Registration Dossier

Administrative data

Description of key information

The oral administration of CGE-PMDA adduct to rats by gavage, at dose levels of 25, 75 and 150 mg/kg bw/day, resulted in a reduction in body weight gain and food consumption in animals of either sex at 150 and 75 mg/kg bw/day. Following the reduction of the high dose level to 100 mg/kg bw/day on Day 15, recovery was evident in these animals and body weight gains also improved at 75 mg/kg bw/day from Week 3 onwards. Treated animals also showed clinical observations that can be associated with the oral administration of an irritant/unpalatable test item formulation and as such, the initial reduced body weight development at 150 and 75 mg/kg bw/day may be the result of the irritant nature of the test item rather than evidence of true systemic toxicity. The microscopic changes evident in the mesenteric lymph nodes were considered to be non-adverse and although the significance of the microscopic seminal vesicle and coagulating gland changes were unclear, there was no evidence of any degeneration or any effect on fertility and complete reversibility was evident. Therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day for both males and females.

The `No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 100 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
designed as dose range finding study
Deviations:
yes
Remarks:
designed as dose range finding study, thus only 14 d administration and only limited examinations (Clinical signs, body weight development, food consumption, water intake, gross necropsy)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han'": Rccl-lant'': WIST
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Ltd., Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 12 weeks
- Housing: in groups of five per sex in solid floor polypropylene cage with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): Rodent 2018C Teklad Global Certified Pelleted Diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 d
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Preliminary sighting work was performed to establish a suitable high dose level for the Phase I of the Repeated Dose Range-Finding Toxicity Study. In the preliminary phase, three consecutive groups of one male and one female were treated with 1000, 500 and 250 mg/kg bw/day for a maximum of six consecutive days.
At 1000 mg/kg bw/day, the female was found dead during the initial mortality checks on Day 2. The male showed clinical signs of noisy respiration 4 hours post dose observations on Day 1 and on Day 2 showed signs of respiratory pattern changes, prostration, cyanosis, dehydration, pallor of the extremities, hypothermia, ptosis and pilo-erection and was therefore sacrificed in extremis. At necropsy both animals showed hemorrhaged and red discolouration of the glandular region of the stomach and gaseous distension in the small and large intestines. The female also had a patchy liver.
At 500 mglkg bw/day the female showed clinical signs of ataxia, hypothermia, hunched posture, pilo-erection, ptosis and diarrhea on Day 2. Therefore, due to these clinical signs both animals were prematurely terminated. At necropsy the female had gaseous distension in the stomach and intestines. No such observations were present in the male.
At 250 mg/kg bw/day both animals received six days oftreatment and were terminated on Day 7. Daily body weights were performed from the start of treatment and a slight reduction in body weight was evident in both animals. Prior to necropsy on Day 7, the male showed clinical signs of hunched posture, pilo-erection and lethargy. The female did not show any clinical signs. At necropsy, the male has a dark liver and no macroscopic findings on the female.
Based on these results, dose levels of 0, 25, 100 and 200 mg/Kg bw/d were chosen for Phase I (non-pregnant animals).

On the basis of the outcome of the Phase I investigations, the following dose levels have been chosen for Phase II (pregnant females): 0, 25, 75, 150 mg/kg bw/d.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
mortality: Twice daily, early and late during the working period.
clinical signs: Individual clinical observations will be performed immediately before dosing, up to 30 minutes after dosing and one hour after dosing. In addition, post dosing observations will also be performed at approximately 4 hours after dosing during the normal working day (excluding weekends and Public Holidays). All observations will be recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights will be recorded on Day 1 (prior to the start of dosing) and then on Days 4, 8,11 and 15.


