Registration Dossier

Administrative data

Description of key information

NOAEL (systemic toxicity) = 3,000 ppm diet (181 mg/kg bw/d); OECD 422; Anon. (2017)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
04 January - 13 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
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: ca. 71 days old
- Weight at study initiation: Control and 7500 ppm groups: males = 343 - 373 g and females = 226 - 273 g; 11000 ppm group: males = 371 - 422 g and females = 244 - 299 g; 3000 ppm group: males = 424 - 504 g and females = 270 - 324 g.
- Fasting period before study: no
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Wood based bedding was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): SDS VRF1 Certified powdered diet provided continuously.
- 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: At least six days prior to the commencement of treatment.

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 January 2017 To: 13 February 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
Remarks:
Conducted under separate study number HK31NR.
Details on analytical verification of doses or concentrations:
The stability and homogenous preparation of the test material in the diet was demonstrated for 28 hours, eight and 22 days following ambient storage (15 to 25 ºC) and 22 days following frozen storage (-10 to -30 ºC).

No formulation analysis was conduced under this study.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Continuous
Dose / conc.:
3 000 ppm
Dose / conc.:
7 500 ppm
Dose / conc.:
11 000 ppm
No. of animals per sex per dose:
5
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:

The acute oral study demonstrated that the acute lethal oral dose (LD50) of the test item was greater than 2000 mg/kg, with no mortality reported. Due to the nature of the test item it is anticipated to have an effect on the palatability of the diet, therefore an initial dose level of 7500 ppm was selected, which was approximately equivalent to 500 mg/kg/day.

After one week of treatment at 7500 ppm there was an indication of treatment related body weight and food consumption reductions in the females. However in the absence of any treatment related clinical signs and absence of any marked treatment related effects in the male animals it was considered acceptable to increase the level of the test item in the diet to 11000 ppm.

After six days of treatment at 11000 ppm there were treatment related body weight and food consumption reductions in the male and female animals. Although there was no change in clinical condition, the treatment related effects seen up to Day 6 for animals receiving 11000 ppm were more marked than that seen up to Day 6 for animals that received 7500 ppm; therefore it was considered appropriate to reduce the level of the test item in the diet. A dietary level of 3000 ppm was chosen in order to try to find either a No Adverse Effect Level or a Low Adverse Effect Level.

- Rationale for animal assignment (if not random): Body weights were reviewed by Study Management and allocation has been adjusted to reduce inter‑/intra-group variation.
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): n/a
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day -3 and daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: Day -3 and daily thereafter

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): No - water intake monitored, but only visually.

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

HAEMATOLOGY: No
- Time schedule for collection of blood: n/a
- Anaesthetic used for blood collection: n/a
- Animals fasted: n/a
- How many animals: n/a

CLINICAL CHEMISTRY: No
- Time schedule for collection of blood: n/a
- Animals fasted: No
- How many animals: n/a

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine: n/a
- Animals fasted: n/a

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

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

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 2)

HISTOPATHOLOGY: No
Other examinations:
no
Statistics:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No treatment realted effects were observed.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment at the high level of 11000 ppm resulted in negative effects on body weight performance (particularly females) and persistent reductions in food intake of both sexes.

At 7500 ppm low food intake was evident during the first 24-48 hours for both sexes and remained slightly lower than control for females to the end of treatment. As a consequence body weight loss was evident during the first 24 hours for the majority of animals; thereafter the body weight gain of males was similar to control, however, for females parity with Day 1 body weight was not achieved until Day 10.

At 3000 ppm effects were minimal, limited to slightly low food intake and minor body weight loss for females on Day 1 and minor body weight loss during Days 1-4 for males, although in the absence of any effect on food intake.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment at the high level of 11000 ppm resulted in persistent reductions in food intake of both sexes.

At 7500 ppm low food intake was evident during the first 24-48 hours for both sexes and remained slightly lower than control for females to the end of treatment.

