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Administrative data

Description of key information

Rat oral NOAEL: 1000 mg/Kg/day (OECD 408)

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-SEP-09 to 2021-NOV-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
June 2018
Deviations:
yes
Remarks:
None of the deviations was considered to have affected the outcome or integrity of the study
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 2008
Deviations:
yes
Remarks:
None of the deviations was considered to have affected the outcome or integrity of the study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
yes
Remarks:
None of the deviations was considered to have affected the outcome or integrity of the study
Qualifier:
according to guideline
Guideline:
other: Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF) Appendix Director General Notification, No. 12-Nousan-8147
Version / remarks:
November 2000
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Valtris Speciality Chemicals (Bridgeport, NJ, USA); Batch Number: 5840
- Purity, including information on contaminants, isomers, etc.: 99.609% (GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneous and stable for 24 hours at room temperature and for seven days when stored refrigerated (2°C to 8°C).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, oily liquid

OTHER SPECIFICS
- Expiration date: 2021-MAR-04
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is a suitable rodent species for toxicity testing, acceptable to regulatory authorities and for which extensive background data are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited (Margate, Kent, CT9 4LT, England)
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: Males: 175 - 223 grams; Females: 118 - 162 grams
- Fasting period before study: Not specified
- Housing: Housed in groups, by sex, in solid-floor cages
- Diet (e.g. ad libitum): Teklad 2014C Rodent Maintenance Diet (Envigo RMS (UK) Limited) ad libitum
- Water (e.g. ad libitum): mains tap water (in bottles) ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
It was considered that none of the contaminants that were monitored was present at a level that might have prevented the study objective from being achieved. Certificates of analysis for diet and water are retained within the CRO Sequani.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C to 24°C
- Humidity (%): 40-70%
- Air changes (per hr): Room airconditioned (number of air changes not specified)
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2020-SEP-03 To: 2020-DEC-22
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration corresponds to a possible route of human exposure during manufacture, use, or handling,
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated within the stability period, for each group separately, as a suspension in corn oil by weighing directly into the final preparation container, with the required quantity of vehicle needed to make up to final weight and stirred until homogeneous. Formulations were divided into daily aliquots and stored refrigerated (2°C to 8°C) and stirred from at least 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (justification not specified)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration Analyses:
Sets of samples (for analysis or for contingency) were taken from each test material formulation prepared for use on the first day of dosing and on one day towards the end of the dosing period. Samples were analysed under Covance Reference Number 8451750 using the method validated in Covance Study Number: LB14VL (2020).

Homogeneity and Stability:
Homogeneity and stability of test material formulations prepared at concentrations of 3.75 and 250 mg/mL, spanning those used in this study (25 to 250 mg/mL), were examined in an earlier formulation validation study (Envigo Study Number: S56026 (2018)).
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control - corn oil)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low dose)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 (Intermediate dose)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected in consultation with the Sponsor after examining existing toxicity data (14-day Repeated Dose Oral (Gavage) Range Finding Toxicity Study in the Rat. Envigo Study Number: S56026 and a 28 Day Oral (Gavage) Toxicity Study in the Rat with a 15 Day Treatment-Free Period. Sequani Study Number: JSK0016). The high dose level of 1000 mg/kg/day selected is the limit dose for this type of study and this dose level was expected to produce some toxicity, such as a reduction in body weight gain or food intake, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level selected was 300 mg/kg/day, which was the approximate geometric mean between the high and low dose, was expected to produce minimal to moderate toxicity. The low dose level of 100 mg/kg/day was selected with the expectation that it would produce no observable indications of toxicity.

- Rationale for animal assignment (if not random):
Allocation to groups was performed using the Provantis stratified randomisation procedure based on individual body weights recorded on arrival. The cages were positioned in the battery using a randomised cage allocation procedure

- Fasting period before blood sampling for clinical biochemistry: No

- Other:
Animals were dosed once daily for at least 90 days (up to 15 weeks), by gavage, using a rubber catheter and disposable syringe at a constant dose volume of 4 mL/kg body weight, until the day before necropsy. Individual doses were adjusted according to the most recent body weight.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: examined twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: examined daily for clinical signs of toxicity or changes in behaviour and appearance from Day 6 onwards. Each animal was given a detailed clinical examination weekly from the start of treatment. From the start of dosing, animals were observed approximately one to two hours after dosing, based on
completion of dosing for the study.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed at the start of dosing and then weekly until necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Amount of food consumed by each cage of animals was recorded at the start of dosing and weekly during the treatment period.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes of all animals were examined before the start of treatment. All animals from each of the control and high dose groups were also examined in Week 13
- Dose groups that were examined: All dose groups
Examinations were performed using direct and indirect ophthalmoscopy after previous use of a mydriatic agent

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day of necropsy
- Animals fasted: No
- How many animals: All animals
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: Day of necropsy
- Animals fasted: No
- How many animals: all animals
Blood samples (0.2 mL) were taken from the sublingual vein under isoflurane anaesthesia into gel separator tubes and allowed to clot for at least 30 minutes at room temperature. All animals were sampled on the day of necropsy between 09.00 and 11.00; animals of the same sex were bled within one hour during this time period. Animals were not fasted prior to blood sampling and were sampled in a random cage order. All samples were centrifuged (3000 g, 10 minutes, at approximately 4°C) and the resultant serum was aliquoted into two tubes (Aliquot 1 contained 50 μL and Aliquot 2 contained all remaining serum) and stored frozen (< -70°C) until analysis. Samples were analysed for thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH), using validated method BMK062MA (Sequani Study number SEQ0361 (2019)).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: All animals were observed once weekly, starting pre-dose, for their behaviour both within their cage and then after placement in an open arena .
- Dose groups that were examined: All dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity: During Weeks 13 and 15, sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity were recorded for all animals

IMMUNOLOGY: No

OTHER:
VAGINAL SMEARS:
On the day of necropsy, vaginal smears were taken by lavage. The smear was examined under light microscopy and the stage of the oestrous cycle was recorded.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)

All animals were killed by exposure to carbon dioxide gas in a rising concentration, weighed and examined externally. The abdominal cavity was opened and the animals were exsanguinated from the caudal vena cava. The cranial and thoracic cavities were opened, a full internal examination was performed and all macroscopic abnormalities were recorded. Organs listed in Table no. 4 were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together).

HISTOPATHOLOGY: Yes (see table 5)

For all animals, with the exception of the eyes, brain, adrenals, bone marrow smear, Harderian glands, optic nerves and testes, either whole organs or samples of the tissues listed in Table no. 5 were preserved in 10 % buffered formalin. The eyes, Harderian glands and optic nerves were fixed in Davidson’s solution, the adrenals, brain and the testes were fixed in Modified Davidson’s solution for approximately 24 to 72 hours and then transferred to 10 % buffered formalin. The bone marrow smears were fixed in methanol and stained but, in the absence of any haematological reasons for doing so, they were not examined.

Initially, for all control and high dose animals, the specified tissues were wax embedded, cut at a nominal thickness of 4 μm to 5 μm, stained with haematoxylin and eosin and examined microscopically. Following treatment-related findings seen in high dose animals, examination of the thyroids, liver and urinary bladder was then extended to all toxicity assessment animals.
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1.

Depending on the nature of the data set that was to be analysed, appropriate tests were applied, as indicated in Table 2. Where parametric tests were appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied.

Probability values of less than 5% were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted. Further details are provided in Table no. 6.
Clinical signs:
no effects observed
Description (incidence and severity):
No signs of clinical toxicity were observed through the study period.
Mortality:
no mortality observed
Description (incidence):
No mortality was observed through the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no effect on body weight or body weight gain in female rats at any dose level or in male rats given 100 mg/kg/day or 300 mg/kg/day. A slight (7%), non-statistically significant lowering of overall body weight gain was observed in males given 1000 mg/kg/day, when compared with the corresponding controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related ocular changes or abnormalities were observed after exposure to the test material.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, mean platelet volume (MPV) was statistically significantly higher than controls in males (p≤0.001) and females (p≤0.05), and platelet distribution width was higher than controls in males (p≤0.05). Mean cell volume (MCV) was statistically significantly lower than controls in male rats (p≤0.01) and female rats (p≤0.05) given 1000 mg/kg/day. Reticulocyte haemoglobin content (CHr) was lower than controls in males at all dose levels (p≤0.05 to p≤0.001) and haemoglobin distribution width (HDW) in female rats was statistically significantly higher than controls at 300 or 1000 mg/kg/day (p≤0.01).These changes were marginal, with most or all individual values within the historical control range, and as they had no effect on the primary red cell parameters, were considered not to be adverse.

Mean reticulocyte corpuscular volume (MCVr) in male rats given 1000 mg/kg/day was statistically significantly lower than the corresponding control mean. However, this was due to three high individual control values (i.e. values above the upper limit of the historical control range) rather than an effect of the test material.

White blood cell count in males given 1000 mg/kg/day was statistically significantly higher than controls (p≤0.05), primarily due to slight increases in absolute lymphocyte and monocyte counts. However, all individual values were within the historical control range and since these changes were minor in nature, considered not to be adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
White blood cell count in males given 1000 mg/kg/day was statistically significantly higher than controls (p≤0.05), primarily due to slight increases in absolute lymphocyte and monocyte counts. However, all individual values were within the historical control range and since these changes were minor in nature, considered not to be adverse.

Plasma cholesterol in both sexes was higher compared to controls at 300 (p≤0.05 to p≤0.01) and 1000 mg/kg/day (p≤0.01). All individual values, with the exception of one female given 1000 mg/kg/day, and one male given 300 or 1000 mg/kg/day, were within the historical control background ranges.

Triglyceride concentration was statistically significantly higher in males given 300 or 1000 mg/kg/day (p≤0.05 and p≤0.01, respectively) but most individual values for were within the historical control background ranges.

Plasma calcium was statistically significantly higher than controls in males at 1000 mg/kg/day (p≤0.001). However, all individual values were within the historical control background range. Statistically significant (p≤0.05) increases in plasma sodium concentration at 300 and 1000 mg/kg/day, and lowering of plasma chloride concentrations (p≤0.01) at 1000 mg/kg/day were observed in male rats, however most or all individual values were within the historical control
range.

These changes in plasma cholesterol, triglyceride, calcium, sodium, and chloride levels were relatively minor and, in the absence of corroborative pathology changes, were considered not to be adverse.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
In male rats given 1000 mg/kg/day, group mean TSH concentration was 2.6-fold higher than controls (p≤0.01), where 7 out 10 males were above the mean historical control range. In females at this dose level, there was a similar 2.5-fold increase in TSH concentration when compared with corresponding controls where 4 out of 10 females were above the mean historical control range although, this did not achieve statistical significance.

In female rats, there was a marginal, but statistically significant (p≤0.05) increase in group mean T4 concentration at 1000 mg/kg/day where 9 out 10 females were above the mean historical control range. The increase in TSH concentration was considered to be associated with the rodent-specific adaptive epithelial hypertrophy in the thyroid gland and, in the absence of any marked changes in T3 or T4 concentrations, this finding was considered to be non-adverse.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Several instances of hyper- and hypoactivity in the arena were observed in all dose groups during the weekly functional observations throughout the study in both sexes. However, as the incidence was low and the pattern and frequency of these observations not dose-related, this observation was not considered to be treatment-related. At 100 mg/kg/day on Day 77, two males had a straub tail and one female excessively reared in the arena. On Day 91 of the study, at 300 mg/kg/day, one male displayed an excessive response to tail-pinching. These observations were considered not to be treatment-related due to their infrequent and transient nature. At 1000 mg/kg/day, in males and females, there were two and one instances of slight salivation on removal from the home cage, respectively, and one instance of salivation in the arena in males.

Mean movement counts and distance travelled for all groups, including control rats, generally decreased with time in both sexes. In males, there was a statistically significant increase in movement count and distance travelled during Period 1 at 300 and 1000 mg/kg/day (p≤0.05), but these differences were not dose-related. In female rats given 1000 mg/kg/day, during Period 1, there was a statistically significant increase in distance travelled (p≤0.05), and a decrease in time at rest (p≤0.05). Thereafter, there were no statistically significant intergroup differences in movement counts or distance travelled at any dose level, in either sex. As these inter-group differences were observed in Period 1 only and not at any other time point, they were considered unlikely to be treatment-related.

Fore and hindlimb grip strength was statistically significantly increased in females at 1000 mg/kg/day (p≤0.05) compared with controls. However, as this reduction was not consistent between sexes, it was considered likely to be due to chance rather than any effect of the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver:
Body weight-related liver weight in female rats given 300 or 1000 mg/kg/day and in male rats at all dose levels were statistically significantly higher than controls (p≤0.05 to p≤0.001). Most individual body weight-related liver weights for male rats given 1000 mg/kg/day were above the upper limit of the historical background range, with group mean relative liver weight being 35% and 17% higher than corresponding controls, for males and females respectively, correlating to the adaptive centrilobular hypertrophy of the liver seen microscopically.

Thyroid:
In male rats, mean absolute and body weight-related thyroid weights were statistically significantly higher (p≤0.05 to p≤0.01) than the control mean across all dose groups in a dose-related manner, by up to 2.3-fold, correlating with the adaptive follicular epithelial hypertrophy seen microscopically in the thyroid. Most individual values were, however, within the historical background range.

Kidney:
Body weight-related kidney weights for males (p≤0.001) and females (p≤0.05) given 1000 mg/kg/day were statistically significantly higher than controls, by 12% and 5%, respectively. All individual values were within the historical background range, and there were no microscopic correlates. Therefore, this change was considered not to be of toxicological significance.

Spleen:
In males, absolute and body weight-related spleen weight was higher than controls by up to 24% at 1000 mg/kg/day (p≤0.001). All individual body weight-related spleen weights were within the historical background range, and there were no microscopic correlates, therefore this change was considered not to be of toxicological significance.