FOOD CONSUMPTION:
Dietary intake will be recarded for Days 1 to 4, 4 to 8, 8 to 11 and 11 to 15 for each cage group. Food efficiency (body weight gain/food intake) will also be calculated retrospectively for these periods.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily water intake will be measured gravimetrically for each cage throughout the study.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The majority of animals of either sex treated at 200 or 100 mg/kg bw/day exhibited signs of post dose increased salivation between Days 7 to 13.
During the final week of dosing the males that received 200 mg/kg bw/day, showed instances of noisy respiration; this finding was accompanied in one female on Day 10 by pilo-erection, lethargy and hunched posture. In addition one 200 mg/kg bw/day male was observed to have diarrhoea.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall bodyweight gains of animals of either sex that received 200 mg/kg bw/day were below that of the concurrent control, with individuals of either sex showing actual body weight losses during this period.
Absolute gains in both sexes treated at 25 and 100 mg/kg bw/day were comparable to that of the control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day males showed lower food consumptions throughout the treatment period. The corresponding females showed a lower dietary intake from Day 4 onwards.
At 100 or 25 mg/kg bw/day food consumptions for both sexes remained similar to controls.

Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The inter-group differences in food conversion efficiency (the ratio of body weight gain to dietary intake) of all test groups when compared to that of the controls reflected the associated trends seen in their body weight gains and dietary for food intake during the treatment period.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no adverse treatment-related effects identified in water intake detected between test and control animals.
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:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic examination revealed dark and enlarged spleens in four 200 mg/kg bw/day four females.
Incidental changes involved increased pelvic space in both kidneys of one Control female had increased pelvic space (hydronephrosis) in both kidneys. Such observations represent common sporadic finding amongst rats of the strain and age used and as such this isolated finding was considered not to be associated with treatment. One male at 200 mg/kg bw/day had a fluid filled caecum.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Dose descriptor:
other: upper dose level for Phase II
Effect level:
150 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
food efficiency
gross pathology
Critical effects observed:
not specified

Bodyweights(g)

Day Numbers Relative to Start Date

Group (Sex)

1

4

8

11

15

1(M)

Mean

324.2

331.4

339.6

347.6

351.2

S.D.

9.4

12.1

13.1

15.1

15.6

N

5

5

5

5

5

2(M)

Mean

318.4

325.2

338.6

344.8

352.6

S.D.

6.9

6.5

8.6

8.3

9.1

N

5

5

5

5

5

3(M)

Mean

321.4

322

336

342

346.2

S.D.

7.7

9

11

9.6

8.8

N

5

5

5

5

5

4(M)

Mean

325

323.4

333

327.2

320.2

S.D.

7.8

8.6

8.3

14

22.7

N

5

5

5

5

5

1(F)

Mean

214.4

221

229.2

231

232.2

S.D.

5.4

9

8.9

8.7

10.9

N

5

5

5

5

5

2(F)

Mean

214

217.8

222.2

221.6

229.6

S.D.

3.5

3.3

2.7

8.5

3.8

N

5

5

5

5

5

3(F)

Mean

216.6

220.6

229

227.2

232.8

S.D.

7.7

9.4

11.8

12.8

13.8

N

5

5

5

5

5

4(F)

Mean

210.2

208.6

213.4

214.8

210.8

S.D.

4

9.4

7.6

8.7

11.2

N

5

5

5

5

5

 

 

Increase in Bodyweight (g)

Day Numbers Relative to Start Date

Absolute gain

%gain

Group (Sex)

From:

1

4

8

11

1

1

To:

4

8

11

15

15

15

1(M)

Mean

7.2

8.2

8

3.6   

27.0    

8.3

S.D.

4.5

3.6

3.2

3.5   

10.2    

3.2

N

5

5

5

5

    5

5      

2(M)

Mean

6.8

13.4

6.2

7.8   

34.2   

10.8

S.D.

5.1

2.4

3

2.9   

10.0    

3.3

N

5

5

5

5

5

5

3(M)

Mean

0.6

14

6

4.2   

24.8    

7.7

S.D.

3.2

3.2

1.9

3.0    

4.8    

1.5

N

5

5

5

5

5

5

4(M)

Mean

ƒ1.6

9.6

ƒ5.8

ƒ7.0   

ƒ4.8   

ƒ1.5

S.D.

7.9

6.8

7.7

11.7   

19.4    

6.0

N

5

5

5

5

5

5

1(F)

Mean

6.6

8.2

1.8

1.2   

17.8    

8.4

S.D.

