Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
08 March 2017 - 29 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Range-finding test for the full OECD 422.
Deviations:
yes
Remarks:
The deviations did not affect the outcome of the study
Principles of method if other than guideline:
The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day oral gavage study in the Crl:WI(Han) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 ºC, in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Homogenous suspension formed in corn oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspended in corn oil prior to administration
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Applied as a liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: N/A
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The rat was selected because it is a rodent species known to be accepted by various regulatory authorities.
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: 9-10 weeks
- Weight at study initiation: Males: 299.6 - 327.7 g; Females: 191 - 239.3 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 four 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 5LF2 EU Rodent Diet (International Product Supplies Ltd., London, 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: ≥13 days

DETAILS OF FOOD AND WATER QUALITY: As described above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 10 April 2017 To: 20 June 2017
Route of administration:
oral: gavage
Details on route of administration:
Test article formulations (excluding the control group) were stirred upon arrival to the animal room from at least 30 minutes prior to, and continuously throughout, dosing.

Formulations were dosed within 3 hours of preparation.

The test article was administered orally by gavage. Animals were dosed once daily for up to 14 consecutive days, excluding the day of necropsy. A dose volume of 5 mL/kg was used. Dose volumes were based on the most recent individual body weight for each animal.
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:

Test article formulations (excluding the control group) were stirred upon arrival to the animal room from at least 30 minutes prior to, and continuously throughout, dosing. Formulations were dosed within 3 hours of preparation.

Method of preparation not reported in range-finder.

- DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item formed homogenous formulation in corn oil. Guideline recommended vehicle.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Lot/batch no. (if required): Batch numbers MKBP7039V and MKBZ9899V
- Purity: Not reported, assumed 100 %
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
N/A
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Dosing stopped after 1 day
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Dosing stopped after 2 days due to 5/5 male deaths. Surviving females dosed at 175 mg/kg bw/day for 13 days (total 14 days)
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
300 mg/kg bw/day: 5 males and 5 females
250 mg/kg bw/day: 5 males and 5 females
175 mg/kg bw/day: 3 males and 5 females (5 surviving females from 250 mg/kg bw/day group and 3 spare males used as all died at 250 mg/kg bw/d)
125 mg/kg bw/day: 5 males and 5 females
50 mg/ kg bw/day: 5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A dose level of 300 mg/kg/day was considered an acceptable initial dose level, based on the preliminary findings of the acute toxicity study (Covance Study 8359313).
In the current study, due to adverse clinical observations after the first dose in two females administered 300 mg/kg/day (prostrate, respiratory pattern changes, semi closed eyes, and twitching), which resulted in their early termination, dosing was discontinued for Groups 1 and 2 after the first dose, until a further dose level was explored in the acute toxicity study.

A dose level of 50 mg/kg was well tolerated in the acute toxicity study (Covance Study 8359313), and was therefore used as the dose level to be explored.

In the absence of any effects of test article noted following administration of 50 mg/kg/day in this study, and in the absence of any findings noted in the acute toxicity study (Covance Study 8359313) following administration of 300 mg/kg, the next dose level to be investigated was 250 mg/kg/day.
Adverse effects were noted following the second dose of 250 mg/kg/day (subdued/sluggish behavior, tremors, convulsive episode, and twitching), which resulted in the death of all treated males; as such, dosing was ceased for Group 3 females, and animals were retained for possible future testing at a lower dose level.
An additional intermediate-dose level of 175 mg/kg/day was included to ensure enough information was obtained from this study for successful dose level selection for the subsequent OECD 422 study. This dose was administered for 14 days to observe any adverse effects.

In the absence of effects following administration of 50 mg/kg/day, a dose level of 125 mg/kg/day was chosen as it was anticipated to be tolerated for 14 consecutive days.

- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A
Positive control:
N/A
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Twice Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Weely (Where necessary, unscheduled clinical examinations were performed; these data were reported).