Epididymides:
Absolute epididymis weight at 300 and 1000 mg/kg/day was statistically significantly lower than controls (p≤0.05). However, this was not strictly dose-related, and there were no microscopic correlates, therefore this intergroup difference was considered to be due to chance rather than any effect of the test material.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A variety of spontaneous changes were noted in the control and treated animals with no indication of an effect of treatment. The spectrum of these findings was generally consistent with changes encountered in rats of this age kept under laboratory conditions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathology revealed treatment-related effects in the liver (hypertrophy centrilobular), thyroid glands (hypertrophy of the follicular epithelium and increased basophilia of colloid), and urinary bladder (hyperplasia of the urothelium). These effects in the liver and thyroid were considered to be rodent specific adaptive responses. Hyperplasia of the urothelium of the urinary bladder was considered likely to be a local effect.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
At necropsy, the stage of oestrous was similar across all groups.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic toxicity
Key result
Critical effects observed:
no

The mean concentrations of 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester in test formulations analysed for the study were within 6 % of nominal concentrations which was within applied limits of +10/-15% from nominal, confirming accurate formulation. The coefficient of variation values were less than 4%, which is within applied limits of ≤5 %, confirming the precision of analysis.

Table 7. Results of Formulation analysis

Occasion

Group

Nominal

concentration

(mg/mL)

Analyzed concentration (mg/mL)

RME (%)

CV (%)

Procedural

Recoveries (%)

Top

Middle

Bottom

Mean

First

occasion

1

0

-

ND, ND

-

-

-

-

-

2

25

25.5

25.4

25.6

25.5

+2.0

0.33

101.5

3

75

74.7

76.1

76.3

75.7

+0.9

1.14

102.9

4

250

255

251

260

255

+2.0

1.79

10.24

 

One

occasion

towards

end of the

study

1

0

-

ND, ND

-

-

-

-

-

2

25

25.3

26.9

26.8

26.4

+5.6

3.51

110.0

3

75

78.9

79.0

78.3

78.8

+5.1

0.47

104.5

4

250

232, 2311

244, 2401

242, 2541

241

-3.6

3.61

94.12, 95.52, 94.82

RME Relative mean error, representing the deviation from nominal

ND Not detected

CV Coefficient of variation

1 Contingency samples

2 Recovery samples are outside of validation limits (96.1 – 111.1 %), however recovery results were at roughly 95%, which was within 10% of nominal and as samples were not corrected for recovery values, this was considered to have no impact on the integrity of the study, therefore these results were reported.

Table 8. Functional Observations: Motor activity (Group mean values)

Group

 

Movements

Counts Period 1

(Day Number)

Movements

Counts Period 2

(Day Number)

Movements

Counts Period 3

(Day Number)

Movements

Counts Period 4

(Day Number)

Movements

Counts Period 5

(Day Number)

Movements

Counts Period 6

(Day Number)

90 (#)

90 (#)

90 (#)

90 (#)

90 (#)

90 (#)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

1251.3

761.7

663.6

749.1

584.6

477.0

SD

362.0

212.7

225.5

275.0

139.7

350.3

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

1466.0

708.1

602.2

571.1

550.4

391.5

SD

307.5

196.3

168.3

255.0

240.5

301.4

N

10

10

10

10

10

10

Trend

>0.05

-

-

-

-

-

 

Group 3

(P1400

300 mg/kg/day)

Mean

1717.3

901.4

650.4

781.9

595.4

323.3

SD

222.6

182.3

313.2

545.5

281.1

263.5

N

10

10

10

10

10

10

Trend

≤0.05*

-

-

-

-

-

 

Group 4

(P1400

1000 mg/kg/day)

Mean

1545.2

925.6

703.8

739.0

557.6

343.5

SD

428.8

323.9

367.5

447.3

323.7

185.9

N

10

10

10

10

10

10

Trend

≤0.05*

>0.05

>0.05

>0.05

>0.05

>0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05

Note: a period equates to an interval of 10 minutes

Table 9. Functional Observations: Motor activity (Group mean values)

Group

 

Distance Travelled

Period 1 (cm)

(Day Number)

Distance Travelled

Period 2 (cm)

(Day Number)

Distance Travelled

Period 3 (cm)

(Day Number)

Distance Travelled

Period 4 (cm)

(Day Number)

Distance Travelled

Period 5 (cm)

(Day Number)

Distance Travelled

Period 6 (cm)

(Day Number)

90 (#)

90 (# 1)

90 (#)

90 (#)

90 (#)

90 (#; # 2)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

4506.5

2760.3

2418.9

2731.4

2136.3

1685.3

SD

1254.0

748.8

825.2

998.5

473.4

1249.4

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

5207.7

2516.7

2184.6

2052.4

1987.5

1422.4

SD

941.6

588.2

611.4

876.1

772.9

1095.2

N

10

10

10

10

10

10

Trend

>0.05

-

-

-

-

-

 

Group 3

(P1400

300 mg/kg/day)

Mean

5926.4

3125.7

2257.4

2698.0

2120.9

1135.6

SD

761.5

540.5

1114.2

1713.8

1010.0

956.9

N

10

10

10

10

10

10

Trend

≤0.05*

-

-

-

-

-

 

Group 4

(P1400

1000 mg/kg/day)

Mean

5636.9

3343.1

2528.5

2646.9

2088.0

1262.3

SD

1409.3

1122.1

1196.0

1567.0

1199.7

675.0

N

10

10

10

10

10

10

Trend

≤0.05*

>0.05

>0.05

>0.05

>0.05

>0.05

Females

Group 1

(Control – 0 mg/kg/day)

Mean

4932.7

3201.8

2818.6

2532.5

1842.3

1998.9

SD

1503.5

798.9

1168.3

1136.5

983.4

1399.8

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

5472.0

3538.5

2780.7

2462.0

2724.9

2069.3

SD

1119.9

1061.8

982.4

716.1

956.0

396.2

N

10

10

10

10

10

10

 

Group 3

(P1400

300 mg/kg/day)

Mean

5747.9

3794.3

3240.2

2528.6

2378.4

1605.9

SD

1415.3

1259.4

1264.7

1105.4

1278.6

1319.3

N

10

10

10

10

10

10

Trend

>0.05

-

-

-

-

-

 

Group 4

(P1400

1000 mg/kg/day)

Mean

6361.8

3465.2

2051.8

1948.4

2024.7

1558.5

SD

1521.9

1264.1

1256.8

1308.0

1392.2

1557.4

N

10

10

10

10

10

10

Trend

≤0.05*

>0.05

>0.05

>0.05

>0.05

>0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05

(#1) - Williams, Anova & Dunnett(Log)

(#2) - Shirley, Kruskal-Wallis & Steel (Females)

Note: a period equates to an interval of 10 minutes

Table 10. Functional Observations: Motor activity (Group mean values)

Group

 

Time at Rest

Period 1 (sec)

(Day Number)

Time at Rest

Period 2 (sec)

(Day Number)

Time at Rest

Period 3 (sec)

(Day Number)

Time at Rest

Period 4 (sec)

(Day Number)

Time at Rest

Period 5 (sec)

(Day Number)

Time at Rest

Period 6 (sec)

(Day Number)

90 (#)

90 (#)

90 (#)

90 (#)

90 (# 1)

90 (# 1)

Females

Group 1

(Control – 0 mg/kg/day)

Mean

244.58

334.84

360.27

390.63

438.42

434.04

SD

52.59

52.59

83.26

78.05

88.59

114.73

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

208.73

312.08

368.36

398.39

382.79

423.86

SD

36.40

57.02

49.43

55.08

60.41

38.78

N

10

10

10

10

10

10

 

Group 3

(P1400

300 mg/kg/day)

Mean

213.12

311.86

365.61

399.92

400.14

453.98

SD

41.35

50.96

68.77

70.04

95.92

114.93

N

10

10

10

10

10

10

Trend

>0.05

-

-

-

-

-

 

Group 4

(P1400

1000 mg/kg/day)

Mean

197.05

339.0

430.51

450.14

443.46

468.96

SD

52.82

53.95

90.29

100.15

99.29

106.08

N

10

10

10

10

10

10

Trend

≤0.05*

>0.05

>0.05

>0.05

>0.05

>0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05

(#1) - Williams, Anova & Dunnett (Log)

Note: a period equates to an interval of 10 minutes

Table 11. Functional observations: Grip strength (Group mean values – Females)

Group

 

Forelimb

Mean (g)

(Day Number)

Hindlimb

Mean (g)

(Day Number)

 

 

103 (#)

103 (#)

Group 1

(Control – 0 mg/kg/day)

Mean

450.07

317.63

SD

121.18

46.52

N

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

509.00

337.90

SD

104.04

55.27

N

10

10

 

Group 3

(P1400

300 mg/kg/day)

Mean

469.70

287.23

SD

114.26

61.65

N

10

10

Trend

>0.05

>0.05

 

Group 4

(P1400

1000 mg/kg/day)

Mean

580.07

379.90

SD

147.38

73.43

N

10

10

Trend

≤0.05*

≤0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05

Table 12. Select Haematology Parameters (Group mean values)

Group

 

Males

MCV

fl

(Week Number)

MPV

fl

(Week Number)

PDW

%

(Week Number)

MCVr

fl

(Week Number)

CHr

pg

(Week Number)

WBC

10^3/uL (Week Number)

Lymph

10^3/uL

(Week Number)

Mono

10^3/uL

(Week Number)

LUC

10^3/uL

(Week Number)

Eosin

%

(Week Number)

15 (#)

15 (# 1)

15 (#)

15 (#)

15 (#)

15 (#)

15 (#)

15 (#)

15 (# 2)

15 (# 1)

Group 1

(Control – 0 mg/kg/day)

Mean

52.96

7.99**

48.78

66.28

19.15

5.358

4.466

0.076

0.019

1.26

SD

1.11

0.20

5.61

0.78

0.37

0.963

0.966

0.025

0.010

0.51

N

10

9

9

10

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

52.09

8.31*

49.84

65.39

18.75

4.978

4.079

0.084

0.019

1.30

SD

1.08

0.27

1.87

1.11

0.42

0.531

0.449

0.020

0.007

0.26

N

10

10

10

10

10

10

10

10

10

10

Trend

-

-

-

-

≤0.05*

-

-

-

-

-

 

Group 3

(P1400

300 mg/kg/day)

Mean

51.99

7.93

49.14

65.24

18.76

5.401

4.529

0.089

0.020

1.26

SD

1.07

0.29

2.90

1.23

0.50

1.241

1.035

0.038

0.011

0.55

N

10

10

10

10

10

10

10

10

10

10

Trend

>0.05

>0.05

>0.05

>0.05

≤0.05*

>0.05

>0.05

>0.05

>0.05

>0.05

 

Group 4

(P1400

1000 mg/kg/day)

Mean

50.72

8.39

53.11

63.55

18.15

6.450

5.440

0.113

0.032

0.77

SD

2.11

0.17

4.60

1.98

0.62

0.953

0.977

0.038

0.018

0.22

N

10

10

10

10

10

10

10

10

10

10

Trend

≤0.01**

≤0.001***

≤0.05*

≤0.001***

≤0.001***

≤0.05*

≤0.05*

≤0.05*

≤0.05*

≤0.05*

(#) & (# 1) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

(#2) - Williams, Anova & Dunnett(Log): * = p ≤ 0.05

Table 13. Select Haematology Parameters (Group mean values)

Group

 

Females

MCV

fl

(Week Number)

MCH

pg

(Week Number)

MCHC

g/dl

(Week Number)

RDW

%

(Week Number)

HDW

g/dl

(Week Number)

MPV

fl

(Week Number)

15 (#)

15 (# 1)

15 (# 1)

15 (#)

15 (#)

15 (# 1)

Group 1

(Control – 0 mg/kg/day)

Mean

55.40

18.93*

34.16**

10.95*

1.958

8.00

SD

1.27

0.46

0.46

0.37

0.066

0.19

N

10

10

10

10

10

9

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

55.39

18.73

33.87

11.16

1.979

8.08

SD

2.01

0.37

0.83

1.64

0.155

0.18

N

10

10

10

10

10

10

Trend

-

-

-

-

>0.05

-

 

Group 3

(P1400

300 mg/kg/day)

Mean

55.01

19.19

34.89*

11.66

2.103

7.97

SD

1.19

0.43

0.55

2.08

0.149

0.18

N

10

10

10

10

10

10

Trend

>0.05

-

-

-

≤0.01**

>0.05

 

Group 4

(P1400

1000 mg/kg/day)

Mean

53.98

18.64

34.54

11.29

2.050

8.28

SD

0.97

0.47

0.50

0.37

0.067

0.36

N

10

10

10

10

10

10

Trend

≤0.05*

>0.05

>0.05

>0.05

≤0.01**

≤0.05*

(#) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05

Table 14. Select Clinical Chemistry Parameters (Group mean values)

Group

 

Males

Females

Na

mmol/l

(Week Number)

Cl

mmol/l

(Week Number)

Ca

mg/dl

(Week Number)

Chol

mg/dl

(Week Number)

Trigs

mg/dl

(Week Number)

Glob

g/dl

(Week Number)

A/G

ratio

(Week Number)

Chol

mg/dl

(Week Number)

15 (#)

15 (#)

15 (#)

15 (#)

15 (#)

15 (# 1)

15 (#)

15 (#)

Group 1

(Control – 0 mg/kg/day)

Mean

141.6

102.6

10.42

65.3

146.6

2.22***

2.21***

56.1

SD

1.3

1.2

0.15

10.5

38.9

0.20

0.24

12.4

N

10

10

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

141.5

102.5

10.34

67.9

137.5

1.91***

2.59***

55.8

SD

1.4

0.7

0.15

8.9

2.9

0.13

0.18

11.5

N

10

10

10

10

10

10

10

10

Trend

>0.05

-

-

>0.05

>0.05

-

-

>0.05

 

Group 3

(P1400

300 mg/kg/day)

