Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2016 - 28 May 2017
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-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2016 - 28 May 2017
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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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 to +8 ºC
- Stability of formulation under test conditions: stable - confirmed at 100 and 20000 ppm.
- Solubility and stability of the test substance in the diet: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

OTHER SPECIFICS: n/a
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: ca. 71 days old and females: ca. 85 days old
- Weight at study initiation: males: 319 - 383 g and females: 239 - 293 g
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. An Aspen chew block was provided for envirnmental enrichment throughout the study period. A plastic shelter was also included in each cage throughout the study period, except during pairing and lactation.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet. A 100 g sample of each batch of diet used were retained frozen (-10 to -30°C) until finalization. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Provided ad libitum.
- Acclimation period: Males: six days prior to the commencement of treatment. Females: six days prior to the commencement of estrous cycle evaluation.

DETAILS OF FOOD AND WATER QUALITY: See above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ºC
- Humidity (%): 40 - 70 %
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 04 April 2017 To: 28 May 2017
Route of administration:
oral: feed
Details on route of administration:
On each occasion of the preparation of the premix the required amount of test substance was weighed into a suitable container. An amount of sieved diet that approximately equaled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: n/a

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Basal diet (SDS VRF1 Certified)
- Storage temperature of food: not reported

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
Minimum of 5 weeks with a 2 week recovery period.
Frequency of treatment:
Continuous
Dose / conc.:
1 200 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
7 500 ppm
Dose / conc.:
73.8 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 1200 ppm dose group
Dose / conc.:
181 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 3000 ppm dose group
Dose / conc.:
438 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 7500 ppm dose group
No. of animals per sex per dose:
Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
1200 ppm (toxicity test) = 10
3000 ppm (toxicity test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
1200 ppm (reproduction test) = 10
1200 ppm (toxicity test) = 5
3000 ppm (reproduction test) = 10
3000 ppm (toxicity test) = 5
7500 ppm (reproduction test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5

Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 1200, 3000 and 7500 ppm) were chosen in conjunction with the sponsor based upon the results obtained in a 14 day preliminary study.
- Rationale for animal assignment (if not random): n/a
- Rationale for selecting satellite groups: determine if toxic effects in F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): n/a
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatisation, before feeding of the treated diets on the Day that treatment commenced (Day 1) and twice weekly thereafter (including termination).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study)
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 14 of lactating (females) and test termination (males and females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: lactating females: 5, test temination: 5 males and 5 females.
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 14 of lactating (females), test termination (male and females) and recovery week 4 (males)
- Animals fasted: Yes
- How many animals: lactating females: 5; test termination: 5 males and 5 females; recovery group: all surviving males.
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6 (day before scheduled termination) and recovery group week 2.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and weekly thereafter.
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

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.
- after pairing until mating (reproductive females only).
- four days before scheduled termination (all females except premature decedents).
- Females showed no evidence of mating: following completion of pairing period female was separated from the male and vaginal smearing continued for up to five days or until the first estrous smear was seen. If a female shows an estrous smear during this period, she was killed and subject to macroscopic examination.

THYROID HORMONE ANALYSIS: Yes
- Time schedule: - Day 4 of age (F1 offspring, two females per litter (where possible) - no pups were eliminated when total litter size dropped below ten/litter).
- one pup for T3/T4 (serum)
- one pup for TSH (plasma)
- Day 13 of age (F1 offspring, two males and two females per litter (where possible)
- two for T3/T4 (serum): where possible one male and one female
- two for TSH (plasma): where possible one male and one female
- Termination (All Toxicity and Recovery phase F0 males and all Reproductive phase F0 females surviving to scheduled termination)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)

HISTOPATHOLOGY: Yes (see table 3)
Statistics:
See attached report
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or dose observations related to treatment were seen in the treatment, gestation, lactation period or recovery period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The overall mean bodyweight change from Day 1 to Day 8 for male animals receiving treated diet at 1200 or 3000 ppm was similar to control. Male animals receiving treated diet at 7500 ppm had a reduced mean bodyweight gain from Day 1 to 8 when compared to controls; this is due to a bodyweight loss in 5 animals between Day 1 and 4. All animals at 7500 ppm gained weight between Days 4 and 8, and exceeded their Day 1 bodyweight. Bodyweight change from Day 8 to 15 for all male treated groups was similar to control. However the body weight changes between Day 15 and 22 for all treated male groups were lower than control, with males receiving 3000 ppm showing a mean body weight loss. Bodyweight changes from Days 22 to 29 and Days 29 to 36 shows that male animals receiving 1200 or 3000 ppm return to comparable performance to control, however the weight gain of males receiving 7500 ppm remain significantly lower. The overall body weight change for the treatment period (Days 1 to 36) for male animals receiving treated diet at 1200 ppm and 3000 ppm was lower than control (82 % and 83 %), however male animals receiving treated diet at 7500 ppm was significantly lower than control (70 %). During the recovery priod, mean weight gain of animals previously treated at 7500 ppm was slightly greater than the control.

The overall mean bodyweight change from Day 1 to Day 8 for all treated female groups was lower than control, with an overall mean bodyweight loss for females receiving 3000 or 7500 ppm. Between Day 1 and Day 4 individual bodyweight losses or bodyweight stasis were seen in all treated groups and control (7/20 for control, 11/15 at 1200 ppm and 9/15 at 3000 ppm and 17/20 at 7500 ppm). By Day 8 several females had not obtained parity with Day 1 (3/20 for control, 2/15 at 1200 ppm, 8/15 at 3000 ppm and 15/20 at 7500 ppm). Bodyweight changes from Day 8 to 29 for all female treated groups was similar to control, with the exception of Day 15 to 22 where an mean body weight loss was seen in the control group. Bodyweight change from Days 29 to 36 for all female treated groups was slightly lower than control. The overall bodyweight change for the Toxicity and Recovery females during the treatment period (Days 1 to 36) was lower than control for all treated groups. Most significantly females receiving treated diet at 7500 ppm, gained only 52% of the control value, this was due to the body weight losses and stasis seen in the first week of study. However after 2 weeks of recovery females previously receiving 7500 ppm had a higher than control overall body weight change (Day R1 to R15). The mean weights of recovery animals that had receved 7500 ppm were higher than controls, but this was because the animals pre-selected for recovery had incidentally higher weights.

In the gestation period, reproductive phase females receiving 7500 ppm had statistically lower than control body weights and body weight gain up to Day 20, overall body weight change from Day 1 to 20 of gestation was 79 % of control. Females receiving 1200 or 3000 ppm had slightly reduced overall body weight gain when compared to controls (95 % and 92 % respectively).

In the lactation period, reproductive phase females receiving 7500 ppm had statistically lower than control body weights and body weight gain up to Day 13 of lactation, overall body weight change from Day 1 to 13 of lactation was 70 % of control. Females receiving 3000 ppm had statistically lower than control mean bodyweight values on Days 4, 7 and 10, however the increased gain between Days 10 and 13 resulted in only a minor difference in overall bodyweight change (Days 1 to 13) compared to control (94 %).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption for male animals receiving treated diet at 7500 ppm was slightly lower than control animals on Days 1 to 3 of treatment; however consumption values were comparable from Days 4 to 7 through to Days 32 to 35. The food intake of male animals treated at 1200 or 3000 ppm were unaffected. During the recovery period food consumption for males previously receiving 7500 ppm was similar to control.

Food consumption for female animals receiving treated diet at 1200 ppm was slightly lower than control on Days 1 to 3, but was comparable by Days 4 to 7. The food intake of female animals receiving 3000 or 7500 ppm treated diet were lower than control on Days 1 to 3, and remained slightly lower on Days 4 to 7 , however by Days 8-10 food intake was comparable to control. In the recovery period females previously receiving treated diet at 7500 ppm had higher food intake than control animals.

In the gestation period, food consumption for reproductive phase female animals receiving treated diet at 1200 or 3000 ppm was slightly lower than control on Days 0 to 2 (note that 2 control females had consumption that was somewhat higher than remaining controls), but was comparable by Days 3 to 6. The food intake of female animals receiving 7500 ppm treated diet was lower than control from Days 0 to 16, but was similar to control on Days 17 to 19.

In the lactation period, food consumption for reproductive phase female animals receiving treated diet at 3000 or 7500 ppm was lower than control from Days 1 to 9 and Day 1 to 12 of lactation, respectively. On days 10 to 12 females at 1200 ppm consumed slightly less food than controls but was not statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of haematological parameters at the end of the treatment period revealed higher than control haematocrit concentration in all groups of Toxicity phase males compared to control, though no dose response was evident and no similar finding was observed in females. In addition reticulocyte and neutrophil counts were lower than control in Toxicity phase females receiving treated diet at 7500 ppm. A non-dose dependent decrease in monocytes was also evident in females receiving 3000 or 7500 ppm.

Assessment of haematological parameters at the end of the lactation period revealed lower than control red cell distribution width and a non-dose dependent decrease in mean cell volume and prothrombin time in reproductive phase females receiving 7500 ppm.

All other inter-group differences from controls in the toxicity phase and reproductive phase animals at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical changes in the plasma of the toxicity phase males at the end of the treatment period revealed higher than control urea concentrations and lower glucose concentrations in all treated groups and lower than control triglyceride concentrations in males receiving 3000 or 7500 ppm. In Week 2 of recovery the biochemical changes in the plasma had demonstrated full recovery.

Biochemical changes in the plasma of the reproductive phase females at the end of the lactation period revealed higher than control urea and triglyceride (not statistically significant) concentrations in females receiving 7500 ppm althoughsimilar findings were not observed in toxicity females at the end of the treatment.

All other inter-group differences from controls in the toxicity phase and reproductive phase animals at the end of treatment were minor in nature, lacked dose-relationship, were due to anomalies in the control data or were confined to one sex; these were consequently attributed to normal biological variation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Assessment of urinary parameters at the end of the treatment period revealed the following differences from Control;

In the toxicity phase male animals receiving treated diet at 3000 or 7500 ppm the pH of the urine was more acidic, the total creatinine and total potassium was lower than control in the males receiving 7500 ppm and the urinary chloride was higher than control.

In the female toxicity phase animals receiving diet at 7500 ppm total creatinine and total chloride was higher than control and urinary potassium was lower than control. Total creatinine was also high among females at 3000 ppm.

In Week 2 of recovery, all of the aforementioned urinary changes had demonstrated full recovery.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Sensory reactivity and grip strength were considered unaffected by treatment. There were no consistent intergroup differences in grip strength between toxicity and reproductive phase females.

Motor activity scores for females given the test item were considered to be unaffected by treatment. Statistical significance was obtained for the overall low and high beam breaks for all groups of treated males (except low beams at 1200 ppm) when compared to controls, and although statistical significance was not attained for the final four 6-minute recording periods, the behaviour of the treated males was atypical, with no general increase in activity levels which should have been apparent towards the end of the recording period. The total high beam scores in all treated groups were slightly below the historical control data (HCD) range but total low beam scores at 7500 ppm were well within the HCD range.

In the recovery period the overall low and high beam breaks for males previously fed 7500 ppm continued to stay lower than control, as seen in the treatment period. No statistical significance was obtained for the overall scores however two recordings in the low beam breaks were statistically significant. As seen in the treatment period there was no general increase in activity levels over the last four 6-minute recording periods.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled termination of the toxicity phase animals after 5 weeks of treatment, decreased terminal body weights were evident in males treated at 7500ppm. Analysis of organ weights for the toxicity phase animals revealed increased adjusted kidney and liver weights in male animals treated at 3000 or 7500 ppm when compared to controls. Female toxicity phase animals treated at 7500 ppm also had decreased terminal body weights at scheduled termination. A non-dose dependent decreased adjusted adrenal weight was also apparent in the toxicity females treated at 7500 ppm.

After two weeks off-dose the recovery phase males previously treated at 7500 ppm had higher than control adjusted thymus weights however as this was not present at the end of the treatment period this is thought to be unrelated to treatment. The adjusted adrenal weights for recovery phase females previously receiving 7500 ppm after two weeks off-dose were higher than control.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed revealed no test item related lesions.

The incidence and distribution of all findings were considered to be incidental and unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test item were seen in the liver of both sexes and the kidneys of males;

Liver - Centrilobular hepatocyte hypertrophy was seen in both sexes given 7500 ppm of the test item. The liver changes (increased bodyweight adjusted liver weights and minimal centrilobular hepatocyte hypertrophy) are deemed as a reversible non-adverse adaptive response.

Kidney - Hyaline droplets were seen in the cortical tubules of males given 7500 ppm of the test item. A slight increase in the incidence and severity of cortical tubular basophilia was seen in males given 7500 ppm of the test item which was considered to be treatment related. The kidney changes detected in the males (hyaline droplets in tubules and cortical basophilic tubules) were consistent with well documented species-specific responses of the male rat in response to the administration with hydrocarbons. This effect is, therefore, not indicative of a hazard to human health.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
181 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no

Table 4       Group mean acheived dose rates for each treatment group (in mg/kg bw/day)

 

Time period

Pre-mating and recovery animals

Gestation

Lactation

1200 ppm

3000 ppm

7500 ppm

1200 ppm

3000 ppm

7500 ppm

1200 ppm

3000 ppm

7500 ppm

Male

Female

Mean

Male

Female

Mean

Male

Female

Mean

Female

Day 1 -35

73.5

74.1

73.8

186

175

181

451

424

438

-

-

-

-

-

-

Day 0 - 19

-

-

-

-

-

-

-

-

-

88

215

521

-

-

-

Day 1 - 12

-

-

-

-

-

-

-

-

-

-

-

-

190

456

994

Table 5       Mean body weights and body weight gains/ losses

Sex / Day

Bodyweight (g)

Control

1200 ppm

3000 ppm

7500 ppm

MALES (TOXICITY TEST GROUP)

Day 1

353

352

351

352

Day 4

366

364

360

353

Day 8

(change from Day 1)

384

(+32)

385

(+32)

385

(+34)

374

(+22)

Day 11

400

400

399

390

Day 15

(change from Day 8)

413

(+28)

409

(+24)

413

(+28)

401

(+27)

Day 18

419

406

408

404

Day 22

(change from Day 15)

432

(+20)

414

(+5**)

411

(-3**)

412

(+11)

Day 25

437

425

419

412

Day 29

(change from Day 22)

451

(+19)

433

(+20)

432

(+21)

422*

(+10)

Day 32

459

440

437

427*

Day 36

(change from Day 29)

471

(+20)

450

(+17)

449

(+17)

435*

(+13)

Overall BW gain Day 1 – Day 36

(% gain/ loss)

+118

+97

+98

+83**

MALES (RECOVERY GROUP)

Day 1

456

-

-

437

Day 4

461

-

-

448

Day 8

469

-

-

458

Day 11

476

-

-

466

Day 15

476

-

-

467

Overall BW gain Day 1 – Day 15 (recovery)

(% gain/ loss)

+20

-

-

+30

FEMALES (TOXICITY TEST GROUP)

Day 1

263

269

268

265

Day 4

268

267

261

256**

Day 8

(change from Day 1)

272

(+9)

274

(+5)

263

(-5**)

260**

(-6**)

Day 11

276

276

269

265*

Day 15

(change from Day 8)

276

(+5)

264

(+3)

270

(+7)

264*

(+4)

Day 18

270

264

268

262

Day 22

(change from Day 15)

273

(-3)

269

(+4*)

273

(+4*)

267

(+4**)

Day 25

274

273

278

273

Day 29

(change from Day 22)

276

(+3)

277

(+8)

279

(+5)

273

(+6)

Day 32

281

278

280

271

Day 36

(change from Day 29)

285

(+9)

280

(+3)

281

(+2)

277

(+4)

Overall BW gain Day 1 – Day 36

(% gain/ loss)

+21

+14

+17

+11

FEMALES (RECOVERY GROUP)

Day 1

282

-

-

290

Day 4

285

-

-

302

Day 8

290

-

-

304

Day 11

292

-

-

309

Day 15

290

-

-

304

Overall BW gain Day 1 – Day 15 (recovery)

(% gain/ loss)

+8

-

-

+14

FEMALES (REPRODUCTION GROUP)

Day 0

Gestation

281

280

272

266*

Day 3

(change from Day 0)

297

(+16)

296

(+16)

286

(+15)

275**

(+10*)

Day 7

(change from Day 3)

309

(+11)

312

(+16)

303

(+17)

288*

(+13)

Day 10

(change from Day 7)

327

(+18)

327

(+15)

316

(+12)

299**

(+11*)

Day 14

(change from Day 10)

341

(+14)

340

(+13)

330

(+14)

313**

(+14)

Day 17

(change from Day 14)

379

(+38)

370

(+30*)

361

(+32*)

434**

(+30)

Day 20

(change from Day 17)

427

(+49)

419

(+48)

407

(+46)

381**

(+38*)

Overall BW gain Day 0 – Day 20 (gestation)

(% gain/ loss)

+146

+138

+135

+115**

Day 1

Lactation

324

314

312

293**

Day 4

(change from Day 1)

335

(+11)

326

(+12)

315*

(+2)

293**

(+1*)

Day 7

(change from Day 4)

351

(+16)

39

(+13)

323*

(+8)

297**

(+3*)

Day 10

(change from Day 7)

365

(+15)

363

(+23)

339*

(+16)

323**

(+27)

Overall BW gain Day 1 – Day 10 (lactation)

(% gain/ loss)

+42

+48

+27*

+30*

Day 13

(change from Day 10)

Lactation

377

(+11)

365

(+3)

362

(+23)

330**

(+6)

Overall BW gain Day 1 – Day 13 (lactation)

(% gain/ loss)

+53

+51

+50

+37*

* statistically significantly different from control values p < 0.05

** statistically significantly different from control values p < 0.01

Table 6       Haematology, clinical chemistry, urinalysis and pathology findings (F0)

 

Doses (ppm)

Control

1200

3000

7500

Control

1200

3000

7500

Control

1200

3000

7500

Control

7500

Control

7500

male (toxicity group)

female (toxicity group)

female (reproduction group)

Male (rec.)