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded once weekly during the predose phase; on Days 1 and 4 (all animals) and 8, 11, and 14 (Control, 500 and 750 mg/kg/day groups only) of the dosing phase; and before each necropsy

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The amount of food consumed by each cage of animals was measured at twice weekly intervals where possible. Consumption was calculated as g/animal/day.

WATER CONSUMPTION: Yes
- Time schedule for examinations:
Daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No
Other examinations:
N/A
Statistics:
Statistical analyses were not performed.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Following administration of 175 mg/kg/day, clinical observations included red staining of the neck for two females and vocalization for two females on the final (14th) dosing occasion (Study Day 35). Postdose observations included mouth rubbing for both sexes; paddling for one female; minimal staggering, subdued/sluggish behaviour, twitching, decreased respiration, and reduced activity for one female following the 3rd and 6th dose (Study Days 24 and 27 respectively); and reduced activity and vocalization for one female after the 1st dose (Study Day 21). Postdose observations were mostly recorded immediately upon return to the cage, with the exception of some 1 animal, for which observations were recorded up to 4 hours postdose.

Clinical observations for animals administered 125 mg/kg/day were limited to mouth rubbing and (occasionally) salivation. These observations were recorded immediately after dosing upon return to the cage.

Clinical observations for animals administered 50 mg/kg/day included vocalization for two females on Day 8 or 15 of the dosing phase and postdose observations of mouth rubbing and (occasionally) paddling and salivation. These observations were recorded immediately postdose (upon return to the cage).

Mouth rubbing was also recorded for controls; however, the incidence was much lower than that observed for test article-treated animals.

Other clinical observations, including thinning fur, were considered incidental as they were observed infrequently or are commonly observed in this species.
Mortality:
mortality observed, treatment-related
Description (incidence):
Test article-related mortality was observed after a single dose for animals administered 300 mg/kg. One male and two females administered 300 mg/kg/day were found dead or were sacrificed in a moribund condition on Day 1 of the dosing phase. Clinical observations for animals administered 300 mg/kg/day included prostrate, twitching, unresponsive, semi-closed to closed eyes; and irregular, rapid, or labored respiration.

Following administration of 250 mg/kg/day, all five males were found dead or were sacrificed in a moribund condition on Day 2 of dosing. Dosing was ceased after Day 2 of the dosing phase for all remaining animals due to these deaths. Clinical observations for animals administered 250 mg/kg/day included convulsions, subdued/sluggish behaviour, prostrate, twitching (body and head), arched gait, and tremors.

Dosing of females previously administered 250 mg/kg/day recommenced on Study Day 21 at a lower dose level of 175 mg/kg/day. An additional set of three males was also added to the study as Group 5 and was administered 175 mg/kg/day. All animals administered 175 mg/kg/day survived following 14 days of dosing.

All animals administered 50 or 125 mg/kg/day survived following 14 days of dosing.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following initial body weight losses following administration of 300 or 250 mg/kg/day, after which dosing was discontinued for these animals, to ensure they were suitable for use following a wash-out period, the mean body weight of animals administered 300 or 250 mg/kg/day was generally comparable with that of controls for the remainder of the observation period. Initial body weight decreases were attributed to general debilitation.

Test article-related body weight losses were observed throughout the dosing phase for males administered 175 mg/kg/day and during the first week of dosing for females administered 175 mg/kg/day, with the body weight gain comparable with that of controls during the second week.

Test article-related body weight losses were observed during the first week of dosing for animals administered 125 mg/kg/day. Although no further body weight loss was recorded during the second week of dosing, the mean body weight gain of animals administered 125 mg/kg/day was still lower than controls.

These reductions in body weight were correlated with reduced food consumption for animals administered 125 mg/kg/day.

No effect on body weight was observed for animals administered 50 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following initial reductions in food consumption following administration of 300 or 250 mg/kg/day, after which dosing was discontinued; the mean food consumption for surviving animals was comparable with that of controls for the remainder of the observation period. The initial decrease in food consumption was attributed to general debilitation.