Mean

142.5

102.5

10.48

80.6

195.9

2.03*

2.35

67.0

SD

0.8

1.3

0.19

13.0

72.3

0.16

0.21

9.1

N

10

10

10

10

10

10

10

10

Trend

≤0.05*

>0.05

>0.05

≤0.01**

≤0.05*

-

-

≤0.05*

 

Group 4

P1400

1000 mg/kg/day

Mean

142.9

101.0

10.79

78.9

226.7

2.28

2.12

72.0

SD

0.9

1.0

0.20

9.8

68.9

0.12

0.17

13.4

N

10

10

10

10

10

10

10

10

Trend

≤0.05*

≤0.01**

≤0.001***

≤0.01**

≤0.01**

>0.05

>0.05

≤0.01**

(#) and (# 1) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01;*** = p ≤ 0.001

Values above or below the limit of quantification have been excluded from the group means

Na: sodium

Ca: calcium

Cl: chlorine

Chol: cholesterol

Trigs: triglyceride

Glob: globulin

A/G: albumin/globulin ratio

Table 15. Thyroid hormone assessment (Group mean values)

Group

 

Males

Females

Mean Total

T3 (ng/mL)

Mean Total

T4 (ng/mL)

Mean Total

TSH (ng/mL)

Mean Total

T3 (ng/mL)

Mean Total

T4 (ng/mL)

Mean Total

TSH (ng/mL)

(#)

(# 1)

(# 2)

(#)

(# 1)

(# 2)

Group 1

(Control – 0 mg/kg/day)

Mean

13.471

475.959

0.797

12.763

343.996

0.621

SD

4.387

37.643

0.479

9.613

58.436

0.243

N

10

10

9

9

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

13.875

478.603

0.701

10.889

331.376

1.134

SD

2.430

27.709

0.435

4.327

46.663

1.639

N

10

10

10

10

10

10

 

Group 3

(P1400

300 mg/kg/day)

Mean

16.150

468.248

1.455

12.173

373.460

0.958

SD

4.522

28.543

1.681

4.785

45.217

0.962

N

10

10

10

9

10

9

Trend

-

-

>0.05

-

>0.05

-

 

Group 4

(P1400

1000 mg/kg/day)

Mean

12.321

462.386

2.057

13.993

396.665

1.542

SD

2.358

20.047

1.030

3.594

43.594

1.719

N

10

10

10

10

10

10

Trend

>0.05

>0.05

≤0.01**

>0.05

≤0.05*

>0.05

(#) - Williams, Anova & Dunnett

(#1) - Shirley, Kruskal-Wallis & Steel

(#2) - Williams, Anova & Dunnett(Log): ** = p ≤ 0.01

(#)- Williams, Anova & Dunnett(Log)

(#1)- Williams, Anova & Dunnett: * = p ≤ 0.05

(#2)- Shirley, Kruskal-Wallis & Steel

Table 16. Select Organ weights (Group mean values) - Males

Group

 

Spleen

(g)

Adjusted

Spleen Wt

Spleen %

Body Weight

Liver

(g)

Adjusted

Liver Wt

Liver %

Body Weight

Adjusted

Kidneys Wt

Kidneys %

Body Weight

Epididymides

(g)

Adjusted

Epididymides Wt

Thyroids

(g)

Adjusted

Thyroids Wt

Thyroids %

Body Weight

(#)

(# 1)

(#)

(#)

(# 1)

(#)

(# 1)

(#)

(#)

(# 1)

(#)

(# 1)

(#)

Group 1

(Control – 0 mg/kg/day)

Mean

0.573

0.570

0.131

13.921

13.766

3.162

2.461

0.565

1.596

1.586

0.0140

0.0140

0.0032

SD

0.049

-

0.013

1.209

-

0.071

-

0.045

0.238

-

0.0040

-

0.0010

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

0.621

0.612

0.140

14.848

14.429

3.313

2.424

0.553

1.521

1.495

0.0182

0.0182

0.0041

SD

0.068

-

0.016

1.930

-

0.180

-

0.022

0.196

-

0.0045

-

0.0012

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Trend

-

-

-

-

≤0.05*

≤0.05*

-

-

>0.05

>0.05

≤0.05*

≤0.05*

>0.05

 

Group 3

(P1400

300 mg/kg/day)

Mean

0.598

0.600

0.138

15.320

15.410

3.538

2.450

0.564

1.379

1.385

0.0259

0.0259

0.0060

SD

0.078

-

0.015

1.862

-

0.256

-

0.046

0.147

-

0.0056

-

0.0013

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Trend

>0.05

>0.05

>0.05

>0.05

≤0.001***

≤0.001***

>0.05

>0.05

≤0.05*

≤0.05*

≤0.001***

≤0.001***

≤0.001***

 

Group 4

P1400

1000 mg/kg/day

Mean

0.684

0.695

0.162

18.031

18.514

4.270

2.723

0.631

1.423

1.453

0.0308

0.0308

0.0074

SD

0.064

-

0.012

2.021

-

0.167

-

0.040

0.125

-

0.0045

-

0.0015

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Trend

≤0.001***

≤0.001***

≤0.001***

≤0.001***

≤0.001***

≤0.001***

≤0.001***

≤0.001***

≤0.05*

≤0.05*

≤0.001***

≤0.001***

≤0.001***

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; *** = p ≤ 0.001

(#1) - Williams, Ancova/Anova & Dunnett: * = p ≤ 0.05; *** = p ≤ 0.001

{Covariate(s): Dead body weight}

Table 17. Select Organ weights (Group mean values) - Females

Group

 

Liver (g)

Adjusted

Liver Wt

Liver %

Body Weight

Kidneys %

Body Weight

(#)

(# 1)

(#)

(#)

Group 1

(Control – 0 mg/kg/day)

Mean

8.567

8.446

3.494

0.651

SD

0.819

-

0.157

0.018

N

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

8.506

8.522

3.515

0.637

SD

0.994

-

0.280

0.045

N

9

9

9

9

Trend

-

>0.05

>0.05

-

 

Group 3

(P1400

300 mg/kg/day)

Mean

8.925

9.007

3.722

0.668

SD

0.719

-

0.231

0.045

N

10

10

10

10

Trend

>0.05

≤0.05*

≤0.05*

>0.05

 

Group 4

P1400

1000 mg/kg/day

Mean

9.901

9.926

4.104

0.686

SD

0.820

-

0.178

0.032

N

10

10

10

10

Trend

≤0.01**

≤0.001***

≤0.001***

≤0.05*

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001 (#1) - Williams, Ancova/Anova & Dunnett: * = p ≤ 0.05; *** = p ≤ 0.001

{Covariate(s): Dead body weight}

Table 18. Select Histopathology Findings

 

Males

Females

Group

1

2

3

4

1

2

3

4

Dose Level

(mg/kg/day)

0

100

300

1000

0

100

300

1000

Number of rats examined

10

10

10

10

10

10

10

10

Liver

 

Hypertrophy, centrilobular

Minimal

0

0

4

8

0

0

2

0

Slight

0

0

0

0

0

0

0

0

Total

0

0

4

8

0

0

2

0

Thyroid Glands

 

Hypertrophy, follicular

epithelium

Minimal

0

1

2

5

0

0

0

0

Slight

0

3

5

3

0

0

1

0

Total

0

4

7

8

0

0

1

0

Colloid, increased

basophilic concretions

Slight

4

4

6

3

0

0

1

3

Moderate

0

0

1

7

0

0

0

0

Total

4

4

7

10

0

0

1

3

Urinary Bladder

 

Hyperplasia, urothelium

Slight

0

0

0

5

1

0

0

2

Total

0

0

0

5

1

0

0

2


Conclusions:
Based on the lack of adverse treatment-related effects observed through the study period, the No Observed Adverse Effect Level (NOAEL) for 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester was determined to be 1000 mg/kg/day.
Executive summary:

A key OECD Guideline 408 sub-chronic toxicity study was conducted to assess the toxicity of the test material (1, 2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester), when administered by gavage to the rat once daily for at least 90 days.

 

The test material was administered to Crl:WI(Han) Wistar rats (5/sex/dose) once daily via oral gavage for a period of 90 days at dose levels of 0, 100, 300, or 1000 mg/kg/day in a corn oil vehicle. Animals were subsequently observed for the following parameters: clinical observations, ophthalmoscopy, body weight, food intake, behavioural observations and functional observation battery, vaginal smear on the day of necropsy, haematology, blood chemistry, thyroid hormone assessment, organ weights, macroscopic and microscopic pathology.

 

No mortality or signs of clinical toxicity were observed through the study period. There was no effect on body weight or body weight gain in female rats at any dose level or in male rats given 100 mg/kg/day or 300 mg/kg/day. A slight (7%), non-statistically significant lowering of overall body weight gain was observed in males given 1000 mg/kg/day, when compared with the corresponding controls. Food consumption was unaffected by treatment. No treatment-related ocular changes or abnormalities were observed after exposure to the test material.

 

At 1000 mg/kg/day, mean platelet volume (MPV) was statistically significantly higher than controls in males (p≤0.001) and females (p≤0.05), and platelet distribution width was higher than controls in males (p≤0.05). Mean cell volume (MCV) was statistically significantly lower than controls in male rats (p≤0.01) and female rats (p≤0.05) given 1000 mg/kg/day. Reticulocyte haemoglobin content (CHr) was lower than controls in males at all dose levels (p≤0.05 top≤0.001) and haemoglobin distribution width (HDW) in female rats was statistically significantly higher than controls at 300 or 1000 mg/kg/day (p≤0.01).These changes were marginal, with most or all individual values within the historical control range, and as they had no effect on the primary red cell parameters, were considered not to be adverse. Mean reticulocyte corpuscular volume (MCVr) in male rats given 1000 mg/kg/day was statistically significantly lower than the corresponding control mean. However, this was due to three high individual control values (i.e. values above the upper limit of the historical control range) rather than an effect of the test material.

 

White blood cell count in males given 1000 mg/kg/day was statistically significantly higher than controls (p≤0.05), primarily due to slight increases in absolute lymphocyte and monocyte counts. However, all individual values were within the historical control range and since these changes were minor in nature, considered not to be adverse. Plasma cholesterol in both sexes was higher compared to controls at 300 (p≤0.05 top≤0.01) and 1000 mg/kg/day (p≤0.01). All individual values, with the exception of one female given 1000 mg/kg/day, and one male given 300 or 1000 mg/kg/day, were within the historical control background ranges. Triglyceride concentration was statistically significantly higher in males given 300 or 1000 mg/kg/day (p≤0.05 andp≤0.01, respectively) but most individual values for were within the historical control background ranges. Plasma calcium was statistically significantly higher than controls in males at 1000 mg/kg/day (p≤0.001). However, all individual values were within the historical control background range. Statistically significant (p≤0.05) increases in plasma sodium concentration at 300 and 1000 mg/kg/day, and lowering of plasma chloride concentrations (p≤0.01) at 1000 mg/kg/day were observed in male rats, however most or all individual values were within the historical control range. These changes in plasma cholesterol, triglyceride, calcium, sodium, and chloride levels were relatively minor and, in the absence of corroborative pathology changes, were considered not to be adverse.

 

In male rats given 1000 mg/kg/day, group mean TSH concentration was 2.6-fold higher than controls (p≤0.01), where 7 out 10 males were above the mean historical control range. In females at this dose level, there was a similar 2.5-fold increase in TSH concentration when compared with corresponding controls where 4 out of 10 females were above the mean historical control range although, this did not achieve statistical significance. In female rats, there was a marginal, but statistically significant (p≤0.05) increase in group mean T4 concentration at 1000 mg/kg/day where 9 out 10 females were above the mean historical control range. The increase in TSH concentration was considered to be associated with the rodent-specific adaptive epithelial hypertrophy in the thyroid gland and, in the absence of any marked changes in T3 or T4 concentrations, this finding was considered to be non-adverse.

 

Several instances of hyper- and hypoactivity in the arena were observed in all dose groups during the weekly functional observations throughout the study in both sexes. However, as the incidence was low and the pattern and frequency of these observations not dose-related, this observation was not considered to be treatment-related. At 100 mg/kg/day on Day 77, two males had a straub tail and one female excessively reared in the arena. On Day 91 of the study, at 300 mg/kg/day, one male displayed an excessive response to tail-pinching. These observations were considered not to be treatment-related due to their infrequent and transient nature. At 1000 mg/kg/day, in males and females, there were two and one instances of slight salivation on removal from the home cage, respectively, and one instance of salivation in the arena in males. Mean movement counts and distance travelled for all groups, including control rats, generally decreased with time in both sexes. In males, there was a statistically significant increase in movement count and distance travelled during Period 1 at 300 and 1000 mg/kg/day (p≤0.05), but these differences were not dose-related. In female rats given 1000 mg/kg/day, during Period 1, there was a statistically significant increase in distance travelled (p≤0.05), and a decrease in time at rest (p≤0.05). Thereafter, there were no statistically significant intergroup differences in movement counts or distance travelled at any dose level, in either sex. As these inter-group differences were observed in Period 1 only and not at any other time point, they were considered unlikely to be treatment-related. Fore and hindlimb grip strength was statistically significantly increased in females at 1000 mg/kg/day (p≤0.05) compared with controls. However, as this reduction was not consistent between sexes, it was considered likely to be due to chance rather than any effect of the test material.

 

Gross necropsy did not reveal any remarkable treatment-related findings.