13.4

6.9

7.9

5.6   

11.9    

5.7

N

5

5

5

5

5

5

2(F)

Mean

3.8

4.4

ƒ0.6

8.0   

15.6    

7.3

S.D.

3.6

1.8

9.8

9.7    

3.0    

1.5

N

5

5

5

5

5

5

3(F)

Mean

4

8.4

ƒ1.8

5.6   

16.2    

7.6

S.D.

11.2

4.2

4.1

2.3   

17.1    

8.0

N

5

5

5

5

5

5

4(F)

Mean

ƒ1.6

4.8

1.4

ƒ4.0    

0.6    

0.3

S.D.

7.5

5

10.8

7.5   

12.0    

5.8

N

5

5

5

5

5

5

 

 

Group Mean Weekly Food Consumptions

Day Numbers Relative to Start Date

Group(Sex)

From:

1

4

8    

11

To:

4

8    

11    

15

1(M)

Mean

20.9

19.7  

21.0  

20.9

N

5

5

5     

5

2(M)

Mean

18.3

22.5  

20.2  

21.0

N

5

5

5     

5

3(M)

Mean

22.3

16.6  

19.1  

19.6

N

5

5

5    

5

4(M)

Mean

16.3

17.5  

13.9  

15.4

N

5

5

5     

5

1(F)

Mean

17.3

13.6  

14.7  

16.1

N

5

5

5     

5

2(F)

Mean

14.5

16.2  

15.6  

17.1

N

5

5

5     

5

3(F)

Mean

21.2

9.6  

13.2  

15.6

N

5

5

5     

5

4(F)

Mean

17.3

6.6  

10.9  

13.3

N

5

5

5     

5

 

 

Weekly Food Efficiency

Day Numbers Relative to Start Date

Group(Sex)

From:

1

4

8    

11

To:

4

8    

11    

15

1(M)

Mean

11.5

10.4

12.7

4.3

N

5

5

5

5

2(M)

Mean

12.4

14.9

10.2

9.3

N

5

5

5

5

3(M)

Mean

0.9

21.1

10.5

5.4

N

5

5

5

5

4(M)

Mean

ƒ2.8

13.7

ƒ13.9

ƒ11.4

N

5

5

5

5

1(F)

Mean

12.7

15.1

4.1

1.9

N

5

5

5

5

2(F)

Mean

8.7

6.8

ƒ1.3

11.8

N

5

5

5

5

3(F)

Mean

6.3

22

ƒ4.5

9.0

N

5

5

5

5

4(F)

Mean

ƒ3.1

18.2

4.3

ƒ7.5

N

5

5

5

5

 

 

Summary Incidence of Necropsy Findings

Males

 

control

25 mg/kgbw/d

100 mg/kgbw/d

200mg/kgbw/d

Number of Animals Examined:

5

5

5

5

Caecum

 

 

 

 

Submitted

(0)

(0)

(0)

(1)

No Visible Lesions

0

0

0

0

Fluid Filled

0

0

0

1

 

Females

 

control

25 mg/kgbw/d

100 mg/kgbw/d

200mg/kgbw/d

Number of Animals Examined:

5

5

5

5

Kidneys

 

 

 

 

Submitted

(5)

(5)

(5)

(5)

No Visible Lesions

4

5

5

5

Increased Pelvic Space; both

1

0

0

0

Spleen

 

 

 

 

Submitted

(5)

(5)

(5)

(5)

No Visible Lesions

5

5

5

1

Dark

0

0

0

4

Enlarged

0

0

0

4

 