Female (rec.)

Number of animals/group

5

5

5

5

5

5

5

5

10

10

10

9

5

5

5

5

Haematology(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Hb (g/dl)

15.0

15.7

15.3

15.2

15.2

15.0

15.6

15.1

14.9

15.0

14.5

15.3

-

-

-

-

- RBC (1012/L)

7.78

7.93

7.95

8.02

7.72

7.80

7.77

7.84

7.19

7.37

7.22

7.51

-

-

-

-

- Hct (%)

0.415

0.432*

0.432*

0.432*

0.418

0.414

0.419

0.421

0.424

0.423

0.423

0.428

-

-

-

-

- Retic (1012/L)

0.174

0.138

0.168

0.156

0.142

0.120

0.115

0.096*

0.227

0.219

0.254

0.229

-

-

-

-

- MCH (pg)

19.2

19.8

19.2

19.0

19.7

19.3

20.0

19.3

20.7

20.3

20.1

20.4

-

-

-

-

- MCV (fl)

53.4

54.5

54.4

53.9

54.1

53.1

54.0

53.7

59.1

57.3

58.6

57.0*

-

-

-

-

- MCHC (g/df)

36.1

36.3

35.3

35.3

36.3

36.3

37.1

36.0

35.1

35.4

34.4

35.8

-

-

-

-

- RDW (%)

12.7

11.9

12.2

12.0

11.4

11.5

11.1

11.2

14.3

13.9

13.8

13.1**

-

-

-

-

- WBC (109/L)

8.60

9.43

11.23

8.67

8.08

9.22

10.19

9.07

9.36

8.63

11.84

9.39

-

-

-

-

- Neut (109/L)

1.15

1.38

1.17

1.56

1.19

1.10

0.92

0.64*

4.00

3.76

5.19

4.63

-

-

-

-

- Lymph (109/L)

7.04

7.52

9.54

6.69

6.29

7.55

8.72

7.92

4.79

4.38

5.93

4.22

-

-

-

-

- Mono (109/L)

0.18

0.25

0.26

0.22

0.33

0.28

0.21*

0.23*

0.32

0.25

0.47

0.33

-

-

-

-

- Eos (109/L)

0.08

0.13

0.09

0.08

0.10

0.12

0.14

0.13

0.13

0.14

0.09

0.11

-

-

-

-

- Bas (109/L)

0.05

0.06

0.06

0.04

0.03

0.04

0.05

0.03

0.03

0.03

0.05

0.03

-

-

-

-

- LUC (109/L)

0.09

0.10

0.12

0.08

0.15

0.14

0.16

0.11

0.09

0.07

0.12

0.07

-

-

-

-

- PT (secs)

22.2

23.5

20.7

19.8

20.3

21.6

21.3

22.3

23.4

21.1

21.8

20.1**

-

-

-

-

- PLT (109/L)

795

753

794

721

905

910

854

828

858

783

878

837

-

-

-

-

- APTT (secs)

20.3

18.8

20.3

19.3

19.6

17.9

16.1

19.3

15.9

14.8

15.0

13.6

-

-

-

-

Blood chemistry(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Urea (µmol/L)

4.20

5.42**

5.27**

5.87**

5.96

5.65

5.78

5.99

11.04

11.50

12.09

15.90*

6.68

6.01

-

-

- Glucose (µmol/L)

7.79

6.46**

6.41**

6.16**

7.30

6.85

6.36

3.80

6.22

5.91

6.02

5.71

-

-

-

-

- Tot. Prot. (g/L)

63

61

61

61

70

68

68

69

60

61

60

59

-

-

-

-

- Albumin (g/L)

35

35

35

35

40

39

38

39

33

33

33

33

-

-

-

-

- A/G ratio

1.27

1.33

1.32

1.38*

1.31

1.32

1.29

1.32

1.25

1.19

1.24

1.23

-

-

-

-

- Na+ (mmol/L)

144

145

145

143

143

143

142

144*

137

139

138

138

-

-

-

-

- K+ (mmol/L)

4.0

4.0

4.1

4.2

3.6

3.8

4.2**

3.9**

4.2

4.8

4.6

4.4

-

-

-

-

- Cl- (mmol/L)

101

101

102

100

100

100

101

102

95

97

96

94

-

-

-

-

- Ca++ (mmol/L)

2.51

2.56

2.56

2.56

2.71

2.70

2.74

2.68

2.38

2.44

2.34

2.28

-

-

-

-

- P (mmol/L)

2.19

2.13

2.26

2.24

1.88

1.98

2.06

1.85

3.29

3.41

3.38

3.44

-

-

-

-

- ASAT (U/L)

69

71

78

77

86

61**

62**

62**

162

179

149

124

-

-

-

-

- ALAT (U/L)

35

33

33

42*

33

27

28

48

96

107

100

87

-

-

-

-

- ALP (U/L)

96

117

140*

125*

48

43

44

42

95

107

111

95

-

-

-

-

- Creat (µmol/L)

24

24

20

25

33

31

30

33

34

35

36

45

-

-

-

-

- Chol (µmol/L)

1.29

1.22

0.93

1.23

1.70

1.95

2.01

2.07

2.34

2.23

2.17

2.40

-

-

-

-

- Bili (µmol/L)

1

1

1

1

2

1

1

2

1

1

1

1

-

-

-

-

- Bile ac. (µmol/L)

5.6

21.7

11.3

21.4*

17.1

18.5

11.4

20.2

45.7

57.4

44.2

57.2

-

-

-

-

- Trig (µmol/L)

0.48

0.39

0.26*

0.29*

0.44

0.47

0.46

0.46

1.46

1.30

1.62

2.24

0.39

0.47

-

-

Urinalysis(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Vol (mL)

6.5

8.3

6.5

5.3

4.7

4.3

5.3

6.4

-

-

-

-

7.8

7.8

4.1

4.8

- pH

7.6

7.4

6.9*

6.5**

6.7

6.7

6.7

6.9

-

-

-

-

7.2

7.1

6.4

6.9*

- Sp. gravity (g/L)

1033

1030

1039

1040

1032

1036

1033

1028

-

-

-

-

1037

1032

1039

1035

- T-Prot (mg)

5.658

5.183

4.364

5.146

0.705

0.724

0.811

0.749

-

-

-

-

6.424

5.691

0.994

0.885

-T-Na (mmol)

0.447

0.510

0.330

0.325

0.294

0.237

0.411

0.477

-

-

-

-

0.594

0.459

0.214

0.327

- T-K (mmol)

1.296

1.226

1.321

0.944*

0.639

0.622

0.717

0.567

-

-

-

-

1.393

1.382

0.629

0.709

- T-Cl (mmol)

0.293

0.409

0.309

0.384

0.204

2.447

0.349

0.406*

-

-

-

-

0.346

0.316

0.153

0.221

- T-Glucose (µmol)

5.489

4.464

5.465

5.247

2.947

2.447

2.995

2.749

-

-

-

-

7.174

6.123

3.686

3.561

- T-Creat (µmol)

68.679

69.757

72.899

51.002*

31.320

32.636

39.059*

35.488*

-

-

-

-

88.0

79.5

48.4

44.6

- U-Prot (g/L)

0.88

0.65

0.67

1.01

0.16

0.19

0.16

0.13

-

-

-

-

0.83

0.75

0.25

0.21

- U-Na (mmol/L)

70.8

59.8

48.6

63.6

59.6

64.8

74.8

77.6

-

-

-

-

75.2

64.3

53.4

66.6

- U-K (mmol/L)

202.4

156.2

208.1

189.4

144.4

150.2

137.8

93.5

-

-

-

-

189.0

180.8

154.0

169.1

- U-Cl (mmol/L)

46.0

50.2

47.0

80.8*

38.8

49.0

63.6

64.4

-

-

-

-

43.8

43.2

37.0

46.0

- U-Gluc (mmol/L)

0.85

0.59

0.86

1.05

0.68

0.65

0.57

0.46

-

-

-

-

0.95

0.83

0.92

0.86

- U-Creat (µmol/L)

10576

8897

11487

10877

7197

8580

7493

6063

-

-

-

-

11684

10466

12287

10762

Pathology(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Number of animals/group

5

10

10

5

5

5

5

5

10

10

10

9

5

5

5

5

- Skin

(hair loss)

0

0

2

0

0

0

0

0

0

1

1

0

0

0

0

0

- Skin

(scabs)

0

0

2

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(depression)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(dermal inflammatory cell infiltrate)

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

- Skin

(epidermal ulceration)

0

0

1

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(regenerative hyperplasia)

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

- Skin

(abscessation)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Jejunum

(diverticulum)

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

- LN mesenteric

(dark)

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- LN mesenteric

(sinus erythrocytosis/ erythrophagocytosis)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

- Kidneys

(depression)

0

1

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- Kidneys

(pelvic dilation)

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

- Kidneys

(cortical tubular basophilia)

2

1

2

3

0

0

0

0

0

0

0

0

0

1

0

0

- Kidneys

(cortical tubules with hyaline droplets)

0

0

0

4

0

0

0

0

0

0

0

0

0

0

0

0

- Kidneys

(tubular dilation)

0

0

1

1

0

0

0

0

0

0

0

0

0

0

0

0

- Kidneys

(medullary cysts)

0

0

1

0

0

0

0

0

0

0

0

0

1

1

0

0

- Kidneys

(cortical scarring)

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- Stomach

(corpus depression)

0

0

0

0

0

0

0

0

1

0

0

1

0

0

0

0

- Ovaries

(cysts)

-

-

-

-

1

0

0

0

0

0

0

0

0

0

0

0

- Lungs and bronchi

(pale areas)

0

0

0

1

0

0

0

0

0

1

0

0

0

0

0

1

- Lungs and bronchi

(dark areas)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

- Lungs and bronchi

(incomplete collapse)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Lungs and bronchi

(aggregations of alveolar macrophages)

1

0

0

1

0

0

0

0

0

0

0

0

0

0

0

1

- Lungs and bronchi

(alveolitis)

1

0

0

0

0

0

0

1

0

0

0

0

0

0

0

1

- Lungs and bronchi

(foamy alveolar macrophages)

1

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

- Lungs and bronchi

(perivascular inflammatory cells)

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

- Liver

(hepatocyte hypertrophy, centrilobular)

0

0

0

5

0

0

0

5

0

0

0

0

0

0

0

0

- Liver

(hepatocyte vacuolation, periportal)

3

1

3

3

2

0

3

4

0

0

0

0

0

0

0

1

- Liver

(hepatocyte necrosis, focal)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

- Liver

(inflammatory cell infiltrate)

3

2

1

2

3

0

1

0

0

0

0

0

2

2

1

0

- Liver

(lipidosis, tension)

0

0

0

0

1

1

1

0

0

0

0

0

2

0

0

0

- Heart

(myocardial fibrosis/ inflammatory cells)

1

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

- Adrenals

(cortical hypertrophy)

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

- Skeletal muscle

(inflammatory cell infiltrate)

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

Prostate

(inflammatory cell infiltrate)

2

0

0

2

-

-

-

-

0

0

0

0

0

0

0

0

- Vagina

(dioestrus)

-

-

-

-

1

0

0

3

0

0

0

0

-

-

0

0

- Vagina

(metoestrus)

-

-

-

-

1

0

0

1

0

0

0

0

-

-

0

0

- Vagina

(oestrus)

-

-

-

-

2

0

0

0

0

0

0

0

-

-

0

0

- Vagina

(proestrus)

-

-

-

-

1

0

0

1

0

0

0

0

-

-

0

0

- Testes

(seminiferous tubular vacuolation)

0

0

0

1

-

-

-

-

-

-

-

-

0

0

-

-

- Epididymides

(degenerate spermatogenic cells in duct)

0

0

0

1

-

-

-

-

-

-

-

-

0

0

-

-

- Epididymides

(inflammatory cell infiltrate)

0

0

0

1

-

-

-

-

 

 

 

 

0

0

-

-

- General

(stained fur)

0

0

0

0

1

0

0

0

0

1

0

1

0

0

0

0

* P<0.05, ** P<0.01

Table 7       Absolute and adjusted organ weights (P0)

 

Doses (ppm)

Control

1200

3000

7500

Control

1200

3000

7500

Control

1200

3000

7500

Control

7500

Control

7500

male (toxicity group)

female (toxicity group)

female (reproduction group)

Male (rec.)

Female (rec.)

Number of animals/group

5

5

5

5

5

5

5

5

10

10

10

9

5

5

5

5

BODY WEIGHT (termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

464

430

430

407**

264

265

263

249*

339

333

321

296**

447

444

290

302

ADRENALS (termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.063

0.063

0.065

0.055

0.078

0.064

0.071

0.058

-

-

-

-

0.062

0.064

0.061

0.077

Adjusted weight (g)

0.059

0.061

0.066

0.061

0.077

0.063

0.070

0.062*

-

-

-

-

0.061

0.064

0.062

0.076*

BRAIN(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

2.199

2.195

2.161

2.150

2.077

2.017

2.021

1.964

-

-

-

-

2.214

2.259

2.022

2.015

Adjusted weight (g)

2.178

2.184

2.164

2.178

2.064

2.003

2.011

2.000

-

-

-

-

2.209

2.263

2.040

1.998

EPIDIDYMIDES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.282

1.297a

1.286a

1.207

-

-

-

-

-

-

-

-

1.310

1.290

-

-

Adjusted weight (g)

1.226

1.300a

1.289a

1.251

-

-

-

-

-

-

-

-

1.310

1.291

-

-

HEART(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

1.392

1.400

1.434

1.326

0.937

0.896

0.971

0.912

-

-

-

-

1.417

1.416

0.935

1.060

Adjusted weight (g)

1.330

1.368

1.444

1.410

0.917

0.876

0.957

0.967

-

-

-

-

1.414

1.418

0.963

1.032

KIDNEYS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

3.154

3.142

3.649

3.143

1.945

1.862

1.924

1.753

-

-

-

-

3.179

3.134

1.845

2.076

Adjusted weight (g)

2.960

3.041

3.680*

3.407*

1.922

1.837

1.907

1.819

-

-

-

-

3.169

3.144

1.889

2.032

LIVER(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

13.384

12.510

13.714

13.856

8.114

7.963

8.113

8.574

-

-

-

-

11.586

12.915

9.558

10.491

Adjusted weight (g)

12.283

11.936

13.891*

15.355**

8.023

7.865

8.045

8.831

-

-

-

-

11.535

12.967

9.706

10.343

OVARIES(termination for toxicity females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

-

-

-

-

0.092

0.092

0.086

0.081

0.088

0.088

0.085

0.085

-

-

0.085

0.101

Adjusted weight (g)

-

-

-

-

0.089

0.089

0.084

0.088

0.083

0.084

0.085

0.093

-

-

0.086

0.100

PROSTATE(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.097

1.163a

1.120a

1.016

-

-

-

-

-

-

-

-

1.071

1.042

-

-

Adjusted weight (g)

1.062

1.165a

1.122a

1.043

-

-

-

-

-

-

-

-

1.071

1.042

-

-

SEMINAL VESICLES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.903

2.145a

1.907a

2.095

-

-

-

-

-

-

-

-

2.032

1.944

-

-

Adjusted weight (g)

1.855

2.148a

1.910a

2.131

-

-

-

-

-

-

-

-

2.031

1.944

-

-

SPLEEN(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.811

0.723

0.753

0.679

0.553

0.554

0.507

0.456*

-

-

-

-

0.685

0.712

0.640

0.618

Adjusted weight (g)

0.763

0.698

0.761

0.744

-

-

-

-

-

-

-

-

0.684

0.714

0.624

0.635

TESTES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

3.312

3.492a

3.411a

3.511

-

-

-

-

-

-

-

-

3.646

3.466

-

-

Adjusted weight (g)

3.183

3.500a

3.419a

3.610

-

-

-

-

-

-

-

-

3.645

3.467

-

-

THYMUS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.327

0.254

0271

0.264

0.244

0.292

0.294

0.251

-

-

-

-

0.187

0.271

0.277

0.302

Adjusted weight (g)

0.046

0.077

0.059

0.079

0.233

0.280

0.285

0.283

-

-

-

-

0.185

0.273*

0.257

0.322

THYROID+PARAS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.019

0.020a

0.019a

0.016

0.014

0.014

0.015

0.014

0.016

0.016

0.016

0.015

0.017

0.014

0.015

0.014

Adjusted weight (g)

0.017

0.020a

0.019a

0.017

0.014

0.014

0.015

0.015

0.016

0.016

0.016

0.015

0.017

0.014

0.016

0.014

UTERUS + CERVIX + OVIDUCT(termination for toxicity females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

-

-

-

-

0.864

0.805

0.823

0.680

0.484

0.496

0.433

0.404

-

-

0.834

0.899

Adjusted weight (g)

-

-

-

-

0.857

0.798

0.818

0.699

0.470

0.487

0.435

0.428

-

-

0.884

0849

an=10

* P<0.05, ** P<0.01

Conclusions:
During the treatment period, a dose-dependent decrease in overall mean body weight gain was apparent in all groups, with a greater extent seen in groups treated at 7500 ppm of toxicity and recovery phase males and females given the test item when compared to controls; this decrease in overall weight gain was largely attributable to lower body weight gain and body weight losses during Week 1. This coincides with the reduced food intake seen in the first few days of treatment. A similar trend in body weight performance was evident among the reproductive phase females in the gestation and lactation periods and is considered an adverse effect.