At 3000 ppm effects were minimal, with only slightly low food intake observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no effect of treatment on visual water consumption.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
When compared to control animals’ body weight relative liver weights were slightly increased in both sexes in all treated groups although in the absence of a dose response relationship.

Bodyweight relative kidney weights were slightly increased in males receiving 11000 ppm and bodyweight relative testes weights were slightly increased in males receiving 7500 ppm.

Females receiving 7500 ppm had slightly higher bodyweight relative kidney weights than controls, but in the absence of a similar effect in females receiving 11000 ppm this finding is considered to be fortuitous.

All of the described organ weight findings are to some extent a consequence of the later sacrifice of the controls and are probably fortuitous.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment related macropathology observations were found at the necropsy of animals in any of the test groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Table 3       Mean body weights and body weight gains/ losses

Sex / Day

Bodyweight (g)

Control

7500 ppm

11000 ppm

3000 ppm

MALES

Day -3

340

336

-

-

Day 1

354

356

-

-

Day 2

(change from Day 1)

359

(+5)

349

(-6)

-

-

Day 3

363

354

-

-

Day 4

369

361

-

-

Day 5

374

370

-

-

Day 6

378

370

-

-

Day 7

381

374

-

-

Day 8

388

379

380

-

Day 9

393

385

-

-

Day 10

398

391

-

-

Day 11

404

395

389

-

Day 12

(change from Day 11)

405

(+2)

398

-

380

-

-

Day 13

414

404

385

-

Day 14

415

409

390

-

Day 15

(change from Day 2)

419

(+61)

406

(+57)

389

-

-

Day 16

422

-

395

-

Day 17

422

-

391

-

Day 18

429

-

399

457

Day 19

431

-

404

-

Day 20

429

-

405

-

Day 21

436

-

407

465

Day 22

437

-

408

464

Day 23

451

-

417

470

Day 24

(change from Day 21)

448

(+12)

-

409

-

463

(-1)

Day 25

(change from Day 12)

448

(+43)

-

418

(+38)

467

-

Day 26

451

-

-

475

Day 27

454

-

-

475

Day 28

456

-

-

477

Day 29

461

-

-

482

Day 30

463

-

-

482

Day 31

468

-

-

489

Day 32

467

-

-

489

Day 33

470

-

-

492

Day 34

471

-

-

497

Day 35

(change from Day 24)

475

(+27)

-

-

500

(+37)

FEMALES

Day -3

252

238

-

-

Day 1

257

245

-

-

Day 2

(change from Day 1)

257

(0)

233

(-12)

-

-

Day 3

256

235

-

-

Day 4

256

236

-

-

Day 5

263

240

-

-

Day 6

266

240

-

-

Day 7

263

240

-

-

Day 8

265

239

272

-

Day 9

270

241

-

-

Day 10

272

246

-

-

Day 11

271

247

274

-

Day 12

(change from Day 11)

271

(0)

245

(-2)

264

(-10)

-

Day 13

277

251

262

-

Day 14

280

249

256

-

Day 15

(change from Day 2)

276

(+19)

247

(+15)

259

-

-

Day 16

278

-

265

-

Day 17

278

-

259

-

Day 18

283

-

264

280

Day 19

280

-

259

-

Day 20

280

-

263

-

Day 21

283

-

267

290

Day 22

286

-

267

282

Day 23

289

-

271

290

Day 24

(change from Day 21)

283

-

-

269

-

288

(-2)

Day 25

(change from Day 12)

287

-

-

271

(+7)

288

-

Day 26

291

-

-

287

Day 27

290

-

-

289

Day 28

289

-

-

291

Day 29

293

-

-

291

Day 30

294

-

-

289

Day 31

291

-

-

292

Day 32

292

-

-

293

Day 33

297

-

-

297

Day 34

298

-

-

292

Day 35

(change from Day 24)

294

(+11)

-

-

295

(+8)

Values in bold represent Day 1 of corresponding treatment

 

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

Doses (ppm)