Lower mean food consumption was noted throughout the dosing phase, for males and females administered 175 or 125 mg/kg/day, compared with controls. Reduced mean food consumption was correlated with body weight loss or reduced body weight gain.

Mean food consumption of animals administered 50 mg/kg/day was generally comparable with controls.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
No data was present for animals administered 300 or 250 mg/kg/day due to the cessation of dosing.

Dose-related reductions in water consumption were evident with increasing dose levels for both sexes administered ≥125 mg/kg/day, compared with controls.

Mean water consumption of animals administered 50 mg/kg/day was generally comparable with that of controls.
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test article-related effects were recorded for organ weights.

Compared with concurrent controls, decreased group mean unadjusted and relative (to body weight weight) epididymis and testes weights were recorded for males administered 50 mg/kg/day. These decreased mean values were attributed to small epididymis and testes for one male. All other males of this group had epididymis and testes weights that were similar to those of controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following macroscopic abnormalities were noted:

Thick, cream coloured stomach contents (abnormal contents) were recorded for two females administered 300 mg/kg/day.

Distended jejunum was recorded for one male administered 250 mg/kg/day.

Large seminal vesicle was recorded for one male administered 175 mg/kg/day.

Red mandibular lymph node was recorded for one female administered 125 mg/kg/day.

Following administration of 50 mg/kg/day, pale and/or mottled liver findings were recorded for one male and one female; red thymus was recorded for one female, and small epididymis and testes were recorded for one male.

Findings in these tissues were observed in few animals or lacked a dose-response; therefore, the relationship to the test article was uncertain.
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
N/A
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A

Table 3      Mean body weights and body weight gains/ losses

Sex / Day

Bodyweight (g)

Control

300 / 50 ppm

250 / 175 ppm

125 ppm

175 ppm

MALES

Pre-dose Day 1

280.7

284.9

325.7

-

300.1

Pre-dose Day 2

-

-

-

351.7

-

Pre-dose Day 8

-

-

347.5

-

334.9

Pre-dose Day 15

-

-

369.3

396.5

354.4

Pre-dose Day 22

-

-

-

-

365.7

Pre-dose Day 26

-

-

-

-

372.0

Pre-dose Day 29

-

-

-

-

377.7

Pre-dose Day 30

-

-

-

436.7

-

Pre-dose Day 35

-

-

422.8

448.0

404.0

Dosing Phase

P1

P2

P1

P2

P1

P2

P1

P2

P1

P2

Dosing Day 1

310.3

365.0

314.9

367.7

-

418.8

-

451.4

-

430.7

Dosing Day 2

310.2

-

310.1

-

-

-

-

-

-

-

Dosing Day 3

316.3

-

311.4

-

-

-

-

-

-

-

Dosing Day 4

(Change from Day 1 (P2))

318.4

375.6

(10.6)

314.2

375.0

(7.3)

-

-

-

444.2

(-7.2)

-

409.7

(-21.0)

Dosing Day 5

320.5

-

319.2

-

-

-

-

-

-

-

Dosing Day 6

322.1

-

321.2

-

-

-

-

-

-

-

Dosing Day 7

326.5

-

325.3

-

-

-

-

-

-

405.3

Dosing Day 8

(Change from Day 1 (P1) or Day 4 (P2))

332.0

(21.8)

390.1

(14.5)

331.0

(15.2)

387.7

(12.8)

-

-

-

437.6

(-6.6)

-

404.6

(-5.2)

Dosing Day 9

333.0

-

332.8

-

-

-

-

-

-

-

Dosing Day 10

337.4

-

336.2

-

-

-

-

-

-

-

Dosing Day 11

(Change from Day 8 (P2))

341.6

392.1

(2.1)

343.0

387.5

(-0.2)

-

-

-

432.1

(-5.5)

-

404.0

(-0.5)

Dosing Day 12

344.7

-

349.2

-

-

-

-

-

-

-

Dosing Day 13

343.2

-

345.5

-

-

-

-

-

-

-

Dosing Day 14

(Change from Day 11 (P2))