The kidney changes detected in the males (hyaline droplets in tubules and cortical basophilic tubules) were consistent with well documented species-specific responses of the male rat in response to the administration with hydrocarbons. This effect was, therefore, not indicative of a hazard to human health. The liver changes (increased bodyweight adjusted liver weights and minimal centrilobular hepatocyte hypertrophy) were deemed as a reversible non-adverse adaptive response.

A No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore concluded to be 3000 ppm - where the decrease in overall mean body weight gain in 7500 ppm treatment groups was considered to be an adverse effect.
Executive summary:

OECD 422 (2017) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2 -yl)-1-methylcyclohexene was administered to 30 male and 50 female Crl:CD(SD) strain rats by dietary administration at dose levels of 1200, 3000 and 7500 ppm for up to 5 weeks. In addition, 10 male and 20 female control rats were fed untreated basal diet.

 

A summary of adult responses to the test item are described below;

 

Mortality- No mortality was observed during the test.

 

Clinical signs- No clinical signs or dose observations related to treatment were observed during the test.

 

Behavioural, functional and sensory assessments- Individuals were unaffected by the treatment.

  

Bodyweight- During the treatment period, a dose-dependent decrease in overall mean body weight gain was apparent in all groups, with a greater extent seen in groups treated at 7500 ppm, of toxicity and recovery phase males and females exposed to the test item when compared to controls; this decrease in overall weight gain was largely attributable to lower body weight gain and body weight losses during Week 1. This coincides with the reduced food intake seen in the first few days of treatment. A similar trend in body weight performance was evident among the reproductive phase females in the gestation and lactation period and was considered to be an adverse effect.

  

Food consumption and efficiency- Effects on food consumption were evident during Days 1 to 3 of treatment for toxicity and recovery phase females given 3000 ppm and males and females given 7500 ppm; these differences are thought to be the reason for the reduced weight gain/weight loss during Week 1 of the study. During the remainder of the study, food consumption effects were limited to reproductive phase females given 3000 or 7500 ppm.  In the gestation period, food consumption of females given 1200 or 3000 ppm was slightly lower than control on Days 0 to 2, at 7500 ppm food intake was lower than control from Days 0 to 16.   In the lactation period food consumption for female animals receiving treated diet at 3000 or 7500 ppm was lower than control from Days 1 to 12 of lactation. This again resulted in a low mean bodyweight gain during these periods.

 

Water consumption- No dose related responses were observed during the test.

 

Haematology, blood chemistry and urinalysis- Minor haematological and biochemical changes were evident at the end of the treatment period: high haematocrit concentration in all treated groups of males; low reticulocyte and neutrophil counts in Toxicity phase females given 7500 ppm; low monocyte count in Toxicity phase females given 3000 and 7500 ppm; high urea concentrations in all treated male groups and low triglyceride concentrations in males receiving 3000 or 7500 ppm.  At the end of lactation period reproductive females given 7500 ppm had low red cell distribution width; mean cell volume; prothrombin time and high urea and triglyceride concentrations.  In addition, some minor changes in urinary parameters were evident: the pH of the urine was more acidic; the urinary chloride was high and the total creatinine and total potassium was low in the males given 7500 ppm. In the toxicity phase females given 7500 ppm total creatinine and total chloride was high and urinary potassium was low.

  

Necropsy- No toxicologically significant effects were detected in terminal kill animals of either sex treated with the test item.

  

Organ weights- Changes in organ weights were limited to an increase in adjusted kidney and liver weights in male animals treated at 3000 or 7500 ppm and a non-dose dependent decrease in adjusted adrenal weight in Toxicity females treated at 7500 ppm.  The increased liver and kidney weights correlate with the histopathological findings, of which have been deemed as non-adverse.

 

Histopathological changes; Test item-related histopathological changes were observed in the liver of both sexes and the kidneys of males exposed to 7500 ppm.

Among males given 7500 ppm, minimal hyaline droplet accumulation was observed in the kidneys, with minimal to slight basophilic tubules in the cortex of the kidney.  These changes correlated with an increase in body weight adjusted kidney weight in the 7500 ppm group.  Hyaline droplet formation in the kidney tubules of male rats is a known effect of hydrocarbons (Greaves, 2012).  The droplets contain α2u globulin and their accumulation is considered to be related to the binding of the hydrocarbon with the globulin within lysosomes, with the resulting complex being poorly catabolised.  Prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover and the appearance of basophilic tubules most likely reflects this change.  These changes are limited to male rats.  Toxicity in humans through this mechanism is considered improbable as little or no α2u globulin is present in humans, and therefore these findings in the male rat kidney were considered to be of no relevance to man.

 

Based on these considerations, a No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore concluded to be 3000 ppm - where the decrease in overall mean body weight gain in 7500 ppm treatment groups was considered to be an adverse effect.

  

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 422 in the rat.

Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2016 - 28 May 2017
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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2016)
Deviations:
no
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 to +8 ºC
- Stability of formulation under test conditions: stable - confirmed at 100 and 20000 ppm.
- Solubility and stability of the test substance in the diet: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

OTHER SPECIFICS: n/a
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: ca. 71 days old and females: ca. 85 days old
- Weight at study initiation: males: 319 - 383 g and females: 239 - 293 g
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. An Aspen chew block was provided for envirnmental enrichment throughout the study period. A plastic shelter was also included in each cage throughout the study period, except during pairing and lactation.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet. A 100 g sample of each batch of diet used were retained frozen (-10 to -30°C) until finalization. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Provided ad libitum.
- Acclimation period: Males: six days prior to the commencement of treatment. Females: six days prior to the commencement of estrous cycle evaluation.

DETAILS OF FOOD AND WATER QUALITY: See above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ºC
- Humidity (%): 40 - 70 %
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 04 April 2017 To: 28 May 2017
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: n/a

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Basal diet (SDS VRF1 Certified)
- Storage temperature of food: not reported

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analyzed for achieved concentration of the test item.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as Day 0 of pregnancy.
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): housed individually in a cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Minimum of 5 weeks with a 2 week recovery period.
Frequency of treatment:
Continuous
Duration of test:
Minimum of 5 weeks with a 2 week recovery period.
Dose / conc.:
1 200 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
7 500 ppm
Dose / conc.:
73.8 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 1200 ppm dose group
Dose / conc.:
181 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 3000 ppm dose group
Dose / conc.:
438 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 7500 ppm dose group
No. of animals per sex per dose:
Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
1200 ppm (toxicity test) = 10
3000 ppm (toxicity test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
1200 ppm (reproduction test) = 10
1200 ppm (toxicity test) = 5
3000 ppm (reproduction test) = 10
3000 ppm (toxicity test) = 5
7500 ppm (reproduction test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 1200, 3000 and 7500 ppm) were chosen in conjunction with the sponsor based upon the results obtained in a 14 day preliminary study.
- Rationale for animal assignment (if not random): n/a
- Rationale for selecting satellite groups: determine if toxic effects in F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): n/a
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatisation, before feeding of the treated diets on the Day that treatment commenced (Day 1) and twice weekly thereafter (including termination).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study)
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 14 of lactating (females) and test termination (males and females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: lactating females: 5, test temination: 5 males and 5 females.
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 14 of lactating (females), test termination (male and females) and recovery week 4 (males)
- Animals fasted: Yes
- How many animals: lactating females: 5; test termination: 5 males and 5 females; recovery group: all surviving males.
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6 (day before scheduled termination) and recovery group week 2.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and weekly thereafter.
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

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.
- after pairing until mating (reproductive females only).
- four days before scheduled termination (all females except premature decedents).
- Females showed no evidence of mating: following completion of pairing period female was separated from the male and vaginal smearing continued for up to five days or until the first estrous smear was seen. If a female shows an estrous smear during this period, she was killed and subject to macroscopic examination.

THYROID HORMONE ANALYSIS: Yes
- Time schedule: - Day 4 of age (F1 offspring, two females per litter (where possible) - no pups were eliminated when total litter size dropped below ten/litter).
- one pup for T3/T4 (serum)
- one pup for TSH (plasma)
- Day 13 of age (F1 offspring, two males and two females per litter (where possible)
- two for T3/T4 (serum): where possible one male and one female
- two for TSH (plasma): where possible one male and one female
- Termination (All Toxicity and Recovery phase F0 males and all Reproductive phase F0 females surviving to scheduled termination)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other:
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
See attched report
Indices:
Offspring viability indices including; viability index, lactation index and sex ratio.
Historical control data:
Historical control data was available to the Study Director.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or dose observations related to treatment were seen in the treatment, gestation, lactation period or recovery period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The overall mean bodyweight change from Day 1 to Day 8 for male animals receiving treated diet at 1200 or 3000 ppm was similar to control. Male animals receiving treated diet at 7500 ppm had a reduced mean bodyweight gain from Day 1 to 8 when compared to controls; this is due to a bodyweight loss in 5 animals between Day 1 and 4. All animals at 7500 ppm gained weight between Days 4 and 8, and exceeded their Day 1 bodyweight. Bodyweight change from Day 8 to 15 for all male treated groups was similar to control. However the body weight changes between Day 15 and 22 for all treated male groups were lower than control, with males receiving 3000 ppm showing a mean body weight loss. Bodyweight changes from Days 22 to 29 and Days 29 to 36 shows that male animals receiving 1200 or 3000 ppm return to comparable performance to control, however the weight gain of males receiving 7500 ppm remain significantly lower. The overall body weight change for the treatment period (Days 1 to 36) for male animals receiving treated diet at 1200 ppm and 3000 ppm was lower than control (82 % and 83 %), however male animals receiving treated diet at 7500 ppm was significantly lower than control (70 %). During the recovery priod, mean weight gain of animals previously treated at 7500 ppm was slightly greater than the control.

The overall mean bodyweight change from Day 1 to Day 8 for all treated female groups was lower than control, with an overall mean bodyweight loss for females receiving 3000 or 7500 ppm. Between Day 1 and Day 4 individual bodyweight losses or bodyweight stasis were seen in all treated groups and control (7/20 for control, 11/15 at 1200 ppm and 9/15 at 3000 ppm and 17/20 at 7500 ppm). By Day 8 several females had not obtained parity with Day 1 (3/20 for control, 2/15 at 1200 ppm, 8/15 at 3000 ppm and 15/20 at 7500 ppm). Bodyweight changes from Day 8 to 29 for all female treated groups was similar to control, with the exception of Day 15 to 22 where an mean body weight loss was seen in the control group. Bodyweight change from Days 29 to 36 for all female treated groups was slightly lower than control. The overall bodyweight change for the Toxicity and Recovery females during the treatment period (Days 1 to 36) was lower than control for all treated groups. Most significantly females receiving treated diet at 7500 ppm, gained only 52% of the control value, this was due to the body weight losses and stasis seen in the first week of study. However after 2 weeks of recovery females previously receiving 7500 ppm had a higher than control overall body weight change (Day R1 to R15). The mean weights of recovery animals that had receved 7500 ppm were higher than controls, but this was because the animals pre-selected for recovery had incidentally higher weights.

In the gestation period, reproductive phase females receiving 7500 ppm had statistically lower than control body weights and body weight gain up to Day 20, overall body weight change from Day 1 to 20 of gestation was 79 % of control. Females receiving 1200 or 3000 ppm had slightly reduced overall body weight gain when compared to controls (95 % and 92 % respectively).

In the lactation period, reproductive phase females receiving 7500 ppm had statistically lower than control body weights and body weight gain up to Day 13 of lactation, overall body weight change from Day 1 to 13 of lactation was 70 % of control. Females receiving 3000 ppm had statistically lower than control mean bodyweight values on Days 4, 7 and 10, however the increased gain between Days 10 and 13 resulted in only a minor difference in overall bodyweight change (Days 1 to 13) compared to control (94 %).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption for male animals receiving treated diet at 7500 ppm was slightly lower than control animals on Days 1 to 3 of treatment; however consumption values were comparable from Days 4 to 7 through to Days 32 to 35. The food intake of male animals treated at 1200 or 3000 ppm were unaffected. During the recovery period food consumption for males previously receiving 7500 ppm was similar to control.

Food consumption for female animals receiving treated diet at 1200 ppm was slightly lower than control on Days 1 to 3, but was comparable by Days 4 to 7. The food intake of female animals receiving 3000 or 7500 ppm treated diet were lower than control on Days 1 to 3, and remained slightly lower on Days 4 to 7 , however by Days 8-10 food intake was comparable to control. In the recovery period females previously receiving treated diet at 7500 ppm had higher food intake than control animals.

In the gestation period, food consumption for reproductive phase female animals receiving treated diet at 1200 or 3000 ppm was slightly lower than control on Days 0 to 2 (note that 2 control females had consumption that was somewhat higher than remaining controls), but was comparable by Days 3 to 6. The food intake of female animals receiving 7500 ppm treated diet was lower than control from Days 0 to 16, but was similar to control on Days 17 to 19.

In the lactation period, food consumption for reproductive phase female animals receiving treated diet at 3000 or 7500 ppm was lower than control from Days 1 to 9 and Day 1 to 12 of lactation, respectively. On days 10 to 12 females at 1200 ppm consumed slightly less food than controls but was not statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of haematological parameters at the end of the treatment period revealed higher than control haematocrit concentration in all groups of Toxicity phase males compared to control, though no dose response was evident and no similar finding was observed in females. In addition reticulocyte and neutrophil counts were lower than control in Toxicity phase females receiving treated diet at 7500 ppm. A non-dose dependent decrease in monocytes was also evident in females receiving 3000 or 7500 ppm.

Assessment of haematological parameters at the end of the lactation period revealed lower than control red cell distribution width and a non-dose dependent decrease in mean cell volume and prothrombin time in reproductive phase females receiving 7500 ppm.

All other inter-group differences from controls in the toxicity phase and reproductive phase animals at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical changes in the plasma of the toxicity phase males at the end of the treatment period revealed higher than control urea concentrations and lower glucose concentrations in all treated groups and lower than control triglyceride concentrations in males receiving 3000 or 7500 ppm. In Week 2 of recovery the biochemical changes in the plasma had demonstrated full recovery.

Biochemical changes in the plasma of the reproductive phase females at the end of the lactation period revealed higher than control urea and triglyceride (not statistically significant) concentrations in females receiving 7500 ppm althoughsimilar findings were not observed in toxicity females at the end of the treatment.

All other inter-group differences from controls in the toxicity phase and reproductive phase animals at the end of treatment were minor in nature, lacked dose-relationship, were due to anomalies in the control data or were confined to one sex; these were consequently attributed to normal biological variation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Assessment of urinary parameters at the end of the treatment period revealed the following differences from Control;

In the toxicity phase male animals receiving treated diet at 3000 or 7500 ppm the pH of the urine was more acidic, the total creatinine and total potassium was lower than control in the males receiving 7500 ppm and the urinary chloride was higher than control.

In the female toxicity phase animals receiving diet at 7500 ppm total creatinine and total chloride was higher than control and urinary potassium was lower than control. Total creatinine was also high among females at 3000 ppm.

In Week 2 of recovery, all of the aforementioned urinary changes had demonstrated full recovery.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Sensory reactivity and grip strength were considered unaffected by treatment. There were no consistent intergroup differences in grip strength between toxicity and reproductive phase females.

Motor activity scores for females given the test item were considered to be unaffected by treatment. Statistical significance was obtained for the overall low and high beam breaks for all groups of treated males (except low beams at 1200 ppm) when compared to controls, and although statistical significance was not attained for the final four 6-minute recording periods, the behaviour of the treated males was atypical, with no general increase in activity levels which should have been apparent towards the end of the recording period. The total high beam scores in all treated groups were slightly below the historical control data (HCD) range but total low beam scores at 7500 ppm were well within the HCD range.

In the recovery period the overall low and high beam breaks for males previously fed 7500 ppm continued to stay lower than control, as seen in the treatment period. No statistical significance was obtained for the overall scores however two recordings in the low beam breaks were statistically significant. As seen in the treatment period there was no general increase in activity levels over the last four 6-minute recording periods.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At scheduled termination of the toxicity phase animals after 5 weeks of treatment, decreased terminal body weights were evident in males treated at 7500ppm. Analysis of organ weights for the toxicity phase animals revealed increased adjusted kidney and liver weights in male animals treated at 3000 or 7500 ppm when compared to controls. Female toxicity phase animals treated at 7500 ppm also had decreased terminal body weights at scheduled termination. A non-dose dependent decreased adjusted adrenal weight was also apparent in the toxicity females treated at 7500 ppm.

After two weeks off-dose the recovery phase males previously treated at 7500 ppm had higher than control adjusted thymus weights however as this was not present at the end of the treatment period this is thought to be unrelated to treatment. The adjusted adrenal weights for recovery phase females previously receiving 7500 ppm after two weeks off-dose were higher than control.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed revealed no test item related lesions.

The incidence and distribution of all findings were considered to be incidental and unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test item were seen in the liver of both sexes and the kidneys of males;

Liver - Centrilobular hepatocyte hypertrophy was seen in both sexes given 7500 ppm of the test item. The liver changes (increased bodyweight adjusted liver weights and minimal centrilobular hepatocyte hypertrophy) are deemed as a reversible non-adverse adaptive response.

Kidney - Hyaline droplets were seen in the cortical tubules of males given 7500 ppm of the test item. A slight increase in the incidence and severity of cortical tubular basophilia was seen in males given 7500 ppm of the test item which was considered to be treatment related. The kidney changes detected in the males (hyaline droplets in tubules and cortical basophilic tubules) were consistent with well documented species-specific responses of the male rat in response to the administration with hydrocarbons. This effect is, therefore, not indicative of a hazard to human health.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 7500 ppm, there was a statistically lower mean implantation count, which reflected in part one litter with only 3 implantations. At 3000 ppm, mean implantation count was slightly lower than control, but at 1200 ppm the mean implantation count was similar to control. The mean litter sizes reflected the number of implantations, with values at 7500 ppm being statistically different until Day 4.