Body weight-related liver weight in female rats given 300 or 1000 mg/kg/day and in male rats at all dose levels were statistically significantly higher than controls (p≤0.05 top≤0.001). Most individual body weight-related liver weights for male rats given 1000 mg/kg/day were above the upper limit of the historical background range, with group mean relative liver weight being 35% and 17% higher than corresponding controls, for males and females respectively, correlating to the adaptive centrilobular hypertrophy of the liver seen microscopically. In male rats, mean absolute and body weight-related thyroid weights were statistically significantly higher (p≤0.05 top≤0.01) than the control mean across all dose groups in a dose-related manner, by up to 2.3-fold, correlating with the adaptive follicular epithelial hypertrophy seen microscopically in the thyroid. Most individual values were, however, within the historical background range. Body weight-related kidney weights for males (p≤0.001) and females (p≤0.05) given 1000 mg/kg/day were statistically significantly higher than controls, by 12% and 5%, respectively. All individual values were within the historical background range, and there were no microscopic correlates. Therefore, this change was considered not to be of toxicological significance. In males, absolute and body weight-related spleen weight was higher than controls by up to 24% at 1000 mg/kg/day (p≤0.001). All individual body weight-related spleen weights were within the historical background range, and there were no microscopic correlates, therefore this change was considered not to be of toxicological significance. Absolute epididymis weight at 300 and 1000 mg/kg/day was statistically significantly lower than controls (p≤0.05). However, this was not strictly dose-related, and there were no microscopic correlates, therefore this intergroup difference was considered to be due to chance rather than any effect of the test material.

 

Histopathology revealed treatment-related effects in the liver (hypertrophy centrilobular), thyroid glands (hypertrophy of the follicular epithelium and increased basophilia of colloid), and urinary bladder (hyperplasia of the urothelium). These effects in the liver and thyroid were considered to be rodent specific adaptive responses. Hyperplasia of the urothelium of the urinary bladder was considered likely to be a local effect.

 

Based on the lack of adverse treatment-related effects observed through the study period, the No Observed Adverse Effect Level (NOAEL) for 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester was determined to be 1000 mg/kg/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-JAN-30 to 2020-JUNE-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
3rd October 2008
Deviations:
yes
Remarks:
None of the deviations was considered to have affected the outcome or integrity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Deviations:
yes
Remarks:
None of the deviations was considered to have affected the outcome or integrity of the study.
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050 (Repeated dose 28-day toxicity study in rodents)
Version / remarks:
July 2000
Deviations:
yes
Remarks:
None of the deviations was considered to have affected the outcome or integrity of the study.
Qualifier:
according to guideline
Guideline:
other: Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF) 12-Nousan-8147
Version / remarks:
November 2000
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Valtris Speciality Chemicals (Bridgeport, NJ, USA); Batch Number: 3963
- Purity, including information on contaminants, isomers, etc.: 98.6% (GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15ºC to 25ºC)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: homogenous and stable
for 24 hours when stored at room temperature, and up to seven days when stored refrigerated

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, colourless, oily liquid

OTHER SPECIFICS
- Expiration date: 2020-JULY-16
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat was selected since it is a suitable rodent species for toxicity testing, acceptable to regulatory authorities, and for which extensive background data are available.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited (Margate, Kent, CT9 4LT, England)
- Females (if applicable) nulliparous and non-pregnant: Not specified
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: Males: 200-258 grams; Females: 124-184 grams
- Fasting period before study: Not specified
- Housing: Housed in groups, by sex, in solid-floor cages
- Diet (e.g. ad libitum): Teklad 2014C Rodent Maintenance Diet (Envigo RMS (UK) Limited) ad libitum
- Water (e.g. ad libitum): mains tap water (in bottles) ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 23°C
- Humidity (%): 40-70%
- Air changes (per hr): Air conditioned room (air changes not specified)
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: From: 2020-FEB-13 To: 2020-FEB-27
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration corresponds to a possible route of human exposure.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Doses were formulated separately for each dose group and used within six days of preparation. Formulations were divided into daily aliquots and were stored refrigerated (2°C to 8°C) until at least 15 minutes before dosing on the day of use. They were stirred from 15 minutes before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil (justification not specified)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration Analysis:
Sets of samples (for analysis or for contingency) were taken from each test material formulation prepared for the first day of dosing and on one occasion towards the end of the study. The method of analysis used to analyse the dose formulation was that validated in Covance Study Number LB14VL (2020).

Homogeneity and stability:
Homogeneity and stability of test material formulations prepared at concentrations of 3.75 and 250 mg/mL, spanning those used in this study (25 to 250 mg/mL), were examined in an earlier formulation validation study (Envigo Study Number: S56037 (2018)).
Duration of treatment / exposure:
28-days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control - corn oil)
Following treatment, the five highest numbered surviving animals were allocated to a 15 day treatment-free period.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low dose)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 (Intermediate dose)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 (High dose)
Following treatment, the five highest numbered surviving animals were allocated to a 15 day treatment-free period.
No. of animals per sex per dose:
5/sex/dose
Note: 5 additional animals per sex (Groups 1 and 4) were allocated to a 15 day treatment-free period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor after examining existing toxicity data (14-day Repeated Dose Oral (Gavage) Range Finding Toxicity Study in the Rat. Envigo Study Number: S56026 and Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat with Extension to Evaluate Sexual Maturation of F1 Generation. Envigo Study Number: S56037). The high dose level of 1000 mg/kg/day selected is the limit dose for this type of study and this dose level was expected to produce some toxicity, such as a reduction in body weight gain or food intake, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level selected was 300 mg/kg/day, which was the approximate geometric mean between the high and low dose, was expected to produce minimal to moderate toxicity. The low dose level of 100 mg/kg/day was selected with the expectation that it would produce no observable indications of toxicity.

- Rationale for animal assignment (if not random): Allocation to groups was performed using the Provantis stratified randomisation procedure based on individual body weights recorded on arrival. The cages were positioned in the battery using a randomised cage allocation procedure.

- Fasting period before blood sampling for clinical biochemistry: overnight food deprivation

- Dose range finding studies: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2 (phenylmethyl) ester, CAS no.1200806-67-2: 14-day Repeated Dose Oral (Gavage) Range Finding Toxicity Study in the Rat. Envigo Study Number: S56026

- OTHER: After 28 days treatment, the five highest numbered surviving animals from Groups 1 (Control) and 4 (High dose) were allocated to the 15 day treatment-free period.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: examined twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Initially, from the start of dosing, animals were observed before and one to two hours after dosing. From Day 7 additional post-dose observations were made at 15 minutes after dosing. From Day 9 of the study, additional observations were made at one and around four hours after dosing and at the weekend they were observed either at one hour after dosing or at the end of the working day, whichever was sooner.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed at the start of treatment and then weekly thereafter, up to and including the day of necropsy.

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: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Amount of food consumed by each cage of animals was recorded weekly during the treatment and treatment-free period.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 (all surviving males and females) and towards the end of the treatment-free period (all remaining animals)
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 (all surviving males and females) and towards the end of the treatment-free period (all remaining animals)
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.3] were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 (all males and females) and repeated towards the end of the treatment-free period (all remaining animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight food deprivation)
- Parameters checked in table [No. 4] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

All animals were observed once weekly, starting pre-dose, for their behaviour both within their cage and then after placement in an open arena. Observations were made at approximately the same time of day on each occasion (afternoon).

During Week 4, sensorimotor responses to visual, acoustic, tactile or proprioceptive stimuli, grip strength and motor activity were recorded for all animals. Due to potential treatment-related findings at the end of the treatment period, grip strength and motor activity was assessed for all animals at the end of the treatment-free period.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 5)

All animals were killed by exposure to carbon dioxide gas in a rising concentration, weighed and examined externally. The abdominal cavity was opened and the animals were exsanguinated from the caudal vena cava. The cranial and thoracic cavities were opened, a full internal examination was performed and all macroscopic abnormalities were recorded. The organs listed in Table no. 5 were weighed after trimming of fat and other contiguous tissue (contralateral organs were weighed together).

HISTOPATHOLOGY: Yes (see table 6)

For all animals, with the exception of the brain, adrenals, eyes, bone marrow smear, Harderian glands, optic nerves and testes, either whole organs or samples of the tissues listed in Table no. 6 were preserved in 10 % buffered formalin. The eyes, Harderian glands, and optic nerves were fixed in Davidson’s solution and the brain, adrenals and testes were fixed in Modified Davidson’s solution. The bone marrow smear was fixed in methanol, stained with Wrights/Giemsa stain and myelograms prepared.

Initially, for all Control and high dose animals and premature decedents, the specified tissues were wax embedded, cut at a nominal thickness of 4 μm to 5 μm, stained with haematoxylin and eosin and examined microscopically. However, as treatment-related findings were seen in high dose animals, examination of thymus and thyroid glands was then extended to all toxicity assessment animals, including those assigned to the treatment-free period.
Optional endpoint(s):
Optional endpoints: No
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate. Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1. Depending on the nature of the data set that was to be analysed, appropriate tests were applied, as indicated in Table 2. Where parametric tests were appropriate they were preceded by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied. Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p≤0.05 level. For illustrative purposes, significance levels of p≤0.01 and p≤0.001 were also noted.Table no. 7 provides specific details.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related clinical effects were observed in animals of either sex at 100 mg/kg/day and no clinical signs were observed during the treatment-free period.

At 1000 mg/kg/day, one male displayed prostrate behaviour and decreased activity one hour after dosing on Day 8. This decreased activity continued to be seen at 1 hour post-dosing until Day 11. Sporadic instances of ploughing behaviour were also observed up to one hour after dosing in males given 1000 mg/kg/day between Days 11 and 22 of the study. Instances of excessive salivation were seen in males at both 300 and 1000 mg/kg/day from Day 10 to the end of the treatment period.

From Day 7, females in the 1000 mg/kg/day dose group displayed ploughing behaviour, increased activity and excessive licking of other female genitals at 15 minutes after dosing which ceased one hour post-dosing. On Day 8, these observations were observed again, with the addition of prostration and decreased activity in four females at one hour after dosing. By Day 12, with the exception of some excessive salivation (expected due to the viscous nature of the test material formulation), and sporadic instances of ploughing behaviour, these post-dose observations had generally ceased and the clinical condition of the animals had returned to normal.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two deaths were observed during the study period. Female no. 46 given 300 mg/kg/day and female no. 55 given 1000 mg/kg/day were euthanised on Day 9 due to piloerection and breathing abnormalities. At necropsy, the oesophagus of each of these females had a 5 mm rupture and approximately 2.5 mL to 4 mL of straw coloured clear, oily liquid was found in the thoracic cavity. These findings were considered to be a result of an intubation error and not treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no treatment-related effect on bodyweight gains in males given 100 mg/kg/day and in females at all dose levels.

In males, during Weeks 2 to 5, a slight decrease in body weight gain was observed in animals given 300 or 1000 mg/kg/day when compared to Controls leading to an overall decrease in body weight gains in these animals throughout the course of the treatment period. These inter group differences did not attain statistical significance. There was a statistically significant 27% decrease in body weight gain during the treatment-free period for males previously given 1000 mg/kg/day compared with the Controls (p ≤ 0.05).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption remained unaffected by treatment with the test material.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Mean platelet volume (MPV) and platelet distribution width were marginally, but statistically significantly higher (p ≤0.001 and p≤0.01, respectively) in males given 1000 mg/kg/day compared with Controls. All individual values for MPV were above the upper limit of the historical control range, as were most of the individual Control values. However, four out of five MPV values for males given 1000 mg/kg/day were also above the highest individual Control value. Plateletcrit (PCT) was also statistically significantly increased in males given 300 (p ≤ 0.05) and 1000 mg/kg/day (p ≤ 0.01), with most individual values being above the upper limit of the historical control range. However, as there was no effect on platelet count, these changes are considered not to be adverse.

There was a statistically significant increase in neutrophil count in males given 1000 mg/kg/day, although all individual values were within the historical control range.

Activated partial thromboplastin time was statistically significantly (p ≤ 0.01) longer, by around 1.8 seconds, than the Control group mean in males given 1000 mg/kg/day, however as the individual values for these males were all within the historical control range, this change was considered not to be adverse. In females given 1000 mg/kg/day, prothrombin time was statistically significantly longer (p ≤ 0.05) by around 1.7 seconds compared with Controls. All individual values were above the upper limit of the historical control range, however in the absence of similar findings in the males, this change was considered not to be adverse.

Absolute reticulocytes were increased in females at all dose levels, however, this was not dose-related nor consistent between sexes and all individual values were within the historical control range. In males, there was a statistically significant (p ≤ 0.05), but minor decrease in cellular reticulocyte haemoglobin compared with Controls.

At the end of the treatment-free period, all treatment-related effects had reversed or shown evidence of reversibility.

Red cell count in males previously given 1000 mg/kg/day and absolute monocyte count in females previously given this dose level were statistically significantly higher (p≤0.05) than the respective Control group means at the end of the treatment-free period. However, as these parameters were unaffected at the end of the treatment period, these intergroup differences at the end of the treatment-free period were considered likely to be due to normal biological variation rather than any effect of the test material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes in blood chemistry parameters were considered to be treatment related, but not adverse.

Plasma globulin concentration in both sexes given 1000 mg/kg/day was higher than Controls (p ≤ 0.05), leading to a slight decrease in the albumin/globulin ratio in the females (p ≤ 0.05), and a slight increase in total protein concentration in males (p ≤ 0.01). All individual values with were within the historical Control background ranges with exception to plasma globulin concentrations for one male and one female and for total protein concentration for one female.

Plasma cholesterol was higher compared with Controls in males at all dose levels (p ≤ 0.05), albeit lacking a strict dose-relationship, and at 1000 mg/kg/day in females (p ≤ 0.05). All individual values with exception to one male given 100 mg/kg/day and one female given 1000 mg/kg/day were within historical Control background ranges.

Plasma sodium concentration was statistically significantly higher than the Controls in females given 1000 mg/kg/day (p ≤ 0.05) where all individual values were within historical Control background ranges.

Lowering of plasma creatinine in males and plasma urea in females at all dosages and aspartate aminotransferase activity in females at 1000 mg/kg/day were in the opposite direction to that usually associated with toxicity and were considered to either reflect normal biological variation or to be due to chance rather than an effect of the test material.

At the end of the treatment-free period, all changes to affected parameters had fully reversed.

Statistically significantly higher plasma sodium, calcium and triglyceride concentrations in males previously given 1000 mg/kg/day compared with Controls that were seen at this time were considered to be due to normal biological variation because these parameters were unaffected in this sex at the end of the treatment period.
Endocrine findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine volume in both males and females was unaffected by treatment.