345.0

399.1

(7.0)

346.7

396.7

(9.2)

-

-

-

437.6

(5.5)

-

401.3

(-2.8)

Dosing Day 15

(Change from Day 8 (P1))

351.7

(19.7)

-

353.6

(22.6)

-

-

-

-

-

-

-

Dosing Day 16

348.5

-

357.8

-

-

-

-

-

-

-

Dosing Day 17

354.3

-

358.0

-

-

-

-

-

-

-

Dosing Day 18

356.6

-

357.8

-

-

-

-

-

-

-

Dosing Day 19

363.1

-

362.5

-

-

-

-

-

-

-

Dosing Day 20

359.2

-

364.4

-

-

-

-

-

-

-

Dosing Day 21

(Change from Day 15 (P1))

364.9

(13.2)

-

366.9

(13.3)

-

-

-

-

-

-

-

Dosing Day 22

(Change from Day 21 (P1))

370.1

(5.2)

-

372.1

(5.3)

-

-

-

-

-

-

-

Change over course of dosing

59.8

34.1

56.4

29.0

-

-

-

-2.8

-

-29.5

FEMALES

Pre-dose Day 1

191.0

-

189.4

-

182.3

-

-

-

-

-

Pre-dose Day 2

-

-

-

-

-

-

217.3

-

-

-

Pre-dose Day 8

-

-

-

-

194.3

-

-

-

-

-

Pre-dose Day 15

-

-

-

-

202.2

-

234.4

-

-

-

Pre-dose Day 22

-

-

219.8

-

-

-

-

-

-

-

Pre-dose Day 26

-

-

224.3

-

-

-

-

-

-

-

Pre-dose Day 29

-

-

222.5

-

-

-

-

-

-

-

Pre-dose Day 30

-

-

-

-

-

-

238.2

-

-

-

Pre-dose Day 35

-

-

-

-

225.1

-

242.0

-

-

-

Dosing Phase

P1

P2

P1

P2

P1

P2

P1

P2

P1

P2

Dosing Day 1

201.2

220.5

208.3

221.6

-

222.9

-

243.5

-

-

Dosing Day 2

201.7

-

199.0

-

-

-

-

-

-

-

Dosing Day 3

200.2

-

198.5

-

-

-

-

-

-

-

Dosing Day 4

(Change from Day 1 (P2))

203.6

226.1

(5.7)

203.3

223.1

(1.6)

-

218.0

(-4.9)

-

241.1

(-4.9)

-

-

Dosing Day 5

203.2

-

201.2

-

-

-

-

-

-

-

Dosing Day 6

204.5

-

201.4

-

-

-

-

-

-

-

Dosing Day 7

204.5

-

205.7

-

-

-

-

-

-

-

Dosing Day 8

(Change from Day 1 (P1) or Day 4 (P2))

207.1

(5.9)

231.3

(5.1)

210.7

(8.8)

231.9

(8.8)

-

226.3

(8.3)

-

239.7

(-1.4)

-

-

Dosing Day 9

207.6

-

210.1

-

-

-

-

-

-

-

Dosing Day 10

209.3

-

211.0

-

-

-

-

-

-

-

Dosing Day 11

(Change from Day 8 (P2))

211.7

228.2

(-3.1)

214.5

234.1

(2.1)

-

221.3

(-5.0)

-

238.2

(-1.6)

-

-

Dosing Day 12

213.6

-

214.3

-

-

-

-

-

-

-

Dosing Day 13

212.6

-

213.0

-

-

-

-

-

-

-

Dosing Day 14

(Change from Day 11 (P2))

212.9

235.0

(6.9)

212.4

234.1

(0.1)

-

228.5

(7.3)

(5.6)*

-

239.7

(1.5)

-

-

Dosing Day 15

(Change from Day 8 (P1))

213.0

(5.9)

-

218.5

(7.8)