The lower litter size at 7500 ppm appeared to reflect the lower maternal body weights at conception, an expected relationship.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All pregnant females successfully gave birth to live young.
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
There was considered to be no effect of parental treatment on offspring survival.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days. The gestation index was 100 % in all groups.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days. The gestation index was 100 % in all groups.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Mating performance was unaffected by treatment, with the exception of one female receiving 7500 ppm all females achieved pregnancy.
Details on maternal toxic effects:
During the treatment period, a dose-dependent decrease in overall mean body weight gain was apparent in all maternal groups, with a greater extent seen in groups treated at 7500 ppm, of toxicity and recovery phase males and females given the test item when compared to controls; this decrease in overall weight gain was largely attributable to lower body weight gain and body weight losses during Week 1. This coincides with the reduced food intake seen in the first few days of treatment and is considered to be an adverse effect.

Based on these considerations, the No Observed Adverse Effect Level (NOAEL) for general systemic toxicity was concluded to be 3000 ppm.

There was no effect of treatment on mating performance, fertility, litter size, or offspring survival or clinical condition. The lower litter size at 7500 ppm appeared to reflect the lower maternal body weights at conception, an expected relationship.
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
181 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean body weights for male offspring from the 7500 ppm group were statistically lower than control on Days 7 and 13 of age. Male offspring from the 3000 ppm group were statistically lower than control on Day 13 of age. The overall bodyweight change (Day 1 to 13 of age) for male offspring from groups 3000 or 7500 ppm was statistically lower than control. The 3000 ppm male offspring gained 14 % less than control and the 7500 ppm group gained 18% less than control.

Mean body weights for female offspring from the 7500 ppm group were statistically lower than control on Days 1, 7 and 13 of age. The overall bodyweight change from Days 1 to 13 of age for group 7500 ppm female offspring was 20% lower than control.

The reduction in body weight gain of the offspring derived from parent animals given 7500 ppm was considered to be an adverse effect.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
At 7500 ppm, the mean number of uterine implantation sites and therefore total litter size was lower than control. This likely occurred as a consequence of the lower absolute body weight of the females at mating (which occurred as a result of the low food intake during the first few days of treatment) as there is a strong correlation between absolute body weight and the number of eggs ovulated (Harper, 1964) with lighter females releasing fewer eggs; the mean number of implantations in females in the 7500 ppm group which had Day 0 gestation bodyweights within the concurrent control range was similar to that in weight matched control females. The lower mean numbers of implantations at 1200 or 3000 ppm were not statistically significantly different to controls and within the historical control data range and thus no effect of treatment is inferred.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was considered to be no effect of parental treatment on sex ratio.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
At 7500 ppm, there was a statistically lower mean implantation count, which reflected in part one litter with only 3 implantations. At 3000 ppm, mean implantation count was slightly lower than Control, but at 1200 ppm the mean implantation count was similar to control. The mean litter sizes reflected the number of implantations, with values at 7500 ppm being statistically different until Day 4

This likely occurred as a consequence of the lower absolute body weight of the females at mating (which occurred as a result of the low food intake during the first few days of treatment) as there is a strong correlation between absolute body weight and the number of eggs ovulated (Harper, 1964) with lighter females releasing fewer eggs; the mean number of implantations in females in the 7500 ppm group which had Day 0 gestation bodyweights within the concurrent control range was similar to that in weight matched control females. The lower mean numbers of implantations at 1200 and 3000 ppm were not statistically significantly different to controls and within the historical control data range and thus no effect of treatment is inferred.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was considered to be no effect of parental treatment on offspring survival to Day 13 of age.
External malformations:
no effects observed
Description (incidence and severity):
There were no clinical signs observed among the F1 litters that were clearly attributable to parental treatment of the test item
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
Within the scope of this study it was not possible to establish with certainly the aetiology of the potentially adverse reduction in birth weight and subsequent body weight gain of the male offspring in the 3000 and 7500 ppm group and female offspring in the 7500 ppm group. This may have been as a result of the parent females being smaller and consuming less food (thus there being reduced levels of nutrients available to pass to the fetus in utero and reduced quality milk in lactation), or a direct effect of the test item by cross‑placental in utero exposure followed by exposure post birth in the milk. The magnitude of the reduced offspring body weight gain at 7500 ppm (19-20 %) is considered adverse.

The study also included a screen for endocrine disruptor relevant endpoints. Since there was no conclusive effect of treatment on T4 levels, anogenital distance, nipple counts, external genitals in the offspring, estrus cycles and no macroscopic or microscopic changes in male and female reproductive organs there was no indication that the test item is an endocrine disruptor.
Key result
Dose descriptor:
NOAEL
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other:
Remarks:
Reduction in pup body weight gain may be a consequence of lower parental weight/ food consumption during gestation and lactation periods.
Key result
Dose descriptor:
NOAEL
Effect level:
181 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Remarks on result:
other: Reduction in pup body weight gain may be a consequence of lower parental weight/ food consumption during gestation and lactation periods.
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
There were no macroscopic findings observed in the offspring that died prior to scheduled termination or among those offspring killed on Day 13 of age that were attributable to parental treatment
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
3 000 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
181 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Table 4       Group mean acheived dose rates for each treatment group (in mg/kg bw/day)

 

Time period

Pre-mating and recovery animals

Gestation

Lactation

1200 ppm

3000 ppm

7500 ppm

1200 ppm

3000 ppm

7500 ppm

1200 ppm

3000 ppm

7500 ppm

Male

Female

Mean

Male

Female

Mean

Male

Female

Mean

Female

Day 1 -35

73.5

74.1

73.8

186

175

181

451

424

438

-

-

-

-

-

-

Day 0 - 19

-

-

-

-

-

-

-

-

-

88

215

521

-

-

-

Day 1 - 12

-

-

-

-

-

-

-

-

-

-

-

-

190

456

99

Table 5      Mean body weights and body weight gains/ losses (F0)

Sex / Day

Bodyweight (g)

Control

1200 ppm

3000 ppm

7500 ppm

MALES (TOXICITY TEST GROUP)

Day 1

353

352

351

352

Day 4

366

364

360

353

Day 8

(change from Day 1)

384

(+32)

385

(+32)

385

(+34)

374

(+22)

Day 11

400

400

399

390

Day 15

(change from Day 8)

413

(+28)

409

(+24)

413

(+28)

401

(+27)

Day 18

419

406

408

404

Day 22

(change from Day 15)

432

(+20)

414

(+5**)

411

(-3**)

412

(+11)

Day 25

437

425

419

412

Day 29

(change from Day 22)

451

(+19)

433

(+20)

432

(+21)

422*

(+10)

Day 32

459

440

437

427*

Day 36

(change from Day 29)

471

(+20)

450

(+17)

449

(+17)

435*

(+13)

Overall BW gain Day 1 – Day 36

(% gain/ loss)

+118

+97

+98

+83**

MALES (RECOVERY GROUP)

Day 1

456

-

-

437

Day 4

461

-

-

448

Day 8

469

-

-

458

Day 11

476

-

-

466

Day 15

476

-

-

467

Overall BW gain Day 1 – Day 15 (recovery)

(% gain/ loss)

+20

-

-

+30

FEMALES (TOXICITY TEST GROUP)

Day 1

263

269

268

265

Day 4

268

267

261

256**

Day 8

(change from Day 1)

272

(+9)

274

(+5)

263

(-5**)

260**

(-6**)

Day 11

276

276

269

265*

Day 15

(change from Day 8)

276

(+5)

264

(+3)

270

(+7)

264*

(+4)

Day 18

270

264

268

262

Day 22

(change from Day 15)

273

(-3)

269

(+4*)

273

(+4*)

267

(+4**)

Day 25

274

273

278

273

Day 29

(change from Day 22)

276

(+3)

277

(+8)

279

(+5)

273

(+6)

Day 32

281

278

280

271

Day 36

(change from Day 29)

285

(+9)

280

(+3)

281

(+2)

277

(+4)

Overall BW gain Day 1 – Day 36

(% gain/ loss)

+21

+14

+17

+11

FEMALES (RECOVERY GROUP)

Day 1

282

-

-

290

Day 4

285

-

-

302

Day 8

290

-

-

304

Day 11

292

-

-

309

Day 15

290

-

-

304

Overall BW gain Day 1 – Day 15 (recovery)

(% gain/ loss)

+8

-

-

+14

FEMALES (REPRODUCTION GROUP)

Day 0

Gestation

281

280

272

266*

Day 3

(change from Day 0)

297

(+16)

296

(+16)

286

(+15)

275**

(+10*)

Day 7

(change from Day 3)

309

(+11)

312

(+16)

303

(+17)

288*

(+13)

Day 10

(change from Day 7)

327

(+18)

327

(+15)

316

(+12)

299**

(+11*)

Day 14

(change from Day 10)

341

(+14)

340

(+13)

330

(+14)

313**

(+14)

Day 17

(change from Day 14)

379

(+38)

370

(+30*)

361

(+32*)

434**

(+30)

Day 20

(change from Day 17)

427

(+49)

419

(+48)

407

(+46)

381**

(+38*)

Overall BW gain Day 0 – Day 20 (gestation)

(% gain/ loss)

+146

+138

+135

+115**

Day 1

Lactation

324

314

312

293**

Day 4

(change from Day 1)

335

(+11)

326

(+12)

315*

(+2)

293**

(+1*)

Day 7

(change from Day 4)

351

(+16)

39

(+13)

323*

(+8)

297**

(+3*)

Day 10

(change from Day 7)

365

(+15)

363

(+23)

339*

(+16)

323**

(+27)

Overall BW gain Day 1 – Day 10 (lactation)

(% gain/ loss)

+42

+48

+27*

+30*

Day 13

(change from Day 10)

Lactation

377

(+11)

365

(+3)

362

(+23)

330**

(+6)

Overall BW gain Day 1 – Day 13 (lactation)

(% gain/ loss)

+53

+51

+50

+37*

* statistically significantly different from control values p < 0.05

** statistically significantly different from control values p < 0.01

Table 6      Haematology, clinical chemistry, urinalysis and pathology findings (F0)

 

Doses (ppm)

Control

1200

3000

7500

Control

1200

3000

7500

Control

1200

3000

7500

Control

7500

Control

7500

male (toxicity group)

female (toxicity group)

female (reproduction group)

Male (rec.)

Female (rec.)

Number of animals/group

5

5

5

5

5

5

5

5

10

10

10

9

5

5

5

5

Haematology(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Hb (g/dl)

15.0

15.7

15.3

15.2

15.2

15.0

15.6

15.1

14.9

15.0

14.5

15.3

-

-

-

-

- RBC (1012/L)

7.78

7.93

7.95

8.02

7.72

7.80

7.77

7.84

7.19

7.37

7.22

7.51

-

-

-

-

- Hct (%)

0.415

0.432*

0.432*

0.432*

0.418

0.414

0.419

0.421

0.424

0.423

0.423

0.428

-

-

-

-

- Retic (1012/L)

0.174

0.138

0.168

0.156

0.142

0.120

0.115

0.096*

0.227

0.219

0.254

0.229

-

-

-

-

- MCH (pg)

19.2

19.8

19.2

19.0

19.7

19.3

20.0

19.3

20.7

20.3

20.1

20.4

-

-

-

-

- MCV (fl)

53.4

54.5

54.4

53.9

54.1

53.1

54.0

53.7

59.1

57.3

58.6

57.0*

-

-

-

-

- MCHC (g/df)

36.1

36.3

35.3

35.3

36.3

36.3

37.1

36.0

35.1

35.4

34.4

35.8

-

-

-

-

- RDW (%)

12.7

11.9

12.2

12.0

11.4

11.5

11.1

11.2

14.3

13.9

13.8

13.1**

-

-

-

-

- WBC (109/L)

8.60

9.43

11.23

8.67

8.08

9.22

10.19

9.07

9.36

8.63

11.84

9.39

-

-

-

-

- Neut (109/L)

1.15

1.38

1.17

1.56

1.19

1.10

0.92

0.64*

4.00

3.76

5.19

4.63

-

-

-

-

- Lymph (109/L)

7.04

7.52

9.54

6.69

6.29

7.55

8.72

7.92

4.79

4.38

5.93

4.22

-

-

-

-

- Mono (109/L)

0.18

0.25

0.26

0.22

0.33

0.28

0.21*

0.23*

0.32

0.25

0.47

0.33

-

-

-

-

- Eos (109/L)

0.08

0.13

0.09

0.08

0.10

0.12

0.14

0.13

0.13

0.14

0.09

0.11

-

-

-

-

- Bas (109/L)

0.05

0.06

0.06

0.04

0.03

0.04

0.05

0.03

0.03

0.03

0.05

0.03

-

-

-

-

- LUC (109/L)

0.09

0.10

0.12

0.08

0.15

0.14

0.16

0.11

0.09

0.07

0.12

0.07

-

-

-

-

- PT (secs)

22.2

23.5

20.7

19.8

20.3

21.6

21.3

22.3

23.4

21.1

21.8

20.1**

-

-

-

-

- PLT (109/L)

795

753

794

721

905

910

854

828

858

783

878

837

-

-

-

-

- APTT (secs)

20.3

18.8

20.3

19.3

19.6

17.9

16.1

19.3

15.9

14.8

15.0

13.6

-

-

-

-

Blood chemistry(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Urea (µmol/L)

4.20

5.42**

5.27**

5.87**

5.96

5.65

5.78

5.99

11.04

11.50

12.09

15.90*

6.68

6.01

-

-

- Glucose (µmol/L)

7.79

6.46**

6.41**

6.16**

7.30

6.85

6.36

3.80

6.22

5.91

6.02

5.71

-

-

-

-

- Tot. Prot. (g/L)

63

61

61

61

70

68

68

69

60

61

60

59

-

-

-

-

- Albumin (g/L)

35

35

35

35

40

39

38

39

33

33

33

33

-

-

-

-

- A/G ratio

1.27

1.33

1.32

1.38*

1.31

1.32

1.29

1.32

1.25

1.19

1.24

1.23

-

-

-

-

- Na+ (mmol/L)

144

145

145

143

143

143

142

144*

137

139

138

138

-

-

-

-

- K+ (mmol/L)

4.0

4.0

4.1

4.2

3.6

3.8

4.2**

3.9**

4.2

4.8

4.6

4.4

-

-

-

-

- Cl- (mmol/L)

101

101

102

100

100

100

101

102

95

97

96

94

-

-

-

-

- Ca++ (mmol/L)

2.51

2.56

2.56

2.56

2.71

2.70

2.74

2.68

2.38

2.44

2.34

2.28

-

-

-

-

- P (mmol/L)

2.19

2.13

2.26

2.24

1.88

1.98

2.06

1.85

3.29

3.41

3.38

3.44

-

-

-

-

- ASAT (U/L)

69

71

78

77

86

61**

62**

62**

162

179

149

124

-

-

-

-

- ALAT (U/L)

35

33

33

42*

33

27

28

48

96

107

100

87

-

-

-

-

- ALP (U/L)

96

117

140*

125*

48

43

44

42

95

107

111

95

-

-

-

-

- Creat (µmol/L)

24

24

20

25

33

31

30

33

34

35

36

45

-

-

-

-

- Chol (µmol/L)

1.29

1.22

0.93

1.23

1.70

1.95

2.01

2.07

2.34

2.23

2.17

2.40

-

-

-

-

- Bili (µmol/L)

1

1

1

1

2

1

1

2

1

1

1

1

-

-

-

-

- Bile ac. (µmol/L)

5.6

21.7

11.3

21.4*

17.1

18.5

11.4

20.2

45.7

57.4

44.2

57.2

-

-

-

-

- Trig (µmol/L)

0.48

0.39

0.26*

0.29*

0.44

0.47

0.46

0.46

1.46

1.30

1.62

2.24

0.39

0.47

-

-

Urinalysis(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Vol (mL)

6.5

8.3

6.5

5.3

4.7

4.3

5.3

6.4

-

-

-

-

7.8

7.8

4.1

4.8

- pH

7.6

7.4

6.9*

6.5**

6.7

6.7

6.7

6.9

-

-

-

-

7.2

7.1

6.4

6.9*

- Sp. gravity (g/L)

1033

1030

1039

1040

1032

1036

1033

1028

-

-

-

-

1037

1032

1039

1035

- T-Prot (mg)

5.658

5.183

4.364

5.146

0.705

0.724

0.811

0.749

-

-

-

-

6.424

5.691

0.994

0.885

-T-Na (mmol)

0.447

0.510

0.330

0.325

0.294

0.237

0.411

0.477

-

-

-

-

0.594

0.459

0.214

0.327

- T-K (mmol)

1.296

1.226

1.321

0.944*

0.639

0.622

0.717

0.567

-

-

-

-

1.393

1.382

0.629

0.709

- T-Cl (mmol)

0.293

0.409

0.309

0.384

0.204

2.447

0.349

0.406*

-

-

-

-

0.346

0.316

0.153

0.221

- T-Glucose (µmol)

5.489

4.464

5.465

5.247

2.947

2.447

2.995

2.749

-

-

-

-

7.174

6.123

3.686

3.561

- T-Creat (µmol)

68.679

69.757

72.899

51.002*

31.320

32.636

39.059*

35.488*

-

-

-

-

88.0

79.5

48.4

44.6

- U-Prot (g/L)

0.88

0.65

0.67

1.01

0.16

0.19

0.16

0.13

-

-

-

-

0.83

0.75

0.25

0.21

- U-Na (mmol/L)