In males, there was a dose-related decrease in urine pH in animals given 300 and 1000 mg/kg/day and slight increase in the presence of protein in the urine compared to Controls.

In females given 1000 mg/kg/day at the end of the treatment period, there was a slight decrease in urine pH compared to Controls.

At the end of the treatment-free period, these changes were observed to be reversible.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
During the weekly functional observations, there was an instance of salivation on removal from the home cage and in the arena in males given 1000 mg/kg/day. Furthermore, there was an instance of arousal in the arena in a female given 100 mg/kg/day. These observations are not considered to be treatment-related due to their infrequent nature. There were no other effects on functional observations.

Mean movement counts and distance travelled for all groups, including Controls, generally decreased with time in males. There were a number of statistically significant intergroup differences from Control group means (p≤0.05 to p≤0.01) during periods 4, 5 and 6 but these differences were not dose-related and were considered to be due to chance rather than any effect of the test material.

Time at rest generally increased with time in these groups. There was a dose-related increase in movement counts and distance travelled during Period 1 in females given the test material compared with Controls and, at 1000 mg/kg/day, these differences achieved statistical significance (p≤0.01). Time at rest decreased compared with Controls (p≤0.05) in these animals at this time-point. Thereafter, there were no statistically significant intergroup differences in movement counts or distance travelled at any dose level. Mean time at rest was slightly but significantly increased (p≤0.05) in period 6 for females given 1000 mg/kg/day compared with Controls but, given its minor nature, this isolated finding was considered not to be treatment-related.

Grip strength was statistically significantly decreased in males across all dose levels compared to concurrent Controls (p≤0.05 to p≤0.01). However, these reductions were unrelated to dose and, as there was no statistically significant effect in females, these intergroup differences were considered likely to be due to chance rather than any effect of the test material.

All effects seen appeared to show full or partial reversibility at the end of the treatment-free period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver:
Body weight-related liver weight for both sexes given 1000 mg/kg/day (p≤0.001) and males given 300 mg/kg/day (p≤0.01) were statistically significantly higher than Controls. Most individual body weight-related liver weights for males given 1000 mg/kg/day were above the upper limit of the historical background range, with the group mean relative liver weight being 34% and 19% higher than Controls for males and females respectively, given 1000 mg/kg/day. However, in the absence of histopathological changes correlated with these liver findings, this effect was considered not adverse.

Thyroid:
In males, absolute (p≤0.01) and body weight-related (p≤0.05) thyroid weights were increased across all dose groups compared with Controls, however, this increase was not dose-related. In females given 1000 mg/kg/day, there was an increase in absolute (p≤0.05) and body weight-related (p≤0.01) thyroid weight; body weight-related thyroid weight was 51% higher than Controls. There were microscopic correlates in the thyroid however, all individual values were within the historical background range, therefore this change was considered to be adaptive.

Thymus:
Thymus weight in males (absolute and body weight related) was statistically significantly lower in animals given 1000 mg/kg/day (p≤0.05) compared with Controls. All individual values were within the historical background range and there were microscopic correlates in the thymus however, these changes were considered not adverse.

Kidney:
Body weight-related kidney weights in males given 100, 300, and 1000 mg/kg/day (p ≤0.05 to p ≤0.01) and in females given 1000 mg/kg/day (p ≤ 0.05) were statistically significantly higher than Controls, although individual weights were within the historical background range for both males and females, and there were no microscopic correlates therefore this change was considered not to be adverse and to be due to chance.

Pituitary:
In females, absolute pituitary weight was lowered by 23% at 300 mg/kg/day and by 36% at 1000 mg/kg/day (p ≤0.05 and p ≤0.01 respectively), compared with Controls. Body weight related pituitary weights were similarly lower than Controls. All body weight-related pituitary weights were within the historical background data, and there were no microscopic correlates therefore this change was considered not to be adverse and to be due to chance.

All changes in organ weights were partially or fully reversed at the end of the treatment-free period with the exception of absolute and body weight-related thyroid weight in males given 1000 mg/kg/day, which remained statistically significantly higher than Controls (p≤0.01), however, the effect in the thyroid weight was considered to be adaptive and not treatment-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A variety of spontaneous changes were noted in Control animals and treated animals with no indication of an effect of treatment. The spectrum of findings seen at the end of treatment and after the treatment free period was generally consistent with changes encountered in rats of this age kept under laboratory conditions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Findings considered to be related to treatment were seen in the thyroid gland (hypertrophy of the follicular epithelium) and thymus (atrophy). There were no treatment related microscopic findings recorded in the treatment free animals.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Systemic Toxicity
Key result
Critical effects observed:
no

The mean concentrations of 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester in test formulations analyzed for the study were within 9% of nominal concentrations (within applied limits of +10/-15% from nominal), confirming accurate formulation. The coefficient of variation values were less than 4% (within applied limits of 5%), confirming the precision of analysis.

Table 8. Results of Formulation analysis

Date of

Preparation

Group

Nominal

concentration

(mg/mL)

Analyzed concentration (mg/mL)

RME (%)

CV (%)

Procedural

Recoveries (%)

Top

Middle

Bottom

Mean

2020-JAN-27

(First

formulation

preparation)

1

0

-

ND

-

-

-

-

97.4

2

25

24.2

23.8

23.3

23.8

-4.8

1.96

95.31

3

75

71.0

73.9

73.4

72.8

-2.9

2.14

96.3

4

250

229

222

233

228

-8.8

2.48

97.4

 

2020-FEB-20

(Last

formulation

preparation)

1

0

-

ND, ND

-

-

-

-

 

2

25

25.8

25.5

25.9

25.7

+2.8

0.82

98.1

3

75

75.1

78.5

81.1

78.2

+4.3

3.88

104.6

4

250

250

256

248

251

+0.4

1.54

102.3

RME Relative mean error, representing the deviation from nominal

ND Not detected

CV Coefficient of variation

1 Excluded as outlier as per SOP for recoveries

Table 9. Body weights (g) - Treatment period (Group mean values)

Group

 

Body Weight (Week Number)

1 (#)

2

3

4

5 (#)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

229.5

263.7

289.4

315.5

332.4

SD

18.3

22.0

26.9

30.8

34.1

N

10

10

10

10

10

Trend

-        

-

-

 

Group 2

(P1400

100 mg/kg/day)

Mean

234.8

271.4

298.8

323.2

336.0

SD

9.4

12.4

13.5

13.0

17.0

N

5

5

5

5

5

 

Group 3

(P1400

300 mg/kg/day)

Mean

227.6

259.6

283.6

306.2

314.6

SD

4.8

6.9

9.6

11.8

14.8

N

5

5

5

5

5

 

Group 4

(P1400

1000 mg/kg/day)

Mean

230.4

264.6

287.4

309.5

320.0

SD

18.5

22.5

26.4

34.0

37.4

N

10

10

10

10

10

Trend

>0.05

-

-

-

>0.05

Females

Group 1

(Control – 0 mg/kg/day)

Mean

152.1

168.7

183.4

193.3

197.6

SD

15.2

15.6

17.2

16.3

18.4

N

10

10

10

10

10

Trend

-

-

-

 

Group 2

(P1400

100 mg/kg/day)

Mean

148.4

168.2

179.8

189.2

193.2

SD

4.6

6.3

6.6

11.3

8.7

N

5

5

5

5

5

 

Group 3

(P1400

300 mg/kg/day)

Mean

149.6

164.2

182.0

194.5

200.0

SD

4.4

2.4

3.6

4.1

6.7

N

5

5

4

4

4

 

Group 4

(P1400

1000 mg/kg/day)

Mean

149.9

167.7

180.0

191.9

202.2

SD

13.9

13.9

15.7

15.3

20.7

N

10

10

9

9

9

Trend

>0.05

-

-

-

>0.05

(#) - Williams, Anova & Dunnett

Table 10. Body weights (g) - Treatment-free period (Group mean values)

Group

 

Body Weight (Week Number)

1 (#)

2

3

4

5 (#1)

6

7(#1)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

228.8

263.4

291.6

317.4

340.8

351.4

361.6

SD

20.0

25.2

31.9

37.8

42.4

43.3

41.3

N

5

5

5

5

5

5

5

 

Group 4

P1400

1000 mg/kg/day

Mean

233.4

268.8

289.2

310.4

326.0

334.0

345.7

SD

23.0

27.7

32.3

40.7

45.5

45.9

41.2

N

5

5

5

5

5

5

5

1vs

0.7441

-

-

-

0.6091

-

0.4802

Females

Group 1

(Control – 0 mg/kg/day)

Mean

146.6

163.2

174.0

184.0

192.0

198.2

195.8

SD

16.7

15.4

15.8

15.0

21.1

18.5

15.3

N

5

5

5

5

5

5

5

 

Group 4

P1400

1000 mg/kg/day

Mean

151.4

171.8

181.8

195.2

209.8

215.8

212.4

SD

19.4

18.8

21.0

18.5

25.9

22.9

20.9

N

5

5

5

5

5

5

5

1vs

0.6860

-

-

-

0.2676

-

01.898

(#) - Dunnett, T-Test

(#1) - T-Test


Table 11. Body weight gains (g) - Treatment period (Group mean values)

Group

 

Body Weight Gain (Week Number)

1-2 (#)

2-5 (#)

1-5 (#)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

34.2

68.7

102.9

SD

4.7

16.1

19.7

N

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

36.6

64.6

101.2

SD

5.1

8.9

13.4

N

5

5

5

 

Group 3

(P1400

300 mg/kg/day)

Mean

32.0

55.0

87.0

SD

2.7

9.2

11.8

N

5

5

5

 

Group 4

P1400

1000 mg/kg/day

Mean

34.2

55.4

89.6

SD

5.0

16.7

20.8

N

10

10

10

Trend

>0.05

>0.05

>0.05

Females

Group 1

(Control – 0 mg/kg/day)

Mean

16.6

28.9

45.5

SD

4.7

6.1

5.1

N

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

19.8

25.0

44.8

SD

3.8

5.1

7.2

N

5

5

5

 

Group 3

(P1400

300 mg/kg/day)

Mean

14.6

34.8

48.8

SD

2.6

7.1

7.1

N

5

4

4

 

Group 4

P1400

1000 mg/kg/day

Mean

17.8

34.3

51.9

SD

4.4

10.0

11.1

N

10

9

9

Trend

>0.05

>0.05

>0.05

(#) - Williams, Anova & Dunnett

Table 12. Body weight gains (g) – Treatment-free period (Group mean values)

Group

 

Body Weight Gain (Week Number)

1 to 2 (#)

2 to 5 (#)

1 to 5 (#)

5 to 7 (#)

1 to 7 (#)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

34.6

77.4

112.0

20.8

132.8

SD

6.1

18.5

24.2

4.2

23.0

N

5

5

5

5

5

 

Group 4

(P1400

1000 mg/kg/day)

Mean

35.4

57.2

92.6

15.2

107.8

SD

6.1

18.3

23.2

2.3

23.3

N

5

5

5

5

5

1 vs

0.8411

0.1209

0.2314

0.0308*

0.1262

Females

Group 1

(Control – 0 mg/kg/day)

Mean

16.6

28.8

45.4

3.8

49.2

SD

3.9

6.3

5.7

7.0

6.5

N

5

5

5

5

5

 

Group 4

(P1400

1000 mg/kg/day)

Mean

20.4

38.0

58.4

2.6

61.0

SD

3.7

11.4

10.3

5.4

7.2

N

5

5

5

5

5

1 vs

0.1539

0.1545

0.0390*

0.7683

0.0266*

(#) - T-Test: * = p ≤ 0.05


Table 13. Motor activity – End of treatment period (Group mean values)

Group

 

Movements

Counts Period 1

(Day Number)

Movements

Counts Period 2

(Day Number)

Movements

Counts Period 3

(Day Number)

Movements

Counts Period 4

(Day Number)

Movements

Counts Period 5

(Day Number)

Movements

Counts Period 6

(Day Number)

22 (#)

22 (# 1)

22 (#)

22 (#)

22 (#)

22 (# 2)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

2056.7

1153.8

913.1

652.5**

614.2

571.6

SD

610.4

255.3

319.6

298.6

430.3

379.3

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

1858.4

872.2

819.0

237.2*

269.6

487.6

SD

225.7

202.6

196.7

179.2

322.5

665.0

N

5

5

5

5

5

5

Trend

-

-

-

-

≤0.05*

>0.05

 

Group 3

(P1400

300 mg/kg/day)

Mean

2592.4

1060.6

765.6

346.8

128.8

6.6

SD

935.8

519.8

349.5

234.1

116.2

5.6

N

5

5

5

5

5

5

Trend

-

-

-

-

≤0.05*

≤0.05*

 

Group 4

(P1400

1000 mg/kg/day)

Mean

2262.8

1365.2

681.3

776.8

315.4

121.6

SD

366.1

444.3

348.6

295.0

375.9

81.9

N

10

10

10

10

10

10

Trend

>0.05

>0.05

>0.05

>0.05

≤0.05*

≤0.05*

Females

Group 1

(Control – 0 mg/kg/day)

Mean

1651.2

942.3

619.8

516.2

364.1

600.7

SD

538.5

290.4

345.7

356.2

356.8

511.3

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

1818.6

1011.0

739.0

266.0

262.4

296.0

SD

247.8

194.8

324.4

233.9

258.0

496.0

N

5

5

5

5

5

5

 

Group 3

(P1400

300 mg/kg/day)

Mean

2163.8

1001.3

594.3

711.3

511.8

551.3

SD

743.2

223.0

458.8

185.3

589.0

532.0

N

4

4

4

4

4

4

Trend

>0.05

-

-

-

-

-

 

Group 4

(P1400

1000 mg/kg/day)

Mean

2473.9

1149.4

636.7

833.3

304.9

188.6

SD

462.9

411.0

547.3

580.4

256.5

210.7

N

9

9

9

9

9

9

Trend

≤0.01**

>0.05

>0.05

>0.05

>0.05

>0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01 (#1) - Williams, Anova & Dunnett(Log)