-

-

-

-

-

-

-

Dosing Day 16

212.6

-

221.1

-

-

-

-

-

-

-

Dosing Day 17

214.2

-

219.6

-

-

-

-

-

-

-

Dosing Day 18

218.8

-

218.4

-

-

-

-

-

-

-

Dosing Day 19

216.3

-

223.1

-

-

-

-

-

-

-

Dosing Day 20

218.0

-

223.2

-

-

-

-

-

-

-

Dosing Day 21

(Change from Day 15 (P1))

225.0

(12.0)

-

224.6

(6.1)

-

-

233.6

-

-

-

-

Dosing Day 22

(Change from Day 21 (P1))

228.2

(3.2)

-

221.9

(-2.7)

-

-

-

-

-

-

-

Dosing Day 24

(Change from Day 21 (P2))

-

-

-

227.4

(-6.2)

-

-

Dosing Day 27

-

-

-

226.5

-

-

Dosing Day 28

(Change from Day 24 (P2))

-

-

-

225.4

(-2.0)

-

-

Dosing Day 31

(Change from Day 28 (P2))

-

-

-

224.2

(-1.2)

-

-

Dosing Day 34

(Change from Day 31 (P2))

-

-

-

228.7

(4.4)

(-5.0)^

-

-

Change over course of dosing

27.0

14.6

20.7

12.6

-

-

-

-3.8

-

-

 * change from Day 1 (i.e. 14 days)

^ change from Day 21 (i.e. 14 days)

 

Table 4      Unadjusted organ weights (relative to body weight %)

 

Doses (ppm)

Control

50 ppm

125 ppm

175 ppm

Males

Number of animals/group

5

5

5

3

BODY WEIGHT (termination weight for toxicity males)

Weight (g)

379.0

377.3

433.1

393.9

EPIDIDMYSIS (termination weight for toxicity males)

Weight (g)

(difference vs control %)

1.285

 

1.258

(-2)

1.468

(14)

1.445

(12)

% body weight

(difference vs control %)

0.3397

 

0.3365

(-1)

0.3399

(0)

0.3661

(8)

TESTIS (termination weight for toxicity males)

Weight (g)

(difference vs control %)

3.482

 

3.124

(-10)

3.820

(10)

3.697

(6)

% body weight

(difference vs control %)

0.9220

 

0.8370

(-9)

0.8851

(-4)

0.9380

(2)

Conclusions:
Once daily oral gavage administration of 50, 125, 175, 250, or 300 mg/kg/day Reaction Mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male and female rats resulted in mortality following 250 or 300 mg/kg/day administration.

Dose levels of 125 or 175 mg/kg/day resulted in test article-related effects including body weight losses and reduced food and water consumption, which would not be tolerated over longer dosing duration.

Based on the findings of this 14 day repeated dose study, dose level of 30, 50 and 100 mg/kg/day are suggested for the following OECD 422 study.
Executive summary:

Reaction Mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine was administration to the rat once daily via oral garvage for up to 14 days and to provide the basis for the selection of dose levels for a subsequent OECD 422 study. In dosing phase one, groups of rats (5/sex) were administered 300 mg/k/day test item due to two deaths observed, dosing was stopped allowing the test item formulations to wash out from the remaining animals, phase two dosing was then launched at doses of 50 and 250 mg/kg/day. Dosing stopped after Day 2 at 250 mg/kg/day, following the deaths of all Group 3 males. Females were retained on the study, and dosing recommenced at a lower dose level of 175 mg/kg/day from Day 21 until Day 34 of the dosing phase. Group 5 animals were dosed at the same dose level, using the 3 remaining spare males.

Clinical observations for animals administered 250 or 300 mg/kg/day included one or more of the following: prostrate, twitching, convulsions, tremors, subdued/ sluggish (slow/deliberate movements), unresponsive, semi-closed to closed eyes; and/or irregular, rapid, or labored respiration. In addition, these animals showed body weight loss and low food or water consumption.