70.8

59.8

48.6

63.6

59.6

64.8

74.8

77.6

-

-

-

-

75.2

64.3

53.4

66.6

- U-K (mmol/L)

202.4

156.2

208.1

189.4

144.4

150.2

137.8

93.5

-

-

-

-

189.0

180.8

154.0

169.1

- U-Cl (mmol/L)

46.0

50.2

47.0

80.8*

38.8

49.0

63.6

64.4

-

-

-

-

43.8

43.2

37.0

46.0

- U-Gluc (mmol/L)

0.85

0.59

0.86

1.05

0.68

0.65

0.57

0.46

-

-

-

-

0.95

0.83

0.92

0.86

- U-Creat (µmol/L)

10576

8897

11487

10877

7197

8580

7493

6063

-

-

-

-

11684

10466

12287

10762

Pathology(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Number of animals/group

5

10

10

5

5

5

5

5

10

10

10

9

5

5

5

5

- Skin

(hair loss)

0

0

2

0

0

0

0

0

0

1

1

0

0

0

0

0

- Skin

(scabs)

0

0

2

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(depression)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(dermal inflammatory cell infiltrate)

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

- Skin

(epidermal ulceration)

0

0

1

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(regenerative hyperplasia)

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

- Skin

(abscessation)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Jejunum

(diverticulum)

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

- LN mesenteric

(dark)

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- LN mesenteric

(sinus erythrocytosis/ erythrophagocytosis)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

- Kidneys

(depression)

0

1

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- Kidneys

(pelvic dilation)

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

- Kidneys

(cortical tubular basophilia)

2

1

2

3

0

0

0

0

0

0

0

0

0

1

0

0

- Kidneys

(cortical tubules with hyaline droplets)

0

0

0

4

0

0

0

0

0

0

0

0

0

0

0

0

- Kidneys

(tubular dilation)

0

0

1

1

0

0

0

0

0

0

0

0

0

0

0

0

- Kidneys

(medullary cysts)

0

0

1

0

0

0

0

0

0

0

0

0

1

1

0

0

- Kidneys

(cortical scarring)

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- Stomach

(corpus depression)

0

0

0

0

0

0

0

0

1

0

0

1

0

0

0

0

- Ovaries

(cysts)

-

-

-

-

1

0

0

0

0

0

0

0

0

0

0

0

- Lungs and bronchi

(pale areas)

0

0

0

1

0

0

0

0

0

1

0

0

0

0

0

1

- Lungs and bronchi

(dark areas)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

- Lungs and bronchi

(incomplete collapse)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Lungs and bronchi

(aggregations of alveolar macrophages)

1

0

0

1

0

0

0

0

0

0

0

0

0

0

0

1

- Lungs and bronchi

(alveolitis)

1

0

0

0

0

0

0

1

0

0

0

0

0

0

0

1

- Lungs and bronchi

(foamy alveolar macrophages)

1

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

- Lungs and bronchi

(perivascular inflammatory cells)

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

- Liver

(hepatocyte hypertrophy, centrilobular)

0

0

0

5

0

0

0

5

0

0

0

0

0

0

0

0

- Liver

(hepatocyte vacuolation, periportal)

3

1

3

3

2

0

3

4

0

0

0

0

0

0

0

1

- Liver

(hepatocyte necrosis, focal)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

- Liver

(inflammatory cell infiltrate)

3

2

1

2

3

0

1

0

0

0

0

0

2

2

1

0

- Liver

(lipidosis, tension)

0

0

0

0

1

1

1

0

0

0

0

0

2

0

0

0

- Heart

(myocardial fibrosis/ inflammatory cells)

1

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

- Adrenals

(cortical hypertrophy)

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

- Skeletal muscle

(inflammatory cell infiltrate)

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

Prostate

(inflammatory cell infiltrate)

2

0

0

2

-

-

-

-

0

0

0

0

0

0

0

0

- Vagina

(dioestrus)

-

-

-

-

1

0

0

3

0

0

0

0

-

-

0

0

- Vagina

(metoestrus)

-

-

-

-

1

0

0

1

0

0

0

0

-

-

0

0

- Vagina

(oestrus)

-

-

-

-

2

0

0

0

0

0

0

0

-

-

0

0

- Vagina

(proestrus)

-

-

-

-

1

0

0

1

0

0

0

0

-

-

0

0

- Testes

(seminiferous tubular vacuolation)

0

0

0

1

-

-

-

-

-

-

-

-

0

0

-

-

- Epididymides

(degenerate spermatogenic cells in duct)

0

0

0

1

-

-

-

-

-

-

-

-

0

0

-

-

- Epididymides

(inflammatory cell infiltrate)

0

0

0

1

-

-

-

-

 

 

 

 

0

0

-

-

- General

(stained fur)

0

0

0

0

1

0

0

0

0

1

0

1

0

0

0

0

* P<0.05, ** P<0.01

Table 7     Absolute and adjusted organ weights (F0)

 

Doses (ppm)

Control

1200

3000

7500

Control

1200

3000

7500

Control

1200

3000

7500

Control

7500

Control

7500

male (toxicity group)

female (toxicity group)

female (reproduction group)

Male (rec.)

Female (rec.)

Number of animals/group

5

5

5

5

5

5

5

5

10

10

10

9

5

5

5

5

BODY WEIGHT (termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

464

430

430

407**

264

265

263

249*

339

333

321

296**

447

444

290

302

ADRENALS (termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.063

0.063

0.065

0.055

0.078

0.064

0.071

0.058

-

-

-

-

0.062

0.064

0.061

0.077

Adjusted weight (g)

0.059

0.061

0.066

0.061

0.077

0.063

0.070

0.062*

-

-

-

-

0.061

0.064

0.062

0.076*

BRAIN(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

2.199

2.195

2.161

2.150

2.077

2.017

2.021

1.964

-

-

-

-

2.214

2.259

2.022

2.015

Adjusted weight (g)

2.178

2.184

2.164

2.178

2.064

2.003

2.011

2.000

-

-

-

-

2.209

2.263

2.040

1.998

EPIDIDYMIDES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.282

1.297a

1.286a

1.207

-

-

-

-

-

-

-

-

1.310

1.290

-

-

Adjusted weight (g)

1.226

1.300a

1.289a

1.251

-

-

-

-

-

-

-

-

1.310

1.291

-

-

HEART(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

1.392

1.400

1.434

1.326

0.937

0.896

0.971

0.912

-

-

-

-

1.417

1.416

0.935

1.060

Adjusted weight (g)

1.330

1.368

1.444

1.410

0.917

0.876

0.957

0.967

-

-

-

-

1.414

1.418

0.963

1.032

KIDNEYS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

3.154

3.142

3.649

3.143

1.945

1.862

1.924

1.753

-

-

-

-

3.179

3.134

1.845

2.076

Adjusted weight (g)

2.960

3.041

3.680*

3.407*

1.922

1.837

1.907

1.819

-

-

-

-

3.169

3.144

1.889

2.032

LIVER(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

13.384

12.510

13.714

13.856

8.114

7.963

8.113

8.574

-

-

-

-

11.586

12.915

9.558

10.491

Adjusted weight (g)

12.283

11.936

13.891*

15.355**

8.023

7.865

8.045

8.831

-

-

-

-

11.535

12.967

9.706

10.343

OVARIES(termination for toxicity females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

-

-

-

-

0.092

0.092

0.086

0.081

0.088

0.088

0.085

0.085

-

-

0.085

0.101

Adjusted weight (g)

-

-

-

-

0.089

0.089

0.084

0.088

0.083

0.084

0.085

0.093

-

-

0.086

0.100

PROSTATE(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.097

1.163a

1.120a

1.016

-

-

-

-

-

-

-

-

1.071

1.042

-

-

Adjusted weight (g)

1.062

1.165a

1.122a

1.043

-

-

-

-

-

-

-

-

1.071

1.042

-

-

SEMINAL VESICLES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.903

2.145a

1.907a

2.095

-

-

-

-

-

-

-

-

2.032

1.944

-

-

Adjusted weight (g)

1.855

2.148a

1.910a

2.131

-

-

-

-

-

-

-

-

2.031

1.944

-

-

SPLEEN(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.811

0.723

0.753

0.679

0.553

0.554

0.507

0.456*

-

-

-

-

0.685

0.712

0.640

0.618

Adjusted weight (g)

0.763

0.698

0.761

0.744

-

-

-

-

-

-

-

-

0.684

0.714

0.624

0.635

TESTES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

3.312

3.492a

3.411a

3.511

-

-

-

-

-

-

-

-

3.646

3.466

-

-

Adjusted weight (g)

3.183

3.500a

3.419a

3.610

-

-

-

-

-

-

-

-

3.645

3.467

-

-

THYMUS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.327

0.254

0271

0.264

0.244

0.292

0.294

0.251

-

-

-

-

0.187

0.271

0.277

0.302

Adjusted weight (g)

0.046

0.077

0.059

0.079

0.233

0.280

0.285

0.283

-

-

-

-

0.185

0.273*

0.257

0.322

THYROID+PARAS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.019

0.020a

0.019a

0.016

0.014

0.014

0.015

0.014

0.016

0.016

0.016

0.015

0.017

0.014

0.015

0.014

Adjusted weight (g)

0.017

0.020a

0.019a

0.017

0.014

0.014

0.015

0.015

0.016

0.016

0.016

0.015

0.017

0.014

0.016

0.014

UTERUS + CERVIX + OVIDUCT(termination for toxicity females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

-

-

-

-

0.864

0.805

0.823

0.680

0.484

0.496

0.433

0.404

-

-

0.834

0.899

Adjusted weight (g)

-

-

-

-

0.857

0.798

0.818

0.699

0.470

0.487

0.435

0.428

-

-

0.884

0849

an=10

* P<0.05, ** P<0.01

Table 8       Summary of incidence of mating performance, fertility and gestation lengths (F0)

Group

(ppm)

# of paired males

# of females

Pre-coital interval (days)

Mating index

(%)

Pregnancy index

(%)

Gestation length (days)

Females with live offspring

Gestation index (%)

Paired

Mated

Pregnant

1-4

5-8

9-12

13-14

22

22.5

23

Control

10

10

10

10

10

0

0

0

100

100

2

5

3

10

100

1500

10

10

10

10

10

0

0

0

100

100

2

3

5

10

100

3000

10

10

10

10

10

0

0

0

100

100

3

4

3

10

100

7500

10

10

9

9

10

0

0

0

100

90

2

5

2

9

100

 

Table 9      Group mean litter size (F1)

 

Group

(ppm)

Mean # of corpora lutea

Mean # of implantation sites

Mean total litter size

Number of live offspring

Post implantation survival index (%)

Live birth index

(%)

Viability index

(%)

Lactation index on Day 13

Day 1

Day 4

Day 4

Day 7

Day 13

Pre-blood sampling

Post-blood sampling

Control

21.8

16.3

15.1

14.9

14.5

12.5

12.4

12.4

92.9

98.8

97.2

99.3

1500

19.9

15.9

14.6

14.4

14.0

12.2

12.2

12.2

92.3

98.6

97.2

100.0

3000

19.4

14.8

13.7

13.5

13.4

11.9

11.9

11.9

92.7

98.6

99.2

100.0

7500

20.6

13.0*

12.4*

12.1*

12.0*

10.7

10.7

10.7

96.3

97.8

99.1

100.0

*p<0.05, **p<0.01

Table 10       Group mean litter weights (g) (F1)

 

Group

(ppm)

Day of age

(before blood sampling)

Day of age

(after blood sampling)

Bodyweight change (± g)

1

4

4

7

13

1-4

4-7

7-13

1-13

Males

Control

7.1

10.2

10.2

15.7

29.0

+3.0

+5.5

+13.3

+21.8

1500

7.3

10.2

10.2

15.2

27.5

+2.9

+5.0

+12.3

+20.2

3000

6.9

9.8

9.8

14.3

25.7*

+2.9

+4.5**

+11.4**

+18.8**

7500

6.7

9.6

9.6

13.8*

24.5**

+3.0

+4.1**

+10.7**

+17.8**

Females

Control

6.8

9.5

9.5

14.6

27.1

+2.7

+5.1

+12.5

+20.4

1500

6.8

9.7

9.8

14.7

26.4

+3.0

+4.9

+11.7

+19.6

3000

6.6

9.4

9.4

13.8

25.1

+2.8

+4.4*

+11.3

+18.5

7500

6.1*

8.7

8.7

12.6**

22.4**

+2.6

+3.8**

+9.8**

+16.3**

*p<0.05, **p<0.01

  

Table 11       Group mean sex ratios (F1)

 

Group

(ppm)

Total on Day 1*

(before blood sampling)

Live (before blood sampling) on Day 1

Live (before blood sampling) on Day 4

Live (after blood sampling) on Day 4

Live (after blood sampling) on Day 13

M

F

% M

M

F

% M

M

F

% M

M

F

% M

M

F

% M

Control

6.9

8.2

45.1

6.8

8.1

45.0

6.5

8.0

44.2

6.5

6.0

51.4

6.5

5.9

51.9

1500

6.2

8.3

42.4

6.2

8.2

42.6

6.1

7.9

43.0

6.1

6.1

49.4

6.1

6.1

49.4

3000

7.4

6.3

53.4

7.4

6.1

54.1

7.4

6.0

54.6

7.4

4.5

61.1

7.4

4.5

61.1

7500

7.0

5.3

60.5

6.9

5.2

60.8

6.8

5.2

60.3

6.8

3.9

66.3

6.8

3.9

66.3

*includes offspring that died prior to the designated Day 1 of age. Unsexed offspring missing prior to Day 1 are not accounted for.

 

Table 12       Group mean – Ano-genital distance (g) (F1)

 

Group

(ppm)

Bodyweight at Day 1

(g)

Ano-genital distance

(mm)

Males

Control

7.1

4.2

1500

7.3

4.2

3000

6.9

4.1

7500

6.7

4.1

Females

Control

6.8

2.2

1500

6.8

2.2

3000

6.6

2.2

7500

6.1*

2.1

*p<0.05

Conclusions:
Within the scope of this study it was not possible to establish with certainty the aetiology of the potentially adverse reduction in birth weight and subsequent body weight gain of the male offspring in the 3000 and 7500 ppm group and female offspring in the 7500 ppm group. This may have been as a result of the parent females being smaller and consuming less food (thus there being reduced levels of nutrients available to pass to the fetus in utero and reduced quality milk in lactation), or a direct effect of the test item by cross‑placental in utero exposure followed by exposure post birth in the milk. The magnitude of the reduced offspring body weight gain at 7500 ppm (19-20 %) is considered adverse.

It was therefore concluded that the NOAEL for offspring growth was 3000 ppm.

The study also included a screen for endocrine disruptor relevant endpoints. Since there was no conclusive effect of treatment on T4 levels, anogenital distance, nipple counts, external genitals in the offspring, estrus cycles and no macroscopic or microscopic changes in male and female reproductive organs there was no indication that the test item is an endocrine disruptor.
Executive summary:

OECD 422 (2017) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2 -yl)-1-methylcyclohexene was administered to 30 male and 50 female Crl:CD(SD) strain rats by dietary administration at dose levels of 1200, 3000 and 7500 ppm for up to 5 weeks. In addition, 10 male and 20 female control rats were fed untreated basal diet.

 

A summary of adult responses to the test item are described below;

Systemic toxicity (F0)

 

Mortality- No mortality was observed during the test.

 

Clinical signs- No clinical signs or dose observations related to treatment were observed during the test.

 

Behavioural, functional and sensory assessments- Individuals were unaffected by the treatment.

  

Bodyweight- During the treatment period, a dose-dependent decrease in overall mean body weight gain was apparent in all groups, with a greater extent seen in groups treated at 7500 ppm, of toxicity and recovery phase males and females exposed to the test item when compared to controls; this decrease in overall weight gain was largely attributable to lower body weight gain and body weight losses during Week 1. This coincides with the reduced food intake seen in the first few days of treatment. A similar trend in body weight performance was evident among the reproductive phase females in the gestation and lactation period. The reduction in bodyweight gain in the 7500 ppm maternal groups was considered as an adverse effect.

  

Food consumption and efficiency-Effects on food consumption were evident during Days 1 to 3 of treatment for toxicity and recovery phase females given 3000 ppm and males and females given 7500 ppm; these differences are thought to be the reason for the reduced weight gain/weight loss during Week 1 of the study. During the remainder of the study, food consumption effects were limited to reproductive phase females given 3000 or 7500 ppm.  In the gestation period, food consumption of females given 1200 or 3000 ppm was slightly lower than control on Days 0 to 2, at 7500 ppm food intake was lower than control from Days 0 to 16.   In the lactation period food consumption for female animals receiving treated diet at 3000 or 7500 ppm was lower than control from Days 1 to 12 of lactation. This again resulted in a low mean bodyweight gain during these periods.

 

Water consumption- No dose related responses were observed during the test.

 

Haematology, blood chemistry and urinalysis-Minor haematological and biochemical changes were evident at the end of the treatment period: high haematocrit concentration in all treated groups of males; low reticulocyte and neutrophil counts in Toxicity phase females given 7500 ppm; low monocyte count in Toxicity phase females given 3000 and 7500 ppm; high urea concentrations in all treated male groups and low triglyceride concentrations in males receiving 3000 or 7500 ppm.  At the end of lactation period reproductive females given 7500 ppm had low red cell distribution width; mean cell volume; prothrombin time and high urea and triglyceride concentrations.  In addition, some minor changes in urinary parameters were evident: the pH of the urine was more acidic; the urinary chloride was high and the total creatinine and total potassium was low in the males given 7500 ppm. In the toxicity phase females given 7500 ppm total creatinine and total chloride was high and urinary potassium was low.