Conclusions:
The oral administration of CGE-PMDA adduct was well tolerated by animals treated at 25 and 100 mg/kg bw/day but less so in both sexes that received 200 mg/kg bw/day. In these latter animals exposure to the test item resulted in animals of either sex showing disruptions in body weight development and food consumption/utilisation when compared to controls. On the basis of results from these Phase I study investigations. Therefore, a dose level of 200 mg/kg bw/day is not recommended for use in the next phase of preliminary testing (Phase II) as it may be too stressful for pregnant animals nor for use in longer term treatment periods such as the forthcoming Combined Repeat Dose Toxicity Study with Reproductive/Developmental Toxicity Screening Test in the Rat (Study No. HD72JH).
Based on these results from Phase I of KP08NC, a dose level of 150 mg/kg bw/day as the upper dose level is considered to be suitable as the high dose level for Phase II of KP08NC, along with 25 and 75 mg/kg bw/day as the low and intermediate dose levels, respectively.
Executive summary:

The study was performed to provide information on dose tolerance in nonpregnant rats as the basis for selection of dose levels for Phase II of KP08NC (preliminary investigations in pregnant rats) and the subsequent longer term toxicological evaluation for Combined Repeat Dose Toxicity Study with Reproductive/Developmental Toxicity Screening Test in the Rat (Study No. HD72JH).

 

In Study Phase I CGE-PMDA adduct was administered by gavage to three groups, each of five male and five female Wistar Han: RccHan: WIST strain rats, for fourteen consecutive days, at dose levels of 25, 100 and 200 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400).

 

Clinical signs, body weight development, food consumption and water intake were monitored during the study and all animals were subjected to a gross necropsy examination at termination.

 

Mortality.

There were no unscheduled deaths on the study.

 

Clinical Observations.

The majority of animals of either sex treated at 200 or 100 mg/kg bw/day exhibited signs of post dose increased salivation between Days 7 to 13.

During the final week of dosing the males that received 200 mg/kg bw/day, showed instances of noisy respiration; this finding was accompanied in one female on Day 10 by pilo-erection, lethargy and hunched posture. In addition one 200 mg/kg bw/day male was observed to have diarrhoea.

 

Body weight.

Overall bodyweight gains of animals of either sex that received 200 mg/kg bw/day were below that of the concurrent control, with individuals of either sex showing actual body weight losses during this period.

Absolute gains in both sexes treated at 25 and 100 mg/kg bw/day were comparable to that of the control.

 

Food Consumption.

At 200 mg/kg bw/day males showed lower food consumptions throughout the treatment period. The corresponding females showed a lower dietary intake from Day 4 onwards.

At 100 or 25 mg/kg bw/day food consumptions for both sexes remained similar to controls.

The inter-group differences in food conversion efficiency (the ratio of body weight gain to dietary intake) of all test groups when compared to that of the controls reflected the associated trends seen in their body weight gains and dietary for food intake during the treatment period.

 

Water Consumption.

There were no adverse treatment-related effects identified in water intake detected between test and control animals.

 

Necropsy.

The macroscopic examination revealed dark and enlarged spleens in four 200 mg/kg bw/day four females.

Incidental changes involved increased pelvic space in both kidneys of one Control female had increased pelvic space (hydronephrosis) in both kidneys. Such observations represent common sporadic finding amongst rats of the strain and age used and as such this isolated finding was considered not to be associated with treatment. One male at 200 mg/kg bw/day had a fluid filled caecum.

 

Discussion/Conclusion.

The oral administration of CGE-PMDA adduct was well tolerated by animals treated at 25 and 100 mg/kg bw/day but less so in both sexes that received 200 mglkg bw/day. In these latter animals exposure to the test item resulted in animals of either sex showing disruptions in body weight development and food consumption/utilisation when compared to controls.

On the basis of results from these Phase I study investigations. Therefore, a dose level of 200 mg/kg bw/day is not recommended for use in the next phase of preliminary testing (Phase II) as it may be too stressful for pregnant animals nor for use in longer term treatment periods such as the forthcoming Combined Repeat Dose Toxicity Study with Reproductive/Developmental Toxicity Screening Test in the Rat (Study No. HD72JH). With these taking into considerations in mind of the results from Phase I of KP08NC, a dose level of 100 mg/kg bw/day as the upper dose level is considered to be suitable as the high dose level for Phase II of KP08NC, along with 25 and 50 mg/kg bw/day as the low and intermediate dose levels, respectively.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Dose range-finding study

The range-finding study was designed to investigate the effects of oral administration of the test item on embryonic and foetal development during the period of organogenesis. The results provide supporting information to assist in the selection of dose levels for the subsequent longer term toxicological evaluation of the test item in a Combined Repeat Dose Toxicity Study with Reproductive/Developmental Toxicity Screening Test in the Rat (OECD 422); Study No. HD72JH.