All animals administered 50, 125, or 175 mg/kg/day survived to their scheduled sacrifice. Postdose observations for animals administered 50, 125, or 175 mg/kg/day included mouth rubbing, (occasionally) salivation, and paddling. For one female administered 175 mg/kg/day, minimal staggering, subdued/sluggish behavior, twitching, and decreased respiration were observed on one occasion. Reduced activity was observed on one occasion for two females administered 175 mg/kg/day. Other clinical observations included vocalization or red staining of the neck for animals administered 50 or 175 mg/kg/day; these observations were transient and considered not adverse. Test article-related body weight loss was observed throughout the dosing phase for males administered 175 mg/kg/day and during the first week of dosing for females administered 175 mg/kg/day. During the second week of dosing, body weight gain was comparable with controls for females administered 175 mg/kg/day.

For animals administered 125 mg/kg/day, test article-related body weight loss was observed during the first week of dosing, followed by reduced body weight gain during the second week of dosing.

Macroscopic observations were recorded in one or two animals from each test article-treated group (excluding controls). These consisted of changes in the stomach (abnormal contents), jejunum (distended), liver (pale and mottled), mandibular lymph nodes (red), seminal vesicles (large), thymus (red), epididymis (small), and testes (small). In the absence of a dose-response, the relationship to test article was uncertain.

In conclusion, once daily oral gavage administration of 50, 125, 175, 250, or 300 mg/kg/day Reaction Mass of N,N,N',N'-tetrabutylmethylenediamine and dibutylamine to male and female rats resulted in mortality following 250 or 300 mg/kg/day administration. Dose levels of 125 or 175 mg/kg/day resulted in test article-related effects including body weight losses and reduced food and water consumption, which would not be tolerated over longer dosing duration. Based on the findings of this 14 day repeated dose study, dose level of 30, 50 and 100 mg/kg/day are suggested for the following OECD 422 study.

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.
Principles of method if other than guideline:
An integrated standard genotoxicity assessment into a general toxicology study (OECD, 2016) & ICH S2(R1) (2011) methodology was used during this test.
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-18

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
Vehicle:
corn oil
Details on 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.
Details on mating procedure:
Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: Weekly on Day 7 and 15, or until mating is confirmed.
Daily checks for evidence of mating: Ejected copulation plugs. Sperm within vaginal smear or a retained vaginal plug.
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Females which have not shown evidence of mating by Day 10 of the pairing period will be paired with proven males of the same treatment group. Likewise, males not showing any evidence of mating by Day 10 of the pairing period may be paired with untreated females.
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 in Section 7.5.1.
Duration of treatment / exposure:
5-8 weeks
Frequency of treatment:
Once daily
Details on study schedule:
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.
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 (Imtermediate)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Gropu 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

Parental animals: Observations and examinations:
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 examined

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
Oestrous cyclicity (parental animals):
ESTROUS CYCLE: Yes
-Daily vaginal lavage (washings) was conducted for all females during acclimation (predose), from 1 week after arrival until the day prior to dosing. The stage of estrous was recorded, and only females with regular 4- to 5-day cycles were included on study.
- Daily vaginal lavage was performed on females from the start of dosing until the confirmation of mating, and on LD 14, prior to necropsy.
Sperm parameters (parental animals):
Sliterections of testes and epididymides were also stained with periodic acid‑Schiff (PAS) for spermatogenic staging for all Group 1 and 4 males
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED: Yes
The following parameters were examined in [F0 / F1] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other.

GROSS EXAMINATION OF DEAD PUPS: Yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL: Yes
Postmortem examinations (parental animals):
SACRIFICE: Yes
- Male animals: Post-Pairing Day 22 (Day 43).
- Maternal animals: LD 14 (those that littered) or Day 26 post‑coitum (those that did not litter).