  

Necropsy-No toxicologically significant effects were detected in terminal kill animals of either sex treated with the test item.

  

Organ weights-Changes in organ weights were limited to an increase in adjusted kidney and liver weights in male animals treated at 3000 or 7500 ppm and a non-dose dependent decrease in adjusted adrenal weight in Toxicity females treated at 7500 ppm.  The increased liver and kidney weights correlate with the histopathological findings, of which have been deemed as non-adverse.

 

Histopathological changes;Test item-related histopathological changes were observed in the liver of both sexes and the kidneys of males exposed to 7500 ppm.

Among males given 7500 ppm, minimal hyaline droplet accumulation was observed in the kidneys, with minimal to slight basophilic tubules in the cortex of the kidney.  These changes correlated with an increase in body weight adjusted kidney weight in the 7500 ppm group.  Hyaline droplet formation in the kidney tubules of male rats is a known effect of hydrocarbons (Greaves, 2012).  The droplets contain α2u globulin and their accumulation is considered to be related to the binding of the hydrocarbon with the globulin within lysosomes, with the resulting complex being poorly catabolised.  Prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover and the appearance of basophilic tubules most likely reflects this change.  These changes are limited to male rats.  Toxicity in humans through this mechanism is considered improbable as little or no α2u globulin is present in humans, and therefore these findings in the male rat kidney were considered to be of no relevance to man.

 

Reproductive performance (F0)

Mating- No treatment-related effects were detected in mating performance.

Estrous cycle-There was no effect of treatment on estrous cycle regularity during the course of the 2-week pre-pairing assessment period for the Reproductive phase females.  All females showed regular 4-5 day cycles, with the exception of one female in the 3000 ppm group and one female in the 7500 ppm group with irregular cycles; these irregular cycles had no impact upon pre-coital interval. There was no effect of treatment on the stage of the estrous cycle at termination of the toxicity phase, recovery phase or reproductive phase females.

 

Fertility-Pre-coital interval and mating performance was unaffected my treatment, with the exception of one female receiving 7500 ppm all females achieved pregnancy. All pregnant females successfully gave birth to live young.

 

Gestation length-There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days.  The gestation index was 100 % in all groups

 

Litter size, viability-There was no effect of treatment or the mean number of corpora lutea. There is a dose dependent decrease in implantation counts in the treated groups compared to control.  At 7500 ppm, mean implantation counts, total litter size and live litters size on day 1 and 4 were statistically lower than control. This likely occurred as a consequence of the lower absolute body weight of the females at mating (which occurred as a result of the low food intake during the first few days of treatment) as there is a strong correlation between absolute body weight and the number of eggs ovulated (Harper, 1964) with lighter females releasing fewer eggs; the mean number of implantations in females in the 7500 ppm group which had Day 0 gestation bodyweights within the concurrent control range was similar to that in weight matched control females. The lower mean numbers of implantations at 1200 and 3000 ppm were not statistically significantly different to controls and within the historical control data range and thus no effect of treatment is inferred.

Offspring development (F1)

Litter weight/ weight gain- Mean body weights for male offspring from the 7500 ppm group were statistically lower than control on Days 7 and 13 of age.  Male offspring from the 3000 ppm group were statistically lower than control on Day 13 of age. The overall bodyweight change (Day 1 to 13 of age) for male offspring from groups 3000 or 7500 ppm was statistically lower than control.  The 3000 ppm male offspring gained 14% less than control and the 7500 ppm group gained 18 % less than control.  

Mean body weights for female offspring from the 7500 ppm group were statistically lower than control on Days 1, 7 and 13 of age.  The overall bodyweight change from Days 1 to 13 of age for group 7500 ppm female offspring was 20% lower than control.

Within the scope of this study it was not possible to establish with certainly the aetiology of the potentially adverse reduction in birth weight and subsequent body weight gain of the male offspring in the 3000 and 7500 ppm group and female offspring in the 7500 ppm group.  This may have been as a result of the parent females being smaller and consuming less food (thus there being reduced levels of nutrients available to pass to the fetus in utero and reduced quality milk in lactation), or a direct effect ofthe test item by cross‑placental in utero exposure.

The reduction in body weight gain of the offspring derived from parent animals given 7500 ppm was considered adverse.

Ano-genital distance- No treatment related effects were observed.

Sex ratio- No treatment related effects were observed.

Clinical signs- The clinical signs 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 the tested concentrations.

 

Endocrine disruption (F0/F1)

Since there was no conclusive effect of treatment on T4 levels, anogenital distance, nipple counts, external genitals in the offspring, estrus cycles and no macroscopic or microscopic changes in male and female reproductive organs there was no indication that the test item is an endocrine disruptor.

The NOAEL of the test item for developmental toxicity was concluded to be 3000 ppm, based on the adverse effects observed in offspring growth/ bodyweight gain from parental rats treated at 7500 ppm.

 

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 422 in the rat.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
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 to +8 ºC
- Stability of formulation under test conditions: stable - confirmed at 100 and 20000 ppm.
- Solubility and stability of the test substance in the diet: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the diet matrix
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

OTHER SPECIFICS: n/a

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: ca. 71 days old and females: ca. 85 days old
- Weight at study initiation: males: 319 - 383 g and females: 239 - 293 g
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week. An Aspen chew block was provided for envirnmental enrichment throughout the study period. A plastic shelter was also included in each cage throughout the study period, except during pairing and lactation.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet. A 100 g sample of each batch of diet used were retained frozen (-10 to -30°C) until finalization. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Provided ad libitum.
- Acclimation period: Males: six days prior to the commencement of treatment. Females: six days prior to the commencement of estrous cycle evaluation.

DETAILS OF FOOD AND WATER QUALITY: See above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ºC
- Humidity (%): 40 - 70 %
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 04 April 2017 To: 28 May 2017

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: n/a

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Basal diet (SDS VRF1 Certified)
- Storage temperature of food: not reported

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as Day 0 of pregnancy.
- Further matings after unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): housed individually in a cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
Minimum of 5 weeks with a 2 week recovery period.
Frequency of treatment:
Continuous
Doses / concentrationsopen allclose all
Dose / conc.:
1 200 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
7 500 ppm
Dose / conc.:
73.8 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 1200 ppm dose group
Dose / conc.:
181 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 3000 ppm dose group
Dose / conc.:
438 mg/kg bw/day (actual dose received)
Remarks:
Mean for male and female animals (Day 1 - 35) for 7500 ppm dose group
No. of animals per sex per dose:
Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
1200 ppm (toxicity test) = 10
3000 ppm (toxicity test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
1200 ppm (reproduction test) = 10
1200 ppm (toxicity test) = 5
3000 ppm (reproduction test) = 10
3000 ppm (toxicity test) = 5
7500 ppm (reproduction test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dietary inclusion levels selected for investigation in this study (0, 1200, 3000 and 7500 ppm) were chosen in conjunction with the sponsor based upon the results obtained in a 14 day preliminary study.
- Rationale for animal assignment (if not random): n/a
- Rationale for selecting satellite groups: determine if toxic effects in F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): n/a
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during acclimatisation, before feeding of the treated diets on the Day that treatment commenced (Day 1) and twice weekly thereafter (including termination).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study)
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 14 of lactating (females) and test termination (males and females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: lactating females: 5, test temination: 5 males and 5 females.
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 14 of lactating (females), test termination (male and females) and recovery week 4 (males)
- Animals fasted: Yes
- How many animals: lactating females: 5; test termination: 5 males and 5 females; recovery group: all surviving males.
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6 (day before scheduled termination) and recovery group week 2.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and weekly thereafter.
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a

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.
- after pairing until mating (reproductive females only).
- four days before scheduled termination (all females except premature decedents).
- Females showed no evidence of mating: following completion of pairing period female was separated from the male and vaginal smearing continued for up to five days or until the first estrous smear was seen. If a female shows an estrous smear during this period, she was killed and subject to macroscopic examination.

THYROID HORMONE ANALYSIS: Yes
- Time schedule: - Day 4 of age (F1 offspring, two females per litter (where possible) - no pups were eliminated when total litter size dropped below ten/litter).
- one pup for T3/T4 (serum)
- one pup for TSH (plasma)
- Day 13 of age (F1 offspring, two males and two females per litter (where possible)
- two for T3/T4 (serum): where possible one male and one female
- two for TSH (plasma): where possible one male and one female
- Termination (All Toxicity and Recovery phase F0 males and all Reproductive phase F0 females surviving to scheduled termination)
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:

Dry smears:

For 15 days before pairing using cotton swabs (Reproductive phase females only).

Wet smears:

Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to the Reproductive phase of the study.
- After pairing until mating (Reproductive phase females only).
- For four days before scheduled termination (all females except for premature decedents).
- Females showed no evidence of mating: following completion of pairing period female was separated from the male and vaginal smearing continued for up to five days or until the first estrous smear was seen. If a female shows an estrous smear during this period, she was killed and subject to macroscopic examination.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generations:
testis weight, epididymis weight.

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
clinical signs, litter size, sex ratio, survival indices, ano-genital distance, body weight change, presence of nipple/areolae in male offspring , macropathology/ abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
no

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals - Day 36 (+Day 14 recovery animals)
- Maternal animals: All surviving animals - Day 14 (lactating)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table # were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
See study report
Reproductive indices:
Percent mating, conception rate, fertility and gestation index determined.
Offspring viability indices:
Post-implantation survival index, viability index, lactation index and sex ratio determined.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or dose observations related to treatment were seen in the treatment, gestation, lactation period or recovery period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The overall mean bodyweight change from Day 1 to Day 8 for male animals receiving treated diet at 1200 or 3000 ppm was similar to control. Male animals receiving treated diet at 7500 ppm had a reduced mean bodyweight gain from Day 1 to 8 when compared to controls; this is due to a bodyweight loss in 5 animals between Day 1 and 4. All animals at 7500 ppm gained weight between Days 4 and 8, and exceeded their Day 1 bodyweight. Bodyweight change from Day 8 to 15 for all male treated groups was similar to control. However the body weight changes between Day 15 and 22 for all treated male groups were lower than control, with males receiving 3000 ppm showing a mean body weight loss. Bodyweight changes from Days 22 to 29 and Days 29 to 36 shows that male animals receiving 1200 or 3000 ppm return to comparable performance to control, however the weight gain of males receiving 7500 ppm remain significantly lower. The overall body weight change for the treatment period (Days 1 to 36) for male animals receiving treated diet at 1200 ppm and 3000 ppm was lower than control (82 % and 83 %), however male animals receiving treated diet at 7500 ppm was significantly lower than control (70 %). During the recovery priod, mean weight gain of animals previously treated at 7500 ppm was slightly greater than the control.

The overall mean bodyweight change from Day 1 to Day 8 for all treated female groups was lower than control, with an overall mean bodyweight loss for females receiving 3000 or 7500 ppm. Between Day 1 and Day 4 individual bodyweight losses or bodyweight stasis were seen in all treated groups and control (7/20 for control, 11/15 at 1200 ppm and 9/15 at 3000 ppm and 17/20 at 7500 ppm). By Day 8 several females had not obtained parity with Day 1 (3/20 for control, 2/15 at 1200 ppm, 8/15 at 3000 ppm and 15/20 at 7500 ppm). Bodyweight changes from Day 8 to 29 for all female treated groups was similar to control, with the exception of Day 15 to 22 where an mean body weight loss was seen in the control group. Bodyweight change from Days 29 to 36 for all female treated groups was slightly lower than control. The overall bodyweight change for the Toxicity and Recovery females during the treatment period (Days 1 to 36) was lower than control for all treated groups. Most significantly females receiving treated diet at 7500 ppm, gained only 52% of the control value, this was due to the body weight losses and stasis seen in the first week of study. However after 2 weeks of recovery females previously receiving 7500 ppm had a higher than control overall body weight change (Day R1 to R15). The mean weights of recovery animals that had receved 7500 ppm were higher than controls, but this was because the animals pre-selected for recovery had incidentally higher weights.

In the gestation period, reproductive phase females receiving 7500 ppm had statistically lower than control body weights and body weight gain up to Day 20, overall body weight change from Day 1 to 20 of gestation was 79 % of control. Females receiving 1200 or 3000 ppm had slightly reduced overall body weight gain when compared to controls (95 % and 92 % respectively).

In the lactation period, reproductive phase females receiving 7500 ppm had statistically lower than control body weights and body weight gain up to Day 13 of lactation, overall body weight change from Day 1 to 13 of lactation was 70 % of control. Females receiving 3000 ppm had statistically lower than control mean bodyweight values on Days 4, 7 and 10, however the increased gain between Days 10 and 13 resulted in only a minor difference in overall bodyweight change (Days 1 to 13) compared to control (94 %).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption for male animals receiving treated diet at 7500 ppm was slightly lower than control animals on Days 1 to 3 of treatment; however consumption values were comparable from Days 4 to 7 through to Days 32 to 35. The food intake of male animals treated at 1200 or 3000 ppm were unaffected. During the recovery period food consumption for males previously receiving 7500 ppm was similar to control.

Food consumption for female animals receiving treated diet at 1200 ppm was slightly lower than control on Days 1 to 3, but was comparable by Days 4 to 7. The food intake of female animals receiving 3000 or 7500 ppm treated diet were lower than control on Days 1 to 3, and remained slightly lower on Days 4 to 7 , however by Days 8-10 food intake was comparable to control. In the recovery period females previously receiving treated diet at 7500 ppm had higher food intake than control animals.

In the gestation period, food consumption for reproductive phase female animals receiving treated diet at 1200 or 3000 ppm was slightly lower than control on Days 0 to 2 (note that 2 control females had consumption that was somewhat higher than remaining controls), but was comparable by Days 3 to 6. The food intake of female animals receiving 7500 ppm treated diet was lower than control from Days 0 to 16, but was similar to control on Days 17 to 19.

In the lactation period, food consumption for reproductive phase female animals receiving treated diet at 3000 or 7500 ppm was lower than control from Days 1 to 9 and Day 1 to 12 of lactation, respectively. On days 10 to 12 females at 1200 ppm consumed slightly less food than controls but was not statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of haematological parameters at the end of the treatment period revealed higher than control haematocrit concentration in all groups of Toxicity phase males compared to control, though no dose response was evident and no similar finding was observed in females. In addition reticulocyte and neutrophil counts were lower than control in Toxicity phase females receiving treated diet at 7500 ppm. A non-dose dependent decrease in monocytes was also evident in females receiving 3000 or 7500 ppm.

Assessment of haematological parameters at the end of the lactation period revealed lower than control red cell distribution width and a non-dose dependent decrease in mean cell volume and prothrombin time in reproductive phase females receiving 7500 ppm.

All other inter-group differences from controls in the toxicity phase and reproductive phase animals at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Biochemical changes in the plasma of the toxicity phase males at the end of the treatment period revealed higher than control urea concentrations and lower glucose concentrations in all treated groups and lower than control triglyceride concentrations in males receiving 3000 or 7500 ppm. In Week 2 of recovery the biochemical changes in the plasma had demonstrated full recovery.

Biochemical changes in the plasma of the reproductive phase females at the end of the lactation period revealed higher than control urea and triglyceride (not statistically significant) concentrations in females receiving 7500 ppm althoughsimilar findings were not observed in toxicity females at the end of the treatment.

All other inter-group differences from controls in the toxicity phase and reproductive phase animals at the end of treatment were minor in nature, lacked dose-relationship, were due to anomalies in the control data or were confined to one sex; these were consequently attributed to normal biological variation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Assessment of urinary parameters at the end of the treatment period revealed the following differences from Control;

In the toxicity phase male animals receiving treated diet at 3000 or 7500 ppm the pH of the urine was more acidic, the total creatinine and total potassium was lower than control in the males receiving 7500 ppm and the urinary chloride was higher than control.

In the female toxicity phase animals receiving diet at 7500 ppm total creatinine and total chloride was higher than control and urinary potassium was lower than control. Total creatinine was also high among females at 3000 ppm.

In Week 2 of recovery, all of the aforementioned urinary changes had demonstrated full recovery.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Sensory reactivity and grip strength were considered unaffected by treatment. There were no consistent intergroup differences in grip strength between toxicity and reproductive phase females.

Motor activity scores for females given the test item were considered to be unaffected by treatment. Statistical significance was obtained for the overall low and high beam breaks for all groups of treated males (except low beams at 1200 ppm) when compared to controls, and although statistical significance was not attained for the final four 6-minute recording periods, the behaviour of the treated males was atypical, with no general increase in activity levels which should have been apparent towards the end of the recording period. The total high beam scores in all treated groups were slightly below the historical control data (HCD) range but total low beam scores at 7500 ppm were well within the HCD range.

In the recovery period the overall low and high beam breaks for males previously fed 7500 ppm continued to stay lower than control, as seen in the treatment period. No statistical significance was obtained for the overall scores however two recordings in the low beam breaks were statistically significant. As seen in the treatment period there was no general increase in activity levels over the last four 6-minute recording periods.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test item were seen in the liver of both sexes and the kidneys of males;

Liver - Centrilobular hepatocyte hypertrophy was seen in both sexes given 7500 ppm of the test item. The liver changes (increased bodyweight adjusted liver weights and minimal centrilobular hepatocyte hypertrophy) are deemed as a reversible non-adverse adaptive response.

Kidney - Hyaline droplets were seen in the cortical tubules of males given 7500 ppm of the test item. A slight increase in the incidence and severity of cortical tubular basophilia was seen in males given 7500 ppm of the test item which was considered to be treatment related. The kidney changes detected in the males (hyaline droplets in tubules and cortical basophilic tubules) were consistent with well documented species-specific responses of the male rat in response to the administration with hydrocarbons. This effect is, therefore, not indicative of a hazard to human health.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Prior to the start of treatment all females assigned to all study phases showed regular 4-5 day estrous cycles.