In these investigations (Study Phase II) the test item was administered once daily by gavage to three groups, each of five time-mated Wistar Han: RccHan: WIST strain female rats from Day 3 to Day 19 of gestation at dose levels of 25, 75 and 150 mg/kg bw/day. A control group of five time-mated females received vehicle alone (Polyethylene glycol 400) over the same treatment period. All females were killed on Day 20 of gestation.

Clinical signs, bodyweight development and food consumption were monitored during the study and all animals were subjected to gross necropsy examination including examination of the uterine contents and gross external necropsy of foetuses.

One 150 mg/kg bw/day female was found dead shortly after dosing on Day 5 of Gestation, there were no adverse effects on body weight, food consumption orclinical signs prior to death. Necropsy findings revealed darkened liver and kidneys, with dark patches in the lungs.

There were no further unscheduled deaths on the study.

Between Day 5 to 7 of gestation, the majority of females treated with 75 or 150 mg/kg bw/day showed instances of increased salivation and/or noisy respiration.

Incidental findings included one 75 mg/kg bw/day females showing observations of deceased respiratory rate, pilo-erection and diarrhoea between Days 5 to 7 with noisy respiration between Days 5 to 10 and 12 to16.

All female treatment groups exhibited lower body weight gains following the first dose (Day 3), with improvement evident thereafter.

During the first Week of treatment lower overall accumulative body weight gains were evident in females treated with 75 or 150 mg/kg bw/day in relation to controls. However, during the latter stage of gestation, overall body weight gains return to levels similar to controls. Body weight gains when adjusted for gravid uterus weight, revealed lower body weight gain of 17% and 40%, in females treated with 75 and 150 mg/kg bw/day, respectively. No such effect was detected at 25 mg/kg bw/day. 

Instances of marginally lower food consumption were noted in 75 and 150 mg/kg bw/day dose groups between Days 3 to 8 and 3 to 5 of gestation, respectively. Thereafterfood consumptions were generally similar to controls.

Visual inspection of water consumptions revealed no adverse effects.

The macroscopic findings in the decedent female (No. 58) findings revealed darkened liver and kidneys, with dark patches in the lungs.

No macroscopic abnormalities were detected in animals examined at study termination.

Litter data are reported in the toxicity to reproduction section.

In consideration of the marked body weight effects on the males during the fourteen day repeated dose phase with non-pregnant animals (Phase I) at 200 mg/kg bw/day and the observed effects on body weight gains on females at 150 mg/kg bw/day during the repeated dose phase on pregnant animals (Phase II), these effects were considered not to be so significant to exclude 150 mg/kg bw/day as the high dose level for further investigating. Therefore, dose levels of 0, 25, 75 and 150 mg/kg bw/day are recommended for the forthcoming Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422) (Study No.HD72JH).

 

Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test

This study was designed to investigate the systemic toxicity and potential adverse effects of CGE-PMDA adduct on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints, and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016). It also assesses the ability of the animals to recover from any toxicity over twenty-eight days following the withdrawal of treatment.

 

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to ten weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 25, 75 and 150 mg/kg bw/day. After two weeks of treatment at 150 mg/kg bw/day the dose level was reduced to 100 mg/kg bw/day on Day 15 following marked body weight losses in animals of either sex; particularly in the males. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400) over the same period. Two recovery groups, each of five males and five females, were treated with the high dose (150/100 mg/kg bw/day) or the vehicle alone for forty-two consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

Pairing of non-recovery animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights ano-genital distance and visible nipple count (male offspring only).