(#2) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05

Note: a period equates to an interval of 10 minutes


Table 14. Motor activity – End of treatment period (Group mean values)

Group

 

Distance Travelled

Period 1 (cm)

(Day Number)

Distance Travelled

Period 2 (cm)

(Day Number)

Distance Travelled

Period 3 (cm)

(Day Number)

Distance Travelled

Period 4 (cm)

(Day Number)

Distance Travelled

Period 5 (cm)

(Day Number)

Distance Travelled

Period 6 (cm)

(Day Number)

22 (#)

22 (#)

22 (#)

22 (#)

22 (#)

22 (# 1)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

7263.1

4030.1

3218.5

2332.8**

2146.3

2090.6

SD

2029.4

922.5

1080.4

1086.0

1462.5

1352.2

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

6518.8

3047.6

2903.0

818.2*

906.8

1749.6

SD

752.2

707.4

720.6

604.7

1049.2

2383.8

N

5

5

5

5

5

5

Trend

-

-

-

-

≤0.05*

>0.05

 

Group 3

(P1400

300 mg/kg/day)

Mean

8914.2

3655.8

2706.8

1210.8

513.0

19.2

SD

2925.8

1649.6

1276.2

849.7

436.6

14.3

N

5

5

5

5

5

5

Trend

-

-

-

-

≤0.05*

≤0.05*

 

Group 4

(P1400

1000 mg/kg/day)

Mean

7848.3

4770.4

2490.6

2818.9

1140.0

463.7

SD

1050.9

1665.8

1287.0

1068.4

1353.5

311.4

N

10

10

10

10

10

10

Trend

>0.05

>0.05

>0.05

>0.05

≤0.05*

≤0.05*

Females

Group 1

(Control – 0 mg/kg/day)

Mean

5690.6

3226.3

2194.7

1784.7

1294.3

2162.3

SD

1725.6

952.8

1265.6

1239.4

1256.4

1845.8

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

6140.8

3461.6

2480.0

876.0

916.8

1018.0

SD

867.8

746.3

1046.1

790.3

955.3

1657.9

N

5

5

5

5

5

5

 

Group 3

(P1400

300 mg/kg/day)

Mean

7293.0

3842.5

1995.8

2504.3

1766.3

1970.0

SD

2247.6

723.9

1589.1

662.3

2030.4

1887.8

N

4

4

4

4

4

4

Trend

>0.05

-

-

-

-

-

 

Group 4

(P1400

1000 mg/kg/day)

Mean

8307.2

3919.9

2172.8

2867.2

1054.2

665.7

SD

1642.1

1347.5

1888.8

1962.9

904.2

747.2

N

9

9

9

9

9

9

Trend

≤0.01**

>0.05

>0.05

>0.05

>0.05

>0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01 (#1) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05

Note: a period equates to an interval of 10 minutes


Table 15. Motor activity – End of treatment period (Group mean values)

Group

 

Time at Rest

Period 1 (sec)

(Day Number)

Time at Rest

Period 2 (sec)

(Day Number)

Time at Rest

Period 3 (sec)

(Day Number)

Time at Rest

Period 4 (sec)

(Day Number)

Time at Rest

Period 5 (sec)

(Day Number)

Time at Rest

Period 6 (sec)

(Day Number)

22 (#)

22 (#)

22 (#)

22 (#)

22 (#)

22 (# 1)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

150.70

275.18

335.88

385.49**

418.15

416.08

SD

48.83

50.31

56.86

75.25

111.42

112.18

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

163.66

322.36

341.52

503.18*

518.24

479.86

SD

38.53

56.81

48.88

80.81

77.34

163.62

N

5

5

5

5

5

5

Trend

-

-

-

-

≤0.05*

>0.05

 

Group 3

(P1400

300 mg/kg/day)

Mean

121.68

322.98

361.20

474.84

539.42

598.06

SD

25.79

78.85

81.37

77.58

48.97

1.62

N

5

5

5

5

5

5

Trend

-

-

>0.05

-

≤0.05*

≤0.05*

 

Group 4

(P1400

1000 mg/kg/day)

Mean

144.91

274.56

408.99

372.56

498.92

544.49

SD

38.93

71.28

70.90

72.52

100.00

36.42

N

10

10

10

10

10

10

Trend

>0.05

>0.05

≤0.05*

>0.05

≤0.05*

≤0.05*

Females

Group 1

(Control – 0 mg/kg/day)

Mean

210.84

321.05

404.32

442.34

470.38

416.16

SD

58.30

65.94

95.80

105.46

104.96

149.81

N

10

10

10

10

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

202.12

314.80

397.42

505.34

508.96

515.78

SD

47.13

42.63

71.86

80.56

109.55

123.91

N

5

5

5

5

5

5

 

Group 3

(P1400

300 mg/kg/day)

Mean

178.60

337.60

441.05

386.38

449.58

449.68

SD

51.46

23.93

111.72

78.28

167.82

137.63

N

4

4

4

4

4

4

Trend

>0.05

-

-

-

-

>0.05

 

Group 4

(P1400

1000 mg/kg/day)

Mean

150.58

317.91

433.03

396.96

500.04

537.26

SD

45.35

59.29

133.72

130.45

74.70

61.69

N

9

9

9

9

9

9

Trend

≤0.05*

>0.05

>0.05

>0.05

>0.05

≤0.05*

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01 (#1) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05

Note: a period equates to an interval of 10 minutes


Table 16. Grip strength - End of treatment period (Group mean values)

Group

 

Forelimb

Mean (g)

(Day Number)

Hindlimb

Mean (g)

(Day Number)

26 (#)

26 (#)

Males

Group 1

(Control – 0 mg/kg/day)

Mean

404.30

341.73

SD

83.44

93.21

N

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

209.80

241.40

SD

50.19

48.73

N

5

5

Trend

≤0.01**

≤0.05*

 

Group 3

(P1400

300 mg/kg/day)

Mean

282.13

211.73

SD

99.03

63.47

N

5

5

Trend

≤0.01**

≤0.05*

 

Group 4

(P1400

1000 mg/kg/day)

Mean

286.57

281.63

SD

83.09

84.26

N

10

10

Trend

≤0.001***

≤0.05*

Females

Group 1

(Control – 0 mg/kg/day)

Mean

249.33

281.33

SD

65.36

82.26

N

10

10

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

226.60

250.07

SD

76.93

84.40

N

5

5

 

Group 3

(P1400

300 mg/kg/day)

Mean

226.25

195.00

SD

40.74

92.73

N

4

4

 

Group 4

(P1400

1000 mg/kg/day)

Mean

280.93

271.04

SD

84.10

143.75

N

9

9

Trend

>0.05

>0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

Table 17. Grip strength - End of the treatment-free period (Group mean values)

Group

 

Males

Females

Forelimb

Mean (g)

(Day Number)

Hindlimb

Mean (g)

(Day Number)

Forelimb

Mean (g)

(Day Number)

Hindlimb

Mean (g)

(Day Number)

26 (#)

40 (#)

26 (#)

40 (#)

26 (#)

40 (#)

26 (#)

40 (#)

Group 1

(Control – 0 mg/kg/day)

Mean

404.00

288.73

313.80

301.13

214.87

154.53

242.13

205.60

SD

94.96

114.50

107.25

38.68

63.00

54.84

97.22

36.60

N

5

5

5

5

5

5

5

5

 

Group 4

(P1400

1000 mg/kg/day)

Mean

274.07

232.20

239.40

306.67

323.53

236.93

373.67

215.80

SD

80.79

54.72

75.12

85.33

93.13

25.76

106.34

35.97

N

5

5

5

5

5

5

5

5

1 vs

0.0481*

0.3483

0.2396

0.8982

0.0627

0.0160*

0.0755

0.6685

(#) - T-Test: * = p ≤ 0.05


Table 18. Select Haematology Parameters - End of treatment period (Group mean values)

Group

 

Males

Females

MPV

fL

(Day Number)

PDW

%

(Day Number)

PCT

%

(Day Number)

CHr

pg

(Day Number)

Neut

10^3/uL

(Day Number)

APTT

sec

(Day Number)

A retics

10^6/uL

(Day Number)

PT

sec

(Day Number)

29 (# 1)

29 (# 1)

29 (# 1)

29 (#)

29 (# 1)

29 (#)

29 (#)

29 (#)

Group 1

(Control – 0 mg/kg/day)

Mean

9.36

41.82

0.86

19.02

0.600

14.36

0.1620

23.68

SD

0.22

1.20

0.11

0.43

0.080

1.29

0.0479

0.71

N

5

5

5

5

5

5

5

4

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

9.5

42.64

0.88

19.58

0.784

14.72

0.2024

24.04

SD

0.12

0.86

0.04

0.98

0.171

0.43

0.0191

1.53

N

5

5

5

5

5

5

5

5

Trend

-

-

>0.05

-

-

-

≤0.05*

-

 

Group 3

(P1400

300 mg/kg/day)

Mean

9.58

42.34

1.00

18.46

0.884

15.12

0.2038

24.58

SD

0.22

1.85

0.10

0.42

0.862

0.28

0.0069

0.73

N

5

5

5

5

5

5

4

4

Trend

>0.05

>0.05

≤0.05*

>0.05

>0.05

>0.05

≤0.05*

>0.05

 

Group 4

(P1400

1000 mg/kg/day)

Mean

10.00

44.80

1.06

17.94

1.036

16.18

0.2005

25.38

SD

0.32

1.76

0.11

0.46

0.183

0.50

0.0157

0.83

N

5

5

5

5

5

5

4

4

Trend

≤0.001***

≤0.01**

≤0.01**

≤0.05*

≤0.01**

≤0.01

≤0.05*

≤0.05*

(#) - Shirley, Kruskal-Wallis & Steel

(#1) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

MPV: mean platelet volume

PDW: platelet distribution width

PCT: plateletcrit

CHr: cellular haemoglobin retics

Neut: neutrophils

APTT: activated partial thromboplastin time

A Retics: absolute reticulocyte count

PT: prothrombin time

Table 19. Select Haematology Parameters - End of treatment-free period (Group mean values)

Group

 

Males

Females

RBC

10^6/uL

(Day Number)

MPV

fl

(Day Number)

Mono

10^3/ul

(Day Number)

42 (#)

42 (#)

42 (#)

Group 1

(Control – 0 mg/kg/day)

Mean

8.600

9.20

0.088

SD

0.377

0.24

0.030

N

5

5

5

 

Group 4

P1400

1000 mg/kg/day

Mean

9.168

9.52

0.142

SD

0.356

0.13

0.036

N

5

5

5

1 vs

0.0399*

0.0327*

0.0341*

(#) - T-Test: * = p ≤ 0.05

MPV: mean platelet volume

RBC: red blood cell count

Mono: monocytes


Table 20. Select Clinical Chemistry Parameters - End of treatment period (Group mean values)

Group

 

Males

Females

Cren

mg/dl

(Day Number)

T. Prot

g/dl

(Day Number)

Glob

g/dl

(Day Number)

Chol

mg/dl

(Day Number)

Urea

mg/dl

(Day Number)

Na

mmol/l

(Day Number)

AST

U/l

(Day Number)

Glob

g/dl

(Day Number)

A/G

ratio

(Day Number)

Chol

mg/dl

(Day Number)

29 (#)

29 (#)

29 (#)

29 (#)

29 (#)

29 (#)

29 (#)

29 (#)

29 (#)

29 (#)

Group 1

(Control – 0 mg/kg/day)

Mean

0.268

6.12

2.18

56.2

36.52

140.4

62.6

1.94

2.22

49.0

SD

0.022

0.23

0.20

11.5

3.98

0.9

4.6

0.09

0.19

6.0

N

5

5

5

5

5

5

5

5

5

5

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

0.240

6.18

2.30

72.1

31.16

141.4

59.2

2.00

2.10

50.6

SD

0.020

0.08

0.16

17.6

1.86

0.9

5.2

0.21

0.27

8.5

N

5

5

5

5

5

5

5

5

5

5

Trend

≤0.05*

-

-

≤0.05*

≤0.05*

-

-

-

-

-

 

Group 3

(P1400

300 mg/kg/day)

Mean

0.232

6.30

2.34

79.0

33.08

141.3

58.8

2.05

1.99

51.2

SD

0.019

0.12

0.11

2.6

1.24

1.0

4.5

0.10

0.09

4.4

N

5

5

5

5

4

4

4

4

4

4

Trend

≤0.01**

>0.05

>0.05

≤0.05*

≤0.05*

>0.05

>0.05

>0.05

>0.05

>0.05

 

Group 4

P1400

1000 mg/kg/day

Mean

0.224

6.52

2.50

73.2

31.30

142.0

50.3

2.23

1.89

67.2

SD

0.013

0.28

0.19

11.4

3.15

0.8

3.3

0.17

0.23

16.5

N

5

5

5

5

4

4

4

4

4

4

Trend

≤0.01**

≤0.01**

≤0.05*

≤0.05*

≤0.05*

≤0.05*

≤0.01**

≤0.05*

≤0.05*

≤0.05*

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

Values above or below the limit of quantification have been excluded from the group means

Cren: creatinine

T. Prot:total protein

Glob: globulin

Chol: cholesterol

Na: sodium

AST: aspartate aminotransferase

A/G: albumin/globulin ratio

Table 21. Select Clinical Chemistry Parameters - End of treatment-free period (Group mean values)

Group

 

Males

Na

mmol/l

(Day Number)

Ca

mg/dl

(Day Number)

Trigs

mg/dl

(Day Number)

42 (#)

42 (#)

42 (#)

Group 1

(Control – 0 mg/kg/day)

Mean

140.0

10.36

179.1

SD

0.7

0.33

33.1

N

5

5

5

 

Group 4

P1400

1000 mg/kg/day

Mean

141.4

10.94

279.9

SD

0.5

0.30

40.6

N

5

5

5

1 vs

0.0081**

0.0201*

0.0026**

(#) - T-Test: * = p ≤ 0.05; ** = p ≤ 0.01

Values above or below the limit of quantification have been excluded from the group means