There was no effect of treatment on estrous cycle regularity during the course of the 2-week pre-pairing assessment period for the Reproductive phase females. All females showed regular 4-5 day cycles, with the exception of one female in the 3000 ppm group and one female in the 7500 ppm group with irregular cycles; these irregular cycles had no impact upon pre-coital interval.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages.

No cell or stage specific abnormalities were noted.

Minimal seminiferous tubular vacuolation was seen in a single male given 7500 ppm of the test item with degenerate spermatogenic cells seen in the seminiferous tubular lumens of the testes and ducts of the epididymis in this individual. These findings were considered to be incidental as the findings were minimal, affecting only one individual and with all stages in the spermatogenic cycle being present and normal in this animal.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-coital interval and mating performance was unaffected my treatment, with the exception of one female receiving 7500 ppm all females achieved pregnancy. All pregnant females successfully gave birth to live young.

There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days. The gestation index was 100 % in all groups.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 7 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 438 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights for male offspring from the 7500 ppm group were statistically lower than control on Days 7 and 13 of age. Male offspring from the 3000 ppm group were statistically lower than control on Day 13 of age. The overall bodyweight change (Day 1 to 13 of age) for male offspring from groups 3000 or 7500 ppm was statistically lower than control. The 3000 ppm male offspring gained 14 % less than control and the 7500 ppm group gained 18 % less than control.

Mean body weights for female offspring from the 7500 ppm group were statistically lower than control on Days 1, 7 and 13 of age. The overall bodyweight change from Days 1 to 13 of age for group 7500 ppm female offspring was 20 % lower than control.
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:
effects observed, non-treatment-related
Description (incidence and severity):
There were no macroscopic findings observed in the offspring that died prior to scheduled termination or among those offspring killed on Day 13 of age that were attributable to parental treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There was considered to be no effects of parental treatment on the ano-genital distances of offspring.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 7 500 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 438 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 4       Group mean acheived dose rates for each treatment group (in mg/kg bw/day) dd

 

Time period

Pre-mating and recovery animals

Gestation

Lactation

1200 ppm

3000 ppm

7500 ppm

1200 ppm

3000 ppm

7500 ppm

1200 ppm

3000 ppm

7500 ppm

Male

Female

Mean

Male

Female

Mean

Male

Female

Mean

Female

Day 1 -35

73.5

74.1

73.8

186

175

181

451

424

438

-

-

-

-

-

-

Day 0 - 19

-

-

-

-

-

-

-

-

-

88

215

521

-

-

-

Day 1 - 12

-

-

-

-

-

-

-

-

-

-

-

-

190

456

994

Table 5      Mean body weights and body weight gains/ losses (F0)

Sex / Day

Bodyweight (g)

Control

1200 ppm

3000 ppm

7500 ppm

MALES (TOXICITY TEST GROUP)

Day 1

353

352

351

352

Day 4

366

364

360

353

Day 8

(change from Day 1)

384

(+32)

385

(+32)

385

(+34)

374

(+22)

Day 11

400

400

399

390

Day 15

(change from Day 8)

413

(+28)

409

(+24)

413

(+28)

401

(+27)

Day 18

419

406

408

404

Day 22

(change from Day 15)

432

(+20)

414

(+5**)

411

(-3**)

412

(+11)

Day 25

437

425

419

412

Day 29

(change from Day 22)

451

(+19)

433

(+20)

432

(+21)

422*

(+10)

Day 32

459

440

437

427*

Day 36

(change from Day 29)

471

(+20)

450

(+17)

449

(+17)

435*

(+13)

Overall BW gain Day 1 – Day 36

(% gain/ loss)

+118

+97

+98

+83**

MALES (RECOVERY GROUP)

Day 1

456

-

-

437

Day 4

461

-

-

448

Day 8

469

-

-

458

Day 11

476

-

-

466

Day 15

476

-

-

467

Overall BW gain Day 1 – Day 15 (recovery)

(% gain/ loss)

+20

-

-

+30

FEMALES (TOXICITY TEST GROUP)

Day 1

263

269

268

265

Day 4

268

267

261

256**

Day 8

(change from Day 1)

272

(+9)

274

(+5)

263

(-5**)

260**

(-6**)

Day 11

276

276

269

265*

Day 15

(change from Day 8)

276

(+5)

264

(+3)

270

(+7)

264*

(+4)

Day 18

270

264

268

262

Day 22

(change from Day 15)

273

(-3)

269

(+4*)

273

(+4*)

267

(+4**)

Day 25

274

273

278

273

Day 29

(change from Day 22)

276

(+3)

277

(+8)

279

(+5)

273

(+6)

Day 32

281

278

280

271

Day 36

(change from Day 29)

285

(+9)

280

(+3)

281

(+2)

277

(+4)

Overall BW gain Day 1 – Day 36

(% gain/ loss)

+21

+14

+17

+11

FEMALES (RECOVERY GROUP)

Day 1

282

-

-

290

Day 4

285

-

-

302

Day 8

290

-

-

304

Day 11

292

-

-

309

Day 15

290

-

-

304

Overall BW gain Day 1 – Day 15 (recovery)

(% gain/ loss)

+8

-

-

+14

FEMALES (REPRODUCTION GROUP)

Day 0

Gestation

281

280

272

266*

Day 3

(change from Day 0)

297

(+16)

296

(+16)

286

(+15)

275**

(+10*)

Day 7

(change from Day 3)

309

(+11)

312

(+16)

303

(+17)

288*

(+13)

Day 10

(change from Day 7)

327

(+18)

327

(+15)

316

(+12)

299**

(+11*)

Day 14

(change from Day 10)

341

(+14)

340

(+13)

330

(+14)

313**

(+14)

Day 17

(change from Day 14)

379

(+38)

370

(+30*)

361

(+32*)

434**

(+30)

Day 20

(change from Day 17)

427

(+49)

419

(+48)

407

(+46)

381**

(+38*)

Overall BW gain Day 0 – Day 20 (gestation)

(% gain/ loss)

+146

+138

+135

+115**

Day 1

Lactation

324

314

312

293**

Day 4

(change from Day 1)

335

(+11)

326

(+12)

315*

(+2)

293**

(+1*)

Day 7

(change from Day 4)

351

(+16)

39

(+13)

323*

(+8)

297**

(+3*)

Day 10

(change from Day 7)

365

(+15)

363

(+23)

339*

(+16)

323**

(+27)

Overall BW gain Day 1 – Day 10 (lactation)

(% gain/ loss)

+42

+48

+27*

+30*

Day 13

(change from Day 10)

Lactation

377

(+11)

365

(+3)

362

(+23)

330**

(+6)

Overall BW gain Day 1 – Day 13 (lactation)

(% gain/ loss)

+53

+51

+50

+37*

* statistically significantly different from control values p < 0.05

** statistically significantly different from control values p < 0.01

Table 6      Haematology, clinical chemistry, urinalysis and pathology findings (F0)

 

Doses (ppm)

Control

1200

3000

7500

Control

1200

3000

7500

Control

1200

3000

7500

Control

7500

Control

7500

male (toxicity group)

female (toxicity group)

female (reproduction group)

Male (rec.)

Female (rec.)

Number of animals/group

5

5

5

5

5

5

5

5

10

10

10

9

5

5

5

5

Haematology(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Hb (g/dl)

15.0

15.7

15.3

15.2

15.2

15.0

15.6

15.1

14.9

15.0

14.5

15.3

-

-

-

-

- RBC (1012/L)

7.78

7.93

7.95

8.02

7.72

7.80

7.77

7.84

7.19

7.37

7.22

7.51

-

-

-

-

- Hct (%)

0.415

0.432*

0.432*

0.432*

0.418

0.414

0.419

0.421

0.424

0.423

0.423

0.428

-

-

-

-

- Retic (1012/L)

0.174

0.138

0.168

0.156

0.142

0.120

0.115

0.096*

0.227

0.219

0.254

0.229

-

-

-

-

- MCH (pg)

19.2

19.8

19.2

19.0

19.7

19.3

20.0

19.3

20.7

20.3

20.1

20.4

-

-

-

-

- MCV (fl)

53.4

54.5

54.4

53.9

54.1

53.1

54.0

53.7

59.1

57.3

58.6

57.0*

-

-

-

-

- MCHC (g/df)

36.1

36.3

35.3

35.3

36.3

36.3

37.1

36.0

35.1

35.4

34.4

35.8

-

-

-

-

- RDW (%)

12.7

11.9

12.2

12.0

11.4

11.5

11.1

11.2

14.3

13.9

13.8

13.1**

-

-

-

-

- WBC (109/L)

8.60

9.43

11.23

8.67

8.08

9.22

10.19

9.07

9.36

8.63

11.84

9.39

-

-

-

-

- Neut (109/L)

1.15

1.38

1.17

1.56

1.19

1.10

0.92

0.64*

4.00

3.76

5.19

4.63

-

-

-

-

- Lymph (109/L)

7.04

7.52

9.54

6.69

6.29

7.55

8.72

7.92

4.79

4.38

5.93

4.22

-

-

-

-

- Mono (109/L)

0.18

0.25

0.26

0.22

0.33

0.28

0.21*

0.23*

0.32

0.25

0.47

0.33

-

-

-

-

- Eos (109/L)

0.08

0.13

0.09

0.08

0.10

0.12

0.14

0.13

0.13

0.14

0.09

0.11

-

-

-

-

- Bas (109/L)

0.05

0.06

0.06

0.04

0.03

0.04

0.05

0.03

0.03

0.03

0.05

0.03

-

-

-

-

- LUC (109/L)

0.09

0.10

0.12

0.08

0.15

0.14

0.16

0.11

0.09

0.07

0.12

0.07

-

-

-

-

- PT (secs)

22.2

23.5

20.7

19.8

20.3

21.6

21.3

22.3

23.4

21.1

21.8

20.1**

-

-

-

-

- PLT (109/L)

795

753

794

721

905

910

854

828

858

783

878

837

-

-

-

-

- APTT (secs)

20.3

18.8

20.3

19.3

19.6

17.9

16.1

19.3

15.9

14.8

15.0

13.6

-

-

-

-

Blood chemistry(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Urea (µmol/L)

4.20

5.42**

5.27**

5.87**

5.96

5.65

5.78

5.99

11.04

11.50

12.09

15.90*

6.68

6.01

-

-

- Glucose (µmol/L)

7.79

6.46**

6.41**

6.16**

7.30

6.85

6.36

3.80

6.22

5.91

6.02

5.71

-

-

-

-

- Tot. Prot. (g/L)

63

61

61

61

70

68

68

69

60

61

60

59

-

-

-

-

- Albumin (g/L)

35

35

35

35

40

39

38

39

33

33

33

33

-

-

-

-

- A/G ratio

1.27

1.33

1.32

1.38*

1.31

1.32

1.29

1.32

1.25

1.19

1.24

1.23

-

-

-

-

- Na+ (mmol/L)

144

145

145

143

143

143

142

144*

137

139

138

138

-

-

-

-

- K+ (mmol/L)

4.0

4.0

4.1

4.2

3.6

3.8

4.2**

3.9**

4.2

4.8

4.6

4.4

-

-

-

-

- Cl- (mmol/L)

101

101

102

100

100

100

101

102

95

97

96

94

-

-

-

-

- Ca++ (mmol/L)

2.51

2.56

2.56

2.56

2.71

2.70

2.74

2.68

2.38

2.44

2.34

2.28

-

-

-

-

- P (mmol/L)

2.19

2.13

2.26

2.24

1.88

1.98

2.06

1.85

3.29

3.41

3.38

3.44

-

-

-

-

- ASAT (U/L)

69

71

78

77

86

61**

62**

62**

162

179

149

124

-

-

-

-

- ALAT (U/L)

35

33

33

42*

33

27

28

48

96

107

100

87

-

-

-

-

- ALP (U/L)

96

117

140*

125*

48

43

44

42

95

107

111

95

-

-

-

-

- Creat (µmol/L)

24

24

20

25

33

31

30

33

34

35

36

45

-

-

-

-

- Chol (µmol/L)

1.29

1.22

0.93

1.23

1.70

1.95

2.01

2.07

2.34

2.23

2.17

2.40

-

-

-

-

- Bili (µmol/L)

1

1

1

1

2

1

1

2

1

1

1

1

-

-

-

-

- Bile ac. (µmol/L)

5.6

21.7

11.3

21.4*

17.1

18.5

11.4

20.2

45.7

57.4

44.2

57.2

-

-

-

-

- Trig (µmol/L)

0.48

0.39

0.26*

0.29*

0.44

0.47

0.46

0.46

1.46

1.30

1.62

2.24

0.39

0.47

-

-

Urinalysis(termination for toxicity males and females, Day 14 lactation for reproduction females and week 2 of recovery for recovery animals)

- Vol (mL)

6.5

8.3

6.5

5.3

4.7

4.3

5.3

6.4

-

-

-

-

7.8

7.8

4.1

4.8

- pH

7.6

7.4

6.9*

6.5**

6.7

6.7

6.7

6.9

-

-

-

-

7.2

7.1

6.4

6.9*

- Sp. gravity (g/L)

1033

1030

1039

1040

1032

1036

1033

1028

-

-

-

-

1037

1032

1039

1035

- T-Prot (mg)

5.658

5.183

4.364

5.146

0.705

0.724

0.811

0.749

-

-

-

-

6.424

5.691

0.994

0.885

-T-Na (mmol)

0.447

0.510

0.330

0.325

0.294

0.237

0.411

0.477

-

-

-

-

0.594

0.459

0.214

0.327

- T-K (mmol)

1.296

1.226

1.321

0.944*

0.639

0.622

0.717

0.567

-

-

-

-

1.393

1.382

0.629

0.709

- T-Cl (mmol)

0.293

0.409

0.309

0.384

0.204

2.447

0.349

0.406*

-

-

-

-

0.346

0.316

0.153

0.221

- T-Glucose (µmol)

5.489

4.464

5.465

5.247

2.947

2.447

2.995

2.749

-

-

-

-

7.174

6.123

3.686

3.561

- T-Creat (µmol)

68.679

69.757

72.899

51.002*

31.320

32.636

39.059*

35.488*

-

-

-

-

88.0

79.5

48.4

44.6

- U-Prot (g/L)

0.88

0.65

0.67

1.01

0.16

0.19

0.16

0.13

-

-

-

-

0.83

0.75

0.25

0.21

- U-Na (mmol/L)

70.8

59.8

48.6

63.6

59.6

64.8

74.8

77.6

-

-

-

-

75.2

64.3

53.4

66.6

- U-K (mmol/L)

202.4

156.2

208.1

189.4

144.4

150.2

137.8

93.5

-

-

-

-

189.0

180.8

154.0

169.1

- U-Cl (mmol/L)

46.0

50.2

47.0

80.8*

38.8

49.0

63.6

64.4

-

-

-

-

43.8

43.2

37.0

46.0

- U-Gluc (mmol/L)

0.85

0.59

0.86

1.05

0.68

0.65

0.57

0.46

-

-

-

-

0.95

0.83

0.92

0.86

- U-Creat (µmol/L)

10576

8897

11487

10877

7197

8580

7493

6063

-

-

-

-

11684

10466

12287

10762

Pathology(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Number of animals/group

5

10

10

5

5

5

5

5

10

10

10

9

5

5

5

5

- Skin

(hair loss)

0

0

2

0

0

0

0

0

0

1

1

0

0

0

0

0

- Skin

(scabs)

0

0

2

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(depression)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(dermal inflammatory cell infiltrate)

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

- Skin

(epidermal ulceration)

0

0

1

0

0

0

0

0

0

0

0

0

1

0

0

0

- Skin

(regenerative hyperplasia)

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

- Skin

(abscessation)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Jejunum

(diverticulum)

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

- LN mesenteric

(dark)

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- LN mesenteric

(sinus erythrocytosis/ erythrophagocytosis)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

- Kidneys

(depression)

0

1

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- Kidneys

(pelvic dilation)

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

- Kidneys

(cortical tubular basophilia)

2

1

2

3

0

0

0

0

0

0

0

0

0

1

0

0

- Kidneys

(cortical tubules with hyaline droplets)

0

0

0

4

0

0

0

0

0

0

0

0

0

0

0

0

- Kidneys

(tubular dilation)

0

0

1

1

0

0

0

0

0

0

0

0

0

0

0

0

- Kidneys

(medullary cysts)

0

0

1

0

0

0

0

0

0

0

0

0

1

1

0

0

- Kidneys

(cortical scarring)

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

0

- Stomach

(corpus depression)

0

0

0

0

0

0

0

0

1

0

0

1

0

0

0

0

- Ovaries

(cysts)

-

-

-

-

1

0

0

0

0

0

0

0

0

0

0

0

- Lungs and bronchi

(pale areas)

0

0

0

1

0

0

0

0

0

1

0

0

0

0

0

1

- Lungs and bronchi

(dark areas)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

- Lungs and bronchi

(incomplete collapse)

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

- Lungs and bronchi

(aggregations of alveolar macrophages)

1

0

0

1

0

0

0

0

0

0

0

0

0

0

0

1

- Lungs and bronchi

(alveolitis)

1

0

0

0

0

0

0

1

0

0

0

0

0

0

0

1

- Lungs and bronchi

(foamy alveolar macrophages)

1

0

0

0

0

0

0

0

0

1

0

0

0

0

0

0

- Lungs and bronchi

(perivascular inflammatory cells)

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

- Liver

(hepatocyte hypertrophy, centrilobular)

0

0

0

5

0

0

0

5

0

0

0

0

0

0

0

0

- Liver

(hepatocyte vacuolation, periportal)

3

1

3

3

2

0

3

4

0

0

0

0

0

0

0

1

- Liver

(hepatocyte necrosis, focal)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

- Liver

(inflammatory cell infiltrate)

3

2

1

2

3

0

1

0

0

0

0

0

2

2

1

0

- Liver

(lipidosis, tension)

0

0

0

0

1

1

1

0

0

0

0

0

2

0

0

0

- Heart

(myocardial fibrosis/ inflammatory cells)

1

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

- Adrenals

(cortical hypertrophy)

0

0

0

0

1

0

0

1

0

0

0

0

0

0

0

0

- Skeletal muscle

(inflammatory cell infiltrate)

0

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

Prostate

(inflammatory cell infiltrate)

2

0

0

2

-

-

-

-

0

0

0

0

0

0

0

0

- Vagina

(dioestrus)

-

-

-

-

1

0

0

3

0

0

0

0

-

-

0

0

- Vagina

(metoestrus)

-

-

-

-

1

0

0

1

0

0

0

0

-

-

0

0

- Vagina

(oestrus)

-

-

-

-

2

0

0

0

0

0

0

0

-

-

0

0

- Vagina

(proestrus)

-

-

-

-

1

0

0

1

0

0

0

0

-

-

0

0

- Testes

(seminiferous tubular vacuolation)

0

0

0

1

-

-

-

-

-

-

-

-

0

0

-

-

- Epididymides

(degenerate spermatogenic cells in duct)

0

0

0

1

-

-

-

-

-

-

-

-

0

0

-

-

- Epididymides

(inflammatory cell infiltrate)

0

0

0

1

-

-

-

-

 

 

 

 

0

0

-

-

- General

(stained fur)

0

0

0

0

1

0

0

0

0

1

0

1

0

0

0

0

* P<0.05, ** P<0.01

Table 7       Absolute and adjusted organ weights (F0)

 

Doses (ppm)

Control

1200

3000

7500

Control

1200

3000

7500

Control

1200

3000

7500

Control

7500

Control

7500

male (toxicity group)

female (toxicity group)

female (reproduction group)

Male (rec.)