Extensive functional observations were performed on five selected non-recovery males from each dose group after the completion of the pairing phase, and for five selected non-recovery parental females from each dose group on Day 12post partum. Five non-recovery males and females from each dose group were selected for hematology and blood chemistry assessments prior to terminal sacrifice. Additionally, blood samples were taken at terminal sacrifice from all adult animals and from one male and one female offspring per litter (where possible) on Days 4 and 13post partum, for thyroid hormone analysis; samples from non-recovery adult males and Day 13 offspring were analyzed for Thyroxine (T4).

Vaginal smears were performed for all females from the day after arrival (enabling the exclusion of females not showing appropriate estrous cycling from dosing) and for all non-recovery treated females including controls through pre-pairing, pairing and up to confirmation of mating. Vaginal smears were also performed in the morning on the day of terminal sacrifice for all non-recovery females.

Surviving adult non-recovery males were sacrificed on Day 44 or 45, followed by the sacrifice of all surviving offspring and surviving adult non-recovery females on Days 13 and 14post partum, respectively. Any female which did not produce a pregnancy was sacrificed around the same time as littering females. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. All offspring were examined externally; where external observations were detected an internal necropsy was performed.

Following forty-three days of treatment, recovery group animals were maintained without treatment for a further twenty-eight days. In addition, hematological and blood chemical assessments were performed on all recovery group animals at the end of the treatment-free period. These animals were then subjected to a gross necropsy and histopathological examination of selected tissues was performed.

 

Adult Responses

Mortality

There were no unscheduled deaths that were considered to be related to treatment.

There were four premature deaths during the treatment period (Animal numbers 2, 29, 71 and 83) that were considered to be due to dosing trauma.

 

Clinical Observations

Incidences of increased salivation and noisy respiration were evident in animals of either sex treated with 150/100 mg/kg bw/day throughout the treatment period and to a lesser extent in animals of either sex treated with 75 and 25 mg/kg bw/day. Isolated instances of hunched posture, pilo-erection, decreased respiratory rate, lethargy and stained snout were also evident at 150/100 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 25, 75 or 150/100 mg/kg bw/day.

 

Functional Performance Tests

There were no treatment-related changes in functional performance considered to be related to treatment at 25, 75 or 150/100 mg/kg bw/day.

 

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 25, 75 or 150/100 mg/kg bw/day.

 

Body Weight

At 150 mg/kg bw/day males showed body weight losses during Week 1 and 2 of treatment when compared to controls. Following the reduction of the dose level to 100 mg/kg bw/day on Day 15, these animals showed significant improvement in body weight gains. As a consequence of the initial body weight losses, the overall group mean body weight gain was 45% lower than controls.

Males treated with 75 mg/kg bw/day showed reductions in body weight gain during Weeks 1 and 2 of treatment. Improvement was noted from Week 3 onwards and body weight gains were comparable to controls thereafter. The earlier reduction however, resulted in the overall group mean body weight gain being 26% lower than controls.

No such effects were detected for males treated with 25 mg/kg bw/day.

Females treated with 150/100 mg/kg bw/day showed reduced group mean body weight gains during pre-pairing and during the final week of gestation compared to controls. No adverse effects were evident during lactation.

At 75 mg/kg bw/day, females showed reduced body weight gains during pre-pairing. No effects were evident during gestation or lactation.

At 25 mg/kg bw/day, females showed comparable body weight gains with the exception of slightly lower body weight gains during the first week of pre-pairing compared to controls.

 

Food Consumption

Males treated with 150/100 mg/kg bw/day showed a reduction in food intake during the first two weeks of treatment. Improvement was evident thereafter. There were no effects detected for males treated with 25 mg/kg bw/day. During the treatment free period, 150/100 mg/kg bw/day males showed dietary intake that was similar to controls.

Females treated with 75 or 150/100 mg/kg bw/day showed lower food consumptions during pre-pairing. Slight reductions in food consumption were also evident in these females during gestation and lactation. Females at 25 mg/kg bw/day were generally similar to controls. The recovery 150/100 mg/kg bw/day females generally showed similar dietary intake to controls, with the exception of the first two weeks of treatment where food consumption was slightly lower.