Na: sodium

Ca: calcium

Trigs: triglyceride

Table 22. Select Organ weights - Treatment period (Group mean values) - Males

Group

 

Brain

(g)

Adjusted

Brain Wt

Thymus

(g)

Adjusted

Thymus Wt

Thymus %

Body Weight

Liver

(g)

Adjusted

Liver Wt

Liver %

Body Weight

Kidneys

(g)

Adjusted

Kidneys Wt

Kidneys %

Body Weight

Thyroids

(g)

Adjusted

Thyroids Wt

Thyroids %

Body Weight

#

# 1

#

# 1

#

#

# 1

#

#

# 1

# 2

#

# 1

#

Group 1

(Control – 0 mg/kg/day)

Mean

2.102

2.096*

0.6062

0.602

0.1885

12.186

12.018

3.755

2.050

2.041

0.634

0.0138

0.0139

0.0043

SD

0.080

-

0.0494

-

0.0252

1.419

-

0.184

0.117

-

0.032

0.0030

-

0.0012

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

1.994

1.967*

0.5672

0.550

0.1692

13.044

12.311

3.898

2.278

2.240

0.681

0.0232

0.0236

0.0070

SD

0.053

-

0.0937

-

0.0207

1.349

-

0.224

0.191

-

0.034

0.0018

-

0.0007

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Trend

-

-

-

-

-

-

>0.05

>0.05

-

≤0.05*

≤0.05*

≤0.01**

≤0.05*

≤0.05*

 

Group 3

(P1400

300 mg/kg/day)

Mean

2.060

2.072

0.5430

0.551

0.1723

13.312

13.639

4.225

2.126

2.143

0.675

0.0164

0.0162

0.0052

SD

0.063

-

0.0614

-

0.0142

1.373

-

0.318

0.112

-

0.009

0.0038

-

0.0012

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Trend

>0.05

-

>0.05

>0.05

>0.05

>0.05

≤0.01**

≤0.01**

>0.05

≤0.05*

≤0.05*

≤0.01**

≤0.05*

≤0.05*

 

Group 4

P1400

1000 mg/kg/day

Mean

1.976

1.997

0.4700

0.484

0.1518

15.582

16.156

5.028

2.252

2.282

0.735

0.0192

0.0189

0.0063

SD

0.126

-

0.0928

-

0.0252

1.842

-

0.248

0.083

-

0.101

0.0030

-

0.0015

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Trend

≤0.05*

>0.05

≤0.05*

≤0.05*

≤0.05*

≤0.01**

≤0.001***

≤0.001***

≤0.05*

≤0.01**

≤0.01**

≤0.01**

≤0.05*

≤0.05*

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; *** = p ≤ 0.001

(#1) - Williams, Ancova/Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

{Covariate(s): Dead body weight}

(#2) - Shirley, Kruskal-Wallis & Steel: * = p ≤ 0.05; ** = p ≤ 0.01

Table 23. Select Organ weights - Treatment period (Group mean values) - Females

Group

 

Pituitary

(g)

Adjusted

Pituitary Wt

Pituitary %

Body Weight

Liver

(g)

Adjusted

Liver Wt

Liver %

Body Weight

Kidneys %

Body Weight

Thyroids

(g)

Adjusted

Thyroids Wt

Thyroids %

Body Weight

#

# 1

#

(#)

(# 1)

(#)

(#)

(#)

(# 1)

(#)

Group 1

(Control – 0 mg/kg/day)

Mean

0.0114

0.0111

0.0056

7.696

7.525

3.810

0.721

0.0132

0.0128

202.34

SD

0.0015

-

0.0008

0.4740

-

0.213

0.047

0.0029

-

14.86

N

5

5

5

5

5

5

5

5

5

5

Trend

 

Group 2

(P1400

100 mg/kg/day)

Mean

0.0096

0.0098

0.0050

7.276

7.405

3.809

0.770

0.0158

0.0161

191.20

SD

0.0021

-

0.0009

0.265

-

0.162

0.045

0.0025

-

8.10

N

5

5

5

5

5

5

5

5

5

5

Trend

>0.05

>0.05

>0.05

-

-

-

-

-

-

-

 

Group 3

(P1400

300 mg/kg/day)

Mean

0.0088

0.0086

0.0044

7.820

7.699

3.898

0.745

0.0120

0.0117

200.48

SD

0.0017

-

0.0009

0.511

-

0.154

0.030

0.0018

-

6.19

N

4

4

4

4

4

4

4

4

4

4

Trend

≤0.05*

≤0.05*

≤0.05*

>0.05

>0.05

>0.05

>0.05

>0.05

>0.05

-

 

Group 4

P1400

1000 mg/kg/day

Mean

0.0073

0.0075

0.0038

8.590

8.763

4.531

0.794

0.0185

0.0189

189.55

SD

0.0010

-

0.0004

0.618

-

0.262

0.057

0.0031

-

6.30

N

4

4

4

4

4

4

4

4

4

4

Trend

≤0.01**

≤0.01**

≤0.01**

≤0.05*

≤0.001***

≤0.001***

≤0.05*

≤0.05*

≤0.01**

>0.05

(#) - Williams, Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

(#1) - Williams, Ancova/Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001

{Covariate(s): Dead body weight}

Table 24. Select Organ weights - Treatment-free period (Group mean values)

Group

 

Males

Females

Thyroids (g)

Adjusted

Thyroids Wt

Thyroids %

Body Weight

Kidneys %

Body Weight

(#)

(# 1)

(#)

(#)

Group 1

(Control – 0 mg/kg/day)

Mean

0.0178

0.0171

0.0050

0.703

SD

0.0037

-

0.0007

0.022

N

5

5

5

5

 

Group 4

P1400

1000 mg/kg/day

Mean

0.0246

0.0253

0.0073

0.736

SD

0.0042

-

0.0008

0.023

N

5

5

5

5

1 vs

0.0267*

0.0031**

0.0014**

0.0455*

(#) - T-Test: * = p ≤ 0.05; ** = p ≤ 0.01 (#1) - T-Test: ** = p ≤ 0.01

{Covariate(s): Dead body weight}

Table 25. Select Histopathology Findings

 

Males

Females

Group

1

2

3

4

1

2

3

4

Dose Level

(mg/kg/day)

0

100

300

1000

0

100

300

1000

Number of rats examined

5

5

5

5

5

5

5

5

Thyroid Gland

 

Hypertrophy, follicular

epithelium

Minimal

0

1

0

1

0

0

0

2

Slight

0

0

0

2

0

0

0

0

Total

0

1

0

3

0

0

0

2

Thymus

 

Atrophy

Slight

0

0

3

2

0

0

0

0

Moderate

0

0

0

0

0

0

0

1

Total

0

0

3

2

0

0

0

1


Conclusions:
Treatment-related changes were observed at the highest dose 1000 mg/kg/day (restricted to rat specific adaptive responses consisting of secondary thyroid follicular hypertrophy consistent with liver enzyme induction). However, these were considered to be adaptive changes and not adverse and based on the findings, the No Observed Adverse Effect Level (NOAEL) for 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester was determined to be 1000 mg/kg/day.
Executive summary:

A key OECD Guideline 407 study was conducted to assess the toxicity of the test material (1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester), when administered by gavage to the rat once daily for 28 days and to assess the reversibility of any observed effects over a 15 day treatment-free period.

 

The test material was administered to Crl:WI(Han) Wistar rats (5/sex/dose) once daily via oral gavage for a period of 28 days at dose levels of 0, 100, 300, or 1000 mg/kg/day in a corn oil vehicle. An additional five males and five females were dosed with 0 (vehicle) or 1000 mg/kg/day for 28 days and were then allocated to the 15 day treatment-free period. Animals were subsequently observed for the following parameters: clinical observations, body weight, food intake, haematology, blood chemistry, urinalysis, behavioural observations and functional observation battery, organ weights, macroscopic and microscopic pathology.

 

No treatment-related mortality was observed through the study period although two females were euthanised as a result of trauma associated with the gavage dosing procedure. No treatment-related signs of clinical toxicity were observed in either sex following exposure to the test material at 100 and 300 mg/kg/day. Exposure to the test material at 1000 mg/kg/day resulted in sporadic clinical signs of ploughing, increased/decreased activity and prostration although these were seen notably in females. One clinical sign observed in females was excessive licking of other female genitals from Days 7 and 8 of the study at the 1000 mg/kg/day dose level. These clinical signs either ceased or were only observed sporadically throughout the study. Body weight gain was unaffected in males given 100 mg/kg/day or in female rats at any dose level. A slight decrease in body weight gain was observed at 300 or 1000 mg/kg/day in males compared with the control animals and at the end of the treatment-free period, body weight gain in male rats previously given 1000 mg/kg/day remained lower than the corresponding controls.

 

Food consumption remained unaffected by treatment and aside from isolated instances of excessive salivation observed in male rats given 1000 mg/kg/day (consistent with post-dose observations), there was no effect on functional observations following exposure to the test material. Mean locomotor movement counts and distance travelled for all groups, including controls, generally decreased with time in male rats. Time at rest generally increased with time across all of these groups. There was a dose-related increase in movement counts and distance travelled during Period 1 in treated females compared with control animals and, at 1000 mg/kg/day, these differences attained statistical significance (p≤0.01). Time at rest decreased compared with the controls (p≤0.05) in these animals at this time-point. Thereafter, there were no statistically significant intergroup differences in movement counts or distance travelled at any dose level. Mean locomotor movement counts and distance travelled were similar to the corresponding controls at the end of the treatment-free period.

 

Minor changes in haematology and blood chemistry parameters were observed which were either partially or fully reversible by the end of the treatment-free period. Slight increases in plasma globulin, total protein, and cholesterol concentrations observed were considered to be possibly related to the increase in liver weight seen at 1000 mg/kg/day. The haematology and blood chemistry effects observed, although treatment-related, were not considered to be adverse findings. Urine analysis revealed increased protein content in urine in males given 300 or 1000 mg/kg/day when compared with the control animals. Urine pH was decreased in males at all dose levels and in females given

1000 mg/kg/day. However, at the end of the treatment-free period, protein present in the urine and urine pH was observed to be similar to the corresponding control rats.

 

Gross necropsy did not reveal any remarkable treatment-related findings. Body weight-related liver weight for both sexes given 1000 mg/kg/day (p≤0.001) and males given 300 mg/kg/day (p≤0.01) were statistically significantly higher than controls. Most individual body weight-related liver weights for males given 1000 mg/kg/day were above the upper limit of the historical background range, with the group mean relative liver weight being 34% and 19% higher than controls for males and females, respectively, given 1000 mg/kg/day. However, in the absence of histopathological changes correlated with these liver findings, this effect was considered not adverse.

 

In males, absolute (p≤0.01) and body weight-related (p≤0.05) thyroid weights were increased across all dose groups compared with controls, however, this increase was not dose-related. In females given 1000 mg/kg/day, there was an increase in absolute (p≤0.05) and body weight-related (p≤0.01) thyroid weight; body weight-related thyroid weight was 51% higher than controls. There were microscopic correlates in the thyroid, however, all individual values were within the historical background range, therefore this change was considered to be adaptive.

 

The apparent increases in both thyroid and liver weights and observed hypertrophy in the thyroid gland reflect a commonly observed adaptive response in the liver-thyroid axis which is associated with the metabolism of xenobiotics or their metabolites in the rat.

 

Thymus weight in males (absolute and body weight related) was statistically significantly lower in animals given 1000 mg/kg/day (p≤0.05) compared with controls. All individual values were within the historical background range and there were microscopic correlates in the thymus and these changes were considered to be related to stress and not adverse.

 

Body weight-related kidney weights in males given 100, 300, and 1000 mg/kg/day (p≤0.05 top≤0.01) and in females given 1000 mg/kg/day (p ≤ 0.05) were statistically significantly higher than controls, although individual weights were within the historical background range for both males and females, and there were no microscopic correlates therefore this change was considered not to be adverse and to be due to chance.

 

In females, absolute pituitary weight was lowered by 23% at 300 mg/kg/day and by 36% at 1000 mg/kg/day (p≤0.05 andp≤0.01 respectively), compared with controls. Body weight related pituitary weights were similarly lower than controls. All body weight-related pituitary weights were within the historical background data, and there were no microscopic correlates, therefore this change was considered not to be adverse and to be due to chance.

 

All changes in organ weights were partially or fully reversed at the end of the treatment-free period with the exception of absolute and body weight-related thyroid weight in males given 1000 mg/kg/day, which remained statistically significantly higher than controls (p≤0.01). However, the effect in the thyroid weight was considered to be adaptive and not treatment-related.

 

Treatment-related microscopic findings were seen in the thyroid gland (hypertrophy of the follicular epithelium) and thymus (atrophy). The changes in the thyroid gland are generally considered adaptive and rodent specific while the thymus atrophy was likely to be the result of non-specific toxicity.