Female (rec.)

Number of animals/group

5

5

5

5

5

5

5

5

10

10

10

9

5

5

5

5

BODY WEIGHT (termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

464

430

430

407**

264

265

263

249*

339

333

321

296**

447

444

290

302

ADRENALS (termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.063

0.063

0.065

0.055

0.078

0.064

0.071

0.058

-

-

-

-

0.062

0.064

0.061

0.077

Adjusted weight (g)

0.059

0.061

0.066

0.061

0.077

0.063

0.070

0.062*

-

-

-

-

0.061

0.064

0.062

0.076*

BRAIN(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

2.199

2.195

2.161

2.150

2.077

2.017

2.021

1.964

-

-

-

-

2.214

2.259

2.022

2.015

Adjusted weight (g)

2.178

2.184

2.164

2.178

2.064

2.003

2.011

2.000

-

-

-

-

2.209

2.263

2.040

1.998

EPIDIDYMIDES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.282

1.297a

1.286a

1.207

-

-

-

-

-

-

-

-

1.310

1.290

-

-

Adjusted weight (g)

1.226

1.300a

1.289a

1.251

-

-

-

-

-

-

-

-

1.310

1.291

-

-

HEART(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

1.392

1.400

1.434

1.326

0.937

0.896

0.971

0.912

-

-

-

-

1.417

1.416

0.935

1.060

Adjusted weight (g)

1.330

1.368

1.444

1.410

0.917

0.876

0.957

0.967

-

-

-

-

1.414

1.418

0.963

1.032

KIDNEYS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

3.154

3.142

3.649

3.143

1.945

1.862

1.924

1.753

-

-

-

-

3.179

3.134

1.845

2.076

Adjusted weight (g)

2.960

3.041

3.680*

3.407*

1.922

1.837

1.907

1.819

-

-

-

-

3.169

3.144

1.889

2.032

LIVER(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

13.384

12.510

13.714

13.856

8.114

7.963

8.113

8.574

-

-

-

-

11.586

12.915

9.558

10.491

Adjusted weight (g)

12.283

11.936

13.891*

15.355**

8.023

7.865

8.045

8.831

-

-

-

-

11.535

12.967

9.706

10.343

OVARIES(termination for toxicity females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

-

-

-

-

0.092

0.092

0.086

0.081

0.088

0.088

0.085

0.085

-

-

0.085

0.101

Adjusted weight (g)

-

-

-

-

0.089

0.089

0.084

0.088

0.083

0.084

0.085

0.093

-

-

0.086

0.100

PROSTATE(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.097

1.163a

1.120a

1.016

-

-

-

-

-

-

-

-

1.071

1.042

-

-

Adjusted weight (g)

1.062

1.165a

1.122a

1.043

-

-

-

-

-

-

-

-

1.071

1.042

-

-

SEMINAL VESICLES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

1.903

2.145a

1.907a

2.095

-

-

-

-

-

-

-

-

2.032

1.944

-

-

Adjusted weight (g)

1.855

2.148a

1.910a

2.131

-

-

-

-

-

-

-

-

2.031

1.944

-

-

SPLEEN(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.811

0.723

0.753

0.679

0.553

0.554

0.507

0.456*

-

-

-

-

0.685

0.712

0.640

0.618

Adjusted weight (g)

0.763

0.698

0.761

0.744

-

-

-

-

-

-

-

-

0.684

0.714

0.624

0.635

TESTES(termination for toxicity males and week 2 of recovery for recovery animals)

Weight (g)

3.312

3.492a

3.411a

3.511

-

-

-

-

-

-

-

-

3.646

3.466

-

-

Adjusted weight (g)

3.183

3.500a

3.419a

3.610

-

-

-

-

-

-

-

-

3.645

3.467

-

-

THYMUS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.327

0.254

0271

0.264

0.244

0.292

0.294

0.251

-

-

-

-

0.187

0.271

0.277

0.302

Adjusted weight (g)

0.046

0.077

0.059

0.079

0.233

0.280

0.285

0.283

-

-

-

-

0.185

0.273*

0.257

0.322

THYROID+PARAS(termination for toxicity males and females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

0.019

0.020a

0.019a

0.016

0.014

0.014

0.015

0.014

0.016

0.016

0.016

0.015

0.017

0.014

0.015

0.014

Adjusted weight (g)

0.017

0.020a

0.019a

0.017

0.014

0.014

0.015

0.015

0.016

0.016

0.016

0.015

0.017

0.014

0.016

0.014

UTERUS + CERVIX + OVIDUCT(termination for toxicity females, termination for reproduction females and week 2 of recovery for recovery animals)

Weight (g)

-

-

-

-

0.864

0.805

0.823

0.680

0.484

0.496

0.433

0.404

-

-

0.834

0.899

Adjusted weight (g)

-

-

-

-

0.857

0.798

0.818

0.699

0.470

0.487

0.435

0.428

-

-

0.884

0849

an=10

* P<0.05, ** P<0.01

Table 8       Summary of incidence of mating performance, fertility and gestation lengths

Group

(ppm)

# of paired males

# of females

Pre-coital interval (days)

Mating index

(%)

Pregnancy index

(%)

Gestation length (days)

Females with live offspring

Gestation index (%)

Paired

Mated

Pregnant

1-4

5-8

9-12

13-14

22

22.5

23

Control

10

10

10

10

10

0

0

0

100

100

2

5

3

10

100

1500

10

10

10

10

10

0

0

0

100

100

2

3

5

10

100

3000

10

10

10

10

10

0

0

0

100

100

3

4

3

10

100

7500

10

10

9

9

10

0

0

0

100

90

2

5

2

9

100

 

Table 8       Offspring Survival Indices

 

Group

(ppm)

Mean # of corpora lutea

Mean # of implantation sites

Mean total litter size

Number of live offspring

Post implantation survival index (%)

Live birth index

(%)

Viability index

(%)

Lactation index on Day 13

Day 1

Day 4

Day 4

Day 7

Day 13

Pre-blood sampling

Post-blood sampling

Control

21.8

16.3

15.1

14.9

14.5

12.5

12.4

12.4

92.9

98.8

97.2

99.3

1500

19.9

15.9

14.6

14.4

14.0

12.2

12.2

12.2

92.3

98.6

97.2

100.0

3000

19.4

14.8

13.7

13.5

13.4

11.9

11.9

11.9

92.7

98.6

99.2

100.0

7500

20.6

13.0*

12.4*

12.1*

12.0*

10.7

10.7

10.7

96.3

97.8

99.1

100.0

*p<0.05, **p<0.01

Table 9       Estrous cycles before treatment (F0)

 

Group

(ppm)

Number of animals

Regular Cycles

Irregular Cycle§

Acyclic^

4 day

4/5 day

5 day

Toxicity Group

Control

5

4

(80 %)

1

(20 %)

0

(0 %)

0

(0 %)

0

(0 %)

1500

5

4

(80 %)

0

(0 %)

1

(20 %)

0

(0 %)

0

(0 %)

3000

5

5

(100 %)

0

(0 %)

0

(0 %)

0

(0 %)

0

(0 %)

7500

5

4

(80 %)

1

(20 %)

0

(0 %)

0

(0 %)

0

(0 %)

Reproduction Group

Control

10

9

(90 %)

1

(10 %)

0

(0 %)

0

(0 %)

0

(0 %)

1500

10

7

(70 %)

1

(10 %)

2

(20 %)

0

(0 %)

0

(0 %)

3000

10

8

(80 %)

2

(20 %)

0

(0 %)

0

(0 %)

0

(0 %)

7500

10

7

(70 %)

2

(20 %)

1

(10 %)

0

(0 %)

0

(0 %)

Recovery Group

Control

5

4

(80 %)

1

(20 %)

0

(0 %)

0

(0 %)

0

(0 %)

7500

5

5

(100 %)

0

(0 %)

0

(0 %)

0

(0 %)

0

(0 %)

§at least one cycle of two, three or six to ten days

^ at least ten days without estrus

Table 10       Estrous cycles at termination (F0)

 

Group

(ppm)

Number smeared

Estrus before termination

Cycle stage at termination

Diestrus

Estrus

Metestrus

Pro-estrus

Toxicity Group

Control

5

5

(100 %)

0

(0 %)

1

(20 %)

1

(20 %)

3

(60 %)

1500

5

5

(100 %)

1

(20 %)

1

(20 %)

3

(60 %)

0

(0 %)

3000

5

5

(100 %)

2

(40 %)

1

(20 %)

2

(40 %)

0

(0 %)

7500

5

5

(100 %)

1

(20 %)

3

(60 %)

1

(20 %)

0

(0 %)

Reproduction Group

Control

10

0

(0 %)

0

(0 %)

10

(100 %)

0

(0 %)

0

(0 %)

1500

10

0

(0 %)

0

(0 %)

10

(100 %)

0

(0 %)

0

(0 %)

3000

10

0

(0 %)

0

(0 %)

10

(100 %)

0

(0 %)

0

(0 %)

7500

9

0

(0 %)

0

(0 %)

9

(100 %)

0

(0 %)

0

(0 %)

Recovery Group

Control

5

5

(100 %)

0

(0 %)

1

(20 %)

2

(40 %)

2

(40 %)

7500

5

5

(100 %)

0

(0 %)

0

(0 %)

1

(20 %)

4

(80 %)

Applicant's summary and conclusion

Conclusions:
Estrous cycle regularity, mating performance and gestation length were unaffected by treatment of the test item at all dietary inclusion levels investigated. At 7500 ppm, the mean number of uterine implantation sites and therefore total litter size was statistically lower than control on Day 1 and Day 4. This likely occurred as a consequence of the lower absolute body weight of the females at mating (which occurred as a result of the low food intake during the first few days of treatment) as there is a strong correlation between absolute body weight and the number of eggs ovulated (Harper, 1964) with lighter females releasing fewer eggs; the mean number of implantations in females in the 7500 ppm group which had Day 0 of gestation bodyweights within the concurrent control range was similar to that in weight matched control females. The lower mean numbers of implantations at the other tested concentrations were not statistically significantly different to controls.

In conclusion, there was no effect of treatment on mating performance, fertility, litter size, or offspring survival or clinical condition. The lower litter size at 7500 ppm appeared to reflect the lower maternal body weights at conception, an expected relationship.
Executive summary:

OECD 422 (2017) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2 -yl)-1-methylcyclohexene was administered to 30 male and 50 female Crl:CD(SD) strain rats by dietary administration at dose levels of 1200, 3000 and 7500 ppm for up to 5 weeks. In addition, 10 male and 20 female control rats were fed untreated basal diet.

 

A summary of adult responses to the test item are described below;

Systemic toxicity (F0)

 

Mortality- No mortality was observed during the test.

 

Clinical signs- No clinical signs or dose observations related to treatment were observed during the test.

 

Behavioural, functional and sensory assessments- Individuals were unaffected by the treatment.

  

Bodyweight- During the treatment period, a dose-dependent decrease in overall mean body weight gain was apparent in all groups, with a greater extent seen in groups treated at 7500 ppm, of toxicity and recovery phase males and females exposed to the test item when compared to controls; this decrease in overall weight gain was largely attributable to lower body weight gain and body weight losses during Week 1. This coincides with the reduced food intake seen in the first few days of treatment. A similar trend in body weight performance was evident among the reproductive phase females in the gestation and lactation period. The reduction in bodyweight gain of the maternal individuals treated at 7000 ppm is considered an adverse affect.

  

Food consumption and efficiency-Effects on food consumption were evident during Days 1 to 3 of treatment for toxicity and recovery phase females given 3000 ppm and males and females given 7500 ppm; these differences are thought to be the reason for the reduced weight gain/weight loss during Week 1 of the study. During the remainder of the study, food consumption effects were limited to reproductive phase females given 3000 or 7500 ppm.  In the gestation period, food consumption of females given 1200 or 3000 ppm was slightly lower than control on Days 0 to 2, at 7500 ppm food intake was lower than control from Days 0 to 16.   In the lactation period food consumption for female animals receiving treated diet at 3000 or 7500 ppm was lower than control from Days 1 to 12 of lactation. This again resulted in a low mean bodyweight gain during these periods.

 

Water consumption- No dose related responses were observed during the test.

 

Haematology, blood chemistry and urinalysis-Minor haematological and biochemical changes were evident at the end of the treatment period: high haematocrit concentration in all treated groups of males; low reticulocyte and neutrophil counts in Toxicity phase females given 7500 ppm; low monocyte count in Toxicity phase females given 3000 and 7500 ppm; high urea concentrations in all treated male groups and low triglyceride concentrations in males receiving 3000 or 7500 ppm.  At the end of lactation period reproductive females given 7500 ppm had low red cell distribution width; mean cell volume; prothrombin time and high urea and triglyceride concentrations.  In addition, some minor changes in urinary parameters were evident: the pH of the urine was more acidic; the urinary chloride was high and the total creatinine and total potassium was low in the males given 7500 ppm. In the toxicity phase females given 7500 ppm total creatinine and total chloride was high and urinary potassium was low.

  

Necropsy-No toxicologically significant effects were detected in terminal kill animals of either sex treated with the test item.

  

Organ weights-Changes in organ weights were limited to an increase in adjusted kidney and liver weights in male animals treated at 3000 or 7500 ppm and a non-dose dependent decrease in adjusted adrenal weight in Toxicity females treated at 7500 ppm.  The increased liver and kidney weights correlate with the histopathological findings, of which have been deemed as non-adverse.

 

Histopathological changes;Test item-related histopathological changes were observed in the liver of both sexes and the kidneys of males exposed to 7500 ppm.

Among males given 7500 ppm, minimal hyaline droplet accumulation was observed in the kidneys, with minimal to slight basophilic tubules in the cortex of the kidney.  These changes correlated with an increase in body weight adjusted kidney weight in the 7500 ppm group.  Hyaline droplet formation in the kidney tubules of male rats is a known effect of hydrocarbons (Greaves, 2012).  The droplets contain α2u globulin and their accumulation is considered to be related to the binding of the hydrocarbon with the globulin within lysosomes, with the resulting complex being poorly catabolised.  Prolonged accumulation of hyaline droplets is associated with chronic cell damage and increased cell turnover and the appearance of basophilic tubules most likely reflects this change.  These changes are limited to male rats.  Toxicity in humans through this mechanism is considered improbable as little or no α2u globulin is present in humans, and therefore these findings in the male rat kidney were considered to be of no relevance to man.

 

Reproductive performance (F0)

Mating- No treatment-related effects were detected in mating performance.

Estrous cycle- There was no effect of treatment on estrous cycle regularity during the course of the 2-week pre-pairing assessment period for the Reproductive phase females.  All females showed regular 4-5 day cycles, with the exception of one female in the 3000 ppm group and one female in the 7500 ppm group with irregular cycles; these irregular cycles had no impact upon pre-coital interval. There was no effect of treatment on the stage of the estrous cycle at termination of the toxicity phase, recovery phase or reproductive phase females.

 

Fertility- Pre-coital interval and mating performance was unaffected my treatment, with the exception of one female receiving 7500 ppm all females achieved pregnancy. All pregnant females successfully gave birth to live young.

 

Gestation length- There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days.  The gestation index was 100 % in all groups

 

Litter size, viability- There was a dose dependent decrease in implantation counts in the treated groups compared to control.  At 7500 ppm, mean implantation counts, total litter size and live litters size on day 1 and 4 were statistically lower than control.

 

The clinical signs 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 the tested concentrations.

 

In conclusion, there was no effect of treatment on mating performance, fertility, litter size, or offspring survival or clinical condition. The lower litter size at 7500 ppm reflected the lower maternal body weights at conception, an expected relationship.

 

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 422 in the rat.