Control

7500

11000

3000

Control

7500

11000

3000

Males

Females

Number of animals/group

5

5

5

5

5

5

5

5

BODY WEIGHT (termination weight for toxicity males and females)

Weight (g)

474

405

418

496

293

246

271

295

KIDNEYS (termination weight for toxicity males and females)

Weight (g)

0.726

0.755

0.812

0.744

0.730

0.811

0.743

0.753

LIVER (termination weight for toxicity males and females)

Weight (g)

3.61

4.34

4.25

4.03

3.85

4.44

4.41

4.02

SPLEEN (termination weight for toxicity males and females)

Weight (g)

0.167

0.180

0.183

0.153

0.210

0.247

0.226

0.203

TESTES (termination weight for toxicity males)

Weight (g)

0.719

0.860

0.775

0.715

-

-

-

-

Conclusions:
In conclusion, treatment with the test item at 7500, 11000 and 3000 ppm had no definite effect on clinical condition, water consumption, macropathology or organ weights. However, the high level of 11000 ppm is considered too high for further investigation due to the magnitude of effects on body weight performance and persistent reductions in food intake. Therefore a dietary level of 7500 ppm would be a suitable high dose for the OECD 422 screening study.
Executive summary:

OECD 422 (2017) - The purpose of this test was to assess the systemic toxicity potential of Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2 -yl)-1-methylcyclohexene following repeated exposure to 20 male and 20 female Crl:CD(SD) strain rats by dietary administration at dose levels of 3000, 7500 and 11000 ppm for up to 14 days. The findings would be used to select suitable dose levels for a subsequent combined repeat dose toxicity study with reproductive/ developmental toxicity screening study (OECD 422).

 

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.

  

Bodyweight- Treatment at the high level of 11000 ppm resulted in negative effects on body weight performance (particularly females) and persistent reductions in food intake of both sexes. At 7500 ppm low food intake was evident during the first 24-48 hours for both sexes and remained slightly lower than control for females to the end of treatment.  As a consequence body weight loss was evident during the first 24 hours for the majority of animals; thereafter the body weight gain of males was similar to control, however, for females parity with Day 1 body weight was not achieved until Day 10. At 3000 ppm effects were minimal, limited to slightly low food intake and minor body weight loss for females on Day 1 and minor body weight loss during Days 1-4 for males, although in the absence of any effect on food intake.

  

Food consumption and efficiency- Treatment at the high level of 11000 ppm resulted in persistent reductions in food intake of both sexes. At 7500 ppm low food intake was evident during the first 24-48 hours for both sexes and remained slightly lower than control for females to the end of treatment. At 3000 ppm effects were minimal, with only slightly low food intake observed.

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

   

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

  

Organ weights- When compared to control animals’ body weight relative liver weights were slightly increased in both sexes in all treated groups although in the absence of a dose response relationship. Bodyweight relative kidney weights were slightly increased in males receiving 11000 ppm and bodyweight relative testes weights were slightly increased in males receiving 7500 ppm. Females receiving 7500 ppm had slightly higher bodyweight relative kidney weights than controls, but in the absence of a similar effect in females receiving 11000 ppm this finding is considered to be fortuitous.

 

In conclusion, treatment with the test item at 7500, 11000 and 3000 ppm had no definite effect on clinical condition, water consumption, macropathology or organ weights.  However, the high level of 11000 ppm is considered too high for further investigation due to the magnitude of effects on body weight performance and persistent reductions in food intake.  Therefore a dietary level of 7500 ppm would be a suitable high dose for the OECD 422 screening study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
181 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The endpoint is concluded based on a single key study with a Klimish rating of 1.
System:
other: The NOAEL was driven by effects on bodyweight gain, which was considered an adverse effect during the study.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.
Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

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.

Additional information

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.

Justification for classification or non-classification

The substance does not meet the criteria for classification for specific target organ toxicity - repeated exposure (STOT-RE) in accordance with Regulation (EC) No 1272/2008 (CLP).