GROSS NECROPSY: Yes

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
Postmortem examinations (offspring):
SACRIFICE: Yes
- Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 13 were sacrificed

GROSS NECROPSY: Yes

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
Statistics:
Statistical analyses were performed, where appropriate - refer to the attached report.
Reproductive indices:
Number of indices were used, where appropriate, to evaluate reproductive function: mating index, fecundity index, fertility index, mean duration of gestation, mean number of implantation sites
Offspring viability indices:
Number of indices were used, where appropriate, to evaluate reproductive function: Mean number of pups born, Mean number of pups alive PND 1, % male pups PND 1 (litter), % male pups PND 1 (mean), % Post-implantation survival index (litter), % Post-implantation survival index (mean), % Live birth index (litter) and Live birth index.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
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. These three death were unrelated to treatment.
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. This was considered a low incidence and not treatment related.
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, similar to controls.
Significantly 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 were also observed in females at these doses, (approximately -11% in the first 2 weeks), compared with controls. The mean overall food consumption during the study, for females administered 100 mg/kg/day was 12% lower than controls during lactation.
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.

Upon microscopic examination, test article-related microscopic findings were recorded for the kidney . In the kidneys of males administered 100 mg/kg/day, increased severity of hyaline droplets was pr esent, which generally correlated with increased kidney weights. Hyaline droplets were characterized by small, generally round eosinophilic droplets in the proximal tubular epithelium.
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. No test article-related thyroid weight changes were noted.
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:
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.
All remaining intergroup differences were transient in nature or only affected one or two animals and, as such, these observations were considered to have arisen incidentally.
No test article-related effects were evident for either sex administered 30 mg/kg/day.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The number and length of estrous cycles was unaffected by test article administration
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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 test article-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%).

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no test item related effects observed. not determinable due to absence of adverse toxic effects
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup body weights were essentially similar to controls on PND 1 and 4; however, lower mean male body weights were evident on PND 7 (-5%) and PND 13 (-9%) from litters maternally administered 100 mg/kg/day, compared with controls. Female body weights were also lower on PND 7 (‑5%) and PND 13 (-9%), compared with controls.
For litters in the group administered 50 mg/kg/day, lower male body weights were also evident on PND 7 (-5%) and PND 13 (-6%), compared with controls. Following maternal administration of 50 mg/kg/day, female body weights were lower from PND 1 onwards (-4 to -7%) compared with controls.
No effect on pup body weights was evident following maternal administration of 30 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Description (incidence and severity):

Upon macroscopic examination, no findings considered related to the test article were recorded for adults or the subsequent offspring. Tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Thyroid hormone levels were unaffected in offspring maternally administered the test article.
No nipples/areolae were present for male offspring that survived to PND 13.
No effect on ano-genital distance was observed in litters from dams administered any test article dose level, compared with controls.
No test article-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%).

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no test item related effects observed.
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 2: 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 2. 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

16.9

20.8

       19.2

20.3

19.9

       21.0

24.6

       25.4*H

27.5*H

 *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 Mean Estrous Cycles

Group

Pre‑dose

Pre‑pairing

 

No. of Estrous Cycles

Mean Cycle Length (days)

No. of Estrous Cycles

Mean Cycle Length (days)

control

 2.5

4.3

2.2

4.2

2

2.8

4.6

2.6

4.3

3

2.3

4.8

2.2

4.4

4

1.9

4.4

2.2

4.7

Estrous cycle = from 1st Oestrus to the next nonconsecutive stage of the same type

@ = Number examined reduced due to Female R0800 (Group 4) excluded

Table 9. Summary of Functional Observattional Battery in male

Group
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 10. Summary of Functional Observational Battery in female

Group

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

0.0

 

Table 11. 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

Code

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 12. 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 of Section 7.5.1.

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, reproductive performance and offspring development were unaffected as such, the no observed adverse effect level NOAEL for reproductive toxicity was considered as 100 mg/kg/day.
Executive summary:

In an OECD 422 study, following formulation analysis (by analysing dibutylamine), 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.

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 mortalities were observed; one male in the control group, a female in the 50 mg/kg/day group and a male at 100 mg/kg/day group.

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.