 

Water Consumption

Daily visual assessment of water consumption did not reveal any significant intergroup differences.

 

Reproductive Performance

Estrous Cycle

There was no effect of treatment with the test item at any dose level on the nature of estrous cycle with most females showing regular cycles over the pre-pairing phase of the study.

There were also no intergroup differences in the stage of estrous cycle on the day of necropsy.

 

Mating

No treatment-related effects were detected in mating performance.

 

Fertility

No treatment-related effects were detected in fertility.

 

Gestation Lengths

Gestation lengths were essentially similar to control.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no adverse effect of treatment with the test item on the mean number of implantations, post-implantation loss, sex ratio and subsequent offspring survival to Day 13 of age at any dose level.

Litter size at birth and subsequently on Days 1, 4, 7 and 13post partumwas lower for females treated with 150/100 mg/kg bw/day when compared to control litters, without attaining statistical significance. No such effects were noted for offspring from females treated with 75 or 25 mg/kg bw/day.

 

Offspring Growth and Development

As a consequence of the slightly lower litter size at birth in females treated with 150/100 mg/kg bw/day, litter weights were reduced in these females throughout lactation. There was no effect of treatment with the test item indicated by offspring body weight or body weight gain, ano-genital distance on Day 1post partumor visible nipple count in male offspring on Day 13post partumat any dose level.

The clinical signs and necropsy findings apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 25, 75 or 150/100 mg/kg bw/day.

 

Laboratory Investigations

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined.

 

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

 

Pathology

Necropsy

Offspring

No treatment-related macroscopic abnormalities were detected in interim death or terminal sacrifice offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Adults

No toxicologically significant abnormalities were detected in animals of either sex treated with 25, 75 and 150/100 mg/kg bw/day.

 

Thyroid Hormone Analysis

An evaluation of Thyroxine (T4) in adult males and male/female offspring (Day 13 of age) did not identify any treatment-related findings.

 

Organ Weights

There was an increase in seminal vesicle (including coagulating gland) weights, both absolute and relative to terminal body weights. This correlated with the histopathological findings of increased secretion (dilation), the significance of which is unclear.

There were no further toxicologically significant findings on organ weights measured.

 

Histopathology

The premature death of four animals was considered to be due to dosing trauma and was unrelated to treatment with the test item.

Treatment at 75 or 150/100 mg/kg bw/day resulted in histiocyte aggregates in the mesenteric lymph node of males and females. This showed good reversibility and was considered to be non-adverse.

At 75 or 150/100 mg/kg bw/day, increased secretion (dilation) of the seminal vesicles was evident and at 150/100 mg/kg bw/day this was also evident in the coagulating glands. Complete recovery was evident; however, the significance of this finding is unclear.

 

Conclusion

The oral administration of CGE-PMDA adduct to rats by gavage, at dose levels of 25, 75 and 150 mg/kg bw/day, resulted in a reduction in body weight gain and food consumption in animals of either sex at 150 and 75 mg/kg bw/day. Following the reduction of the high dose level to 100 mg/kg bw/day on Day 15, recovery was evident in these animals and body weight gains also improved at 75 mg/kg bw/day from Week 3 onwards. Treated animals also showed clinical observations that can be associated with the oral administration of an irritant/unpalatable test item formulation and as such, the initial reduced body weight development at 150 and 75 mg/kg bw/day may be the result of the irritant nature of the test item rather than evidence of true systemic toxicity. The microscopic changes evident in the mesenteric lymph nodes were considered to be non-adverse and although the significance of the microscopic seminal vesicle and coagulating gland changes were unclear, there was no evidence of any degeneration or any effect on fertility and complete reversibility was evident. Therefore the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 100 mg/kg bw/day for both males and females.

The `No Observed Effect Level' (NOEL) for reproductive toxicity was considered to be 100 mg/kg bw/day. 

Justification for classification or non-classification

Based on the available relevant and reliable data CGE-PMDA adduct does not need to be classified and labelled according to the CLP Regulation (EC) No 1272/2008 with respect to repeated dose toxicity.