did not affect estrous cycles, mating, fertility, pregnancy or parturition. The number and length of estrous cycles was unaffected by test article administration. Mating performance was unaffected by test article administration; all animals mated within 7 days of pairing. Ten, nine, nine, or ten females administered control article (vehicle) or 30, 50, or 100 mg/kg/day, respectively, achieved pregnancy.  Nine, nine, seven or ten dams administered control article (vehicle) or 30, 50, or 100 mg/kg/day successfully reared a live litter to LD 13. One control female (Animal R0410) had no viable fetuses (100% in utero litter losses).

No treatment related effects were noted on mean gestation lengths or the mean number of implantation sites. A slightly smaller litter size was noted following administration of 100 mg/kg/day, compared with controls (-13%).  No treatment related effect on ano-genital distance, no nipples/areolae were present for male offspring, no effects on thyroid weight or thyroid hormone levels were observed in dose groups.

Test item related offspring effects following maternal exposure of 100 mg/kg/day were confined to slightly smaller litter sizes, compared with controls, and lower mean offspring weights in litters of the groups administered 50 or 100 mg/kg/day, although mean values were ± 10% of controls. In the absence of any test article-related offspring mortality noted at this dose level, these findings were considered as non-adverse.  A medulloblastoma was present in one male administered 100 mg/kg/day (decedent Animal R0306). This tumor has been observed as a background finding in short term studies, although it is rare. As only one animal was affected, a relationship to the test article was considered unlikely. Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.

It was concluded that once daily oral gavage administration of 30, 50, or 100 mg/kg/day Reaction mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male rats for 42 consecutive days and to female rats for up to 58 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) resulted in test item related findings at all dose levels which were considered none adverse. The no observed adverse effect level (NOAEL) for male and female systemic toxicity was considered as 100 mg/kg/day.  A no observed effect level (NOEL) was considered as 30 mg/kg/day for the males, although a NOEL for females could not be established, due to the lower thymus weights observed in all dose groups.  Reproductive performance and offspring development were unaffected as such, the no observed adverse effect level (NOEL) and NOAEL for reproductive toxicity was considered as 100 mg/kg/day.