 

Treatment-related changes were observed at the highest dose 1000 mg/kg/day (restricted to rat specific adaptive responses consisting of secondary thyroid follicular hypertrophy consistent with liver enzyme induction). However, these were considered to be adaptive changes and not adverse and based on the findings, the No Observed Adverse Effect Level (NOAEL) for 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester was determined to be 1000 mg/kg/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-06-15 to 2018-02-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
The deviations were considered to have not affected the integrity or validity of the study.
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test, Office of Prevention, Pesticides and Toxic Substances EPA 712–C–00–368, (7101), July 2000.
Deviations:
yes
Remarks:
The deviations were considered to have not affected the integrity or validity of the study.
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
European Community (EC) Council Directive 96/54/EC, Annex IV.D, replacing EC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity and other
Health Effects; B.34: "One generation reproduction toxicity test". Official Journal of the European Communities No. L248, September 1996.
Deviations:
yes
Remarks:
The deviations were considered to have not affected the integrity or validity of the study.
Principles of method if other than guideline:
The study was a modified OECD 422 study with extended F1 phase in which FI pups were followed from birth to sexual maturation (PND 70).
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: supplied by Envigo (Shardlow, UK) on behalf of the Sponsor (Polyadd Limited, U.K.); Batch no. 0900
- Expiration date of the lot/batch: 2017-01-17
- Purity test date: 2015-10-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: No special storage conditions are needed. Store away from incompatible materials (strong oxidizing agents). Keep the material sealed to avoid contact with moisture. Avoid high temperature.
- Stability under storage conditions: not specified
- Stability under test conditions: stable when stored at room temperature for 24 hours and stored refrigerated for 7 days.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The homogeneity and stability was confirmed for the test item in vehicle formulations at nominal concentrations of 3.75 and 250 mg/mL when stored at room temperature for 24 hours and stored refrigerated for 7 days.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Liquid colorless
Species:
rat
Strain:
Wistar
Remarks:
Hannover Wistar rats
Details on species / strain selection:
Species: Rat - Species recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Supplier (Charles River Laboratories, France); Breeder (Charles River Laboratories, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes, females were nulliparous and non-pregnant
- Age at study initiation: 10-11 weeks (males and females)
- Weight at study initiation: Males: 350-394 g; Females: 215-257 g
- Fasting period before study: Not specified
- Housing: Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne)
Premating period: 5 animals/cage - Makrolon cages-IV
Mating period: one male and one female/cage - Makrolon cages-I
Postmating: individually - Makrolon cages-I
- Diet (e.g. ad libitum): Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L., batches no.: 010616MA,
012016MA and 022216MA, expiry dates: 02 October 2016, 16 October 2016 and 18 November 2016, respectively).

Pelleted standard Teklad 2018C rat/mouse maintenance diet ad libitum(supplied by Envigo RMS, S.L., batch no.: 020916MA, expiry date: 5 November 2016), for lactating females and pups (until day 21 post partum).
- Water (e.g. ad libitum): Tap water in bottles ad libitum
- Acclimation period: 12 days prior to estrus cycle evaluation under test conditions

DETAILS OF FOOD AND WATER QUALITY: The diet was analyzed by the manufacturer for contaminants and to check its composition and the water underwent bacteriological, chemical and contaminant analysis. The results were included in the Annex 4 of the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C - monitored continuously
- Humidity (%): 40-70%
- Air changes (per hr): 15-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (artificial fluorescent light)

ENVIRONMENTAL ENRICHMENT
- different types of material specific to the species (e.g. paper wool and sizzlenest) were provided to reduce stress, enhance well-being and improve behaviour.

IN-LIFE DATES: From: 2016-06-15 To: 2017-10-26
Route of administration:
oral: gavage
Details on route of administration:
Oral route selected since oral ingestion is a possible route of human exposure to the test material. Test material was administered orally via gastric gavage using glass syringes.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test material was added in a suitable glass container followed by the vehicle to reach the final concentration. The vial was gently inverted for a minimum five times to mix the formulation completely. This solution was aliquoted for each administration day in glass containers.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil (justification not specified)
- Concentration in vehicle: The amount of test material to be administered was calculated according to the most recently recorded body weight.
- Amount of vehicle (if gavage): 4 mL/Kg body weight
- Lot/batch no. (if required): Sigma-Aldrich Reference MKBV2080V and MKBW9504V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulation concentration was determined twice during the study and once (top/middle/bottom) for homogeneity in samples taken from the formulation to be administered to Groups 2 to 4. One additional sample was also taken from the vehicle prepared. The formulations were quantified by gas chromatography according to the analytical procedure validated in Study YR67PH (DRF).

The homogeneity and concentration of the formulations was analysed prior to the first day of dosing with the 1st analysis and concentration with the 2nd analysis. On both occasions, duplicate samples of the dosing solution (approx. 5 mL each) were transferred to labelled vials. Aliquot 1 was used for analysis and was shipped refrigerated in the dark to the PI (Paul Watson, Envigo Shardlow, UK). This aliquot was discarded after analysis. Aliquot-2 samples will be retained for any possible subsequent needs at Envigo CRS, S.A.U. and will be discarded after issuing the final phase or the final report unless otherwise requested by the Sponsor. Each sample was labelled at least with study number, phase number (YR67HP), aliquot number, treatment, concentration, formulation preparation date, formulation administration date and storage conditions. The test item was used as analytical standard.
Duration of treatment / exposure:
Parental generation: starting two weeks before mating through a minimum of 28 days for males (after confirming female pregnancy) and until day 20 postpartum for females including the day before sacrifice.

Pups for sexual maturation from day 21 postpartum until sexual maturation 70 days postpartum (although direct treatment starts at or soon after weaning, all offspring have potential indirect exposure in uteroand through the milk during lactation).

Remaining pups (sacrificed on day 4 or 13 postpartum): not treated. Potential indirect exposure in uteroand through the milk during lactation
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 - corn oil
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
12/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on a dose-range-finding (DRF) toxicity study in rats (Envigo Study S56026: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2 (phenylmethyl) ester, CAS no.1200806-67-2: 14-day Repeated Dose Oral (Gavage) Range Finding Toxicity Study in the Rat).

The low dose 150 mg/Kg was intended to ensure that the interval between dose levels was within the limits prescribed in the OECD test guideline number 422 and was also above the cut-off for GHS classification as “toxic” according to the criteria for a 28-day repeat dose toxicity study. The intermediate dose 300 mg/Kg was intended to ensure that the interval between dose levels was within the limits prescribed in the OECD test guideline number 422 and might result in safe dose for exposure to the test material. The high dose 600 mg/Kg was based on mild signs of toxicity observed in animals at 1000 and 700 mg/Kg in the 14-day DRF study (Envigo DRF S56026). The clinical findings suggested that more prolonged dosing in the definitive study, together with the potentially greater sensitivity of the pregnant females, may exceed the maximum tolerated dose level and may result in significant effects on the ability of females to maintain offspring postpartum.

- Rationale for animal assignment (if not random): Animals were allocated at random based on mean body weight similarity among groups. Females were evaluated pre-exposure for estrus cyclicity.

- Fasting period before blood sampling for clinical biochemistry: Yes, animals were fasted overnight before blood sampling but allowed ad libitum access to water. The samples were collected early in the morning
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked for twice daily. Any rat sacrificed during the study was subjected to macroscopic examination.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations:
P males: at pretest, on first day of dosing and twice weekly during the pre-pairing until sacrifice day (included).

P females: at pretest, on first day of dosing, twice weekly during the pre-pairing and pairing period and on days 0, 4, 7, 11, 14, 17 and 20 of pregnancy and within 24 hours of parturition (day 0 or 1 postpartum) and on days 4, 7, 14, 17 and 21 (as terminal body weight).

Pups for sexual maturation: twice weekly after weaning

Body weight during mating period is not reported.

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
P males: at pretest, once weekly during the pre-pairing and weekly during pre-treatment, treatment and postpairing period.

P females: at pretest, once weekly during the pre-pairing and treatment periods and days 0-7, 7-14, 14-20 post coitum and days 1-7 and 7-14 postpartum.

Pups for sexual maturation: once weekly

Food consumption was not examined during the mating period.

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males: following at least 28 administrations of the test item/vehicle (on day of sacrifice)
Females: on day 21 postpartum (on day of sacrifice)
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia)
- Animals fasted: Yes (The animals were fasted overnight before blood sampling but allowed ad libitum access to water. The samples were collected early in the morning)
- How many animals: five animals/sex/ group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males: following at least 28 administrations of the test item/vehicle (on day of sacrifice)
Females: on day 21 postpartum (on day of sacrifice)
- Animals fasted: Yes (The animals were fasted overnight before blood sampling but allowed ad libitum access to water. The samples were collected early in the morning)
- How many animals: five animals/sex/ group
- Parameters checked in table [No.3] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before the first exposure to the test item, once weekly thereafter and one day before sacrifice. These observations were performed outside the home cage, in a standard arena, at least one hour after dosing (where applicable) to ensure identification of any transient effects
- Dose groups that were examined: performed on all test and control group animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Grip strength assessment:
Grip strength (fore- and hind limbs) was measured in five randomly selected males per group once during the final week of treatment and at least one hour after dosing. Five randomly selected females per group were similarly evaluated on day 20 postpartum.

Motor activity assessment:
Motor activity was measured in the animals mentioned above with an activity motor system (AMS from Medical Instruments GmbH (FMI) and DeMeTec-Ams version 2.0 GmbH). Activity of the
animals was recorded in 5-minute intervals over a 30-minute period and measured quantitatively (beam counts).

Sensory reactivity assessment:
Sensory reactivity to different stimuli was evaluated in the animals mentioned above. Details presented in Table No. 4.

IMMUNOLOGY: No

OTHER:
Thyroid Hormones: Blood samples were taken based on the following schedule:

• from all two pups per litter on day 4 after birth,
• from all pups per litter on day 13 after birth,
• from all surviving adult males, at termination,
• from all surviving dams on day 21 postpartum, and
• from cohort F1 on PND 70

All samples were stored under appropriate conditions after centrifugation (at 4 ºC and 2000 g for 10 minutes) to obtain serum. Samples from the PND 13 and 70 pups and adults were assessed for plasma levels of TSH and also serum for T4 in adult animals. Samples were shipped frozen to the PI (Envigo CRS Limited Test Site, Huntingdon) for analysis. Further assessment of T4 in serum samples from the adult animals at sacrifice and fromday-4 pups was done when relevant. Pup samples can be pooled by litter for thyroid hormone analyses. If not, an evaluation of potential effects on the thyroid function could be done in F1 offspring. This would comprise detailed microscopic examination of the thyroid glands from Day-13 offspring together with evaluation of potential effects upon plasma TSH levels in Day-4 and Day-13 offspring. Samples specifically intended for hormone determination were obtained at a comparable time of the day.

Analysis of serum samples for T4 was conducted at Envigo CRS Limited (Huntingdon) under the supervision of a Principal Investigator (Hariharasudhan Bose) for this study phase. Samples were analyzed using LC-MS/MS instrumentation, based on the bioanalytical method BM/2016/0632 (Validation study number: FF58YR).

Thyroid Hormones Acceptance Criteria
Since thyroid hormones are endogenous compounds, the acceptance criteria were widened. The accuracy (RE) determined at each QC concentration level was within ±20% (25% for LLOQ). The RE of at least 67% of the QC samples overall within the batch was within ±20% of their nominal concentration, including at least 50% at each concentration level. At least 75% of the calibration standards were within ±20% of the nominal concentration (±25% at LLOQ). When diluted test samples wereincluded in the batch, then 50% of the diluted QC samples in the batch (normally n = 2) were within 20% of their respective nominal concentration for the diluted sample data to be accepted.
Sacrifice and pathology:
SACRIFICE
Animals sacrificed in extremis were necropsied. Their organs were extracted and fixed but not weighed.

PARENTAL FEMALES
Females on day 21 post-partum and those showing total litter loss were deeply anesthetized with sodium pentobarbital administered intraperitoneally, exsanguinated by excision of the axillary vessels and aorta, and necropsied. The mated females that did not give birth or show signs of pregnancy were sacrificed 24-26 days post coitum as they were not mated again.

The post-mortem examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and their contents with emphasis on the uterus, number of implantation sites was performed. The observation of the organs in situ and description of all macroscopic abnormalities are recorded. A vaginal smear was examined to determine the stage of estrus cycle.

When no implantation site was evident, the uterus was placed in an aqueous solution of ammonium sulfide (Salewski, 1964) to accentuate possible hemorrhagic areas of implantation sites

PARENTAL MALES
After at least 29 administrations, all males were deeply anesthetized with sodium pentobarbital administered intraperitoneally, then exsanguinated by excision of the axillary vessels and aorta and necropsied subjected to confirmation of successful mating.

The post-mortem examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and their contents was performed. The observation of the organs in situ and description of all macroscopic abnormalities was recorded.

PATHOLOGY
The organs to be weighed (Table No. 5) were recorded on the scheduled necropsy dates for all surviving parental animals and group mean and adjusted values were calculated. Samples of tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution (10% formalin) unless otherwise indicated as well as specimens of abnormal tissue.

Reproductive organs of all control and high dose animals as well as of animals selected for sexual maturation were also examined

All organ and tissue samples (except for the nose) of selected animals from groups 1 and 4 (5 animals/sex, as indicated in the actual pathology report) to be examined were processed, embedded, cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the study pathologist.

The bone marrow smears were stained using the May Grünwald-Giemsa method and kept at the test facility for possible further investigation.

HISTOLOGY
Slides of organs and tissues collected at necropsies of all animals to be evaluated, except for the bone marrow smear for which no test was planned, were examined by the pathologist at Envigo CRS, S.A.U. A description of all abnormalities is included in the report. Where possible, the microscopic findings were correlated with the gross observations.

As test-item-related morphological changes were detected in thyroids in high-dose animals. The thyroid glands were examined in all adult animals from Group 2 and 3, as well as on day 70 PND from cohort F1.

A histopathology evaluation of interstitial cells of all males from the control and high-dose groups was performed by the histopathologist
Statistics:
Please see the section 'Any other information on materials and methods incl. tables' for information on statistics.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs considered to indicate an immediate or delayed reaction to treatment.

Salivation was observed during postnatal periods in males at 300 and 600 mg/Kg/day as well as during the treatment period at 600 mg/Kg/day. In females, salivation was observed in the same treatment groups during treatment, mating, gestation and lactation periods, as well as occasionally in females receiving 150 mg/Kg/day during gestation and lactation.

Although this was considered a test-item related effect, it could be related to palatability and thus not considered to be an adverse effect, as the animals could taste the test substance when the probe (used for gavage) was removed after administration.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test material-related deaths.

Male no. 66 administered the test item at 600 mg/Kg/day was sacrificed on day 14 of treatment for animal welfare reasons, as there was a problem during the administration handling the previous day (the handler accidently lost hold of the animal as it tried to escape and it fell to the floor, after which it did not recover).

Females no. 109 (0 mg/Kg/day) and 161 (600 mg/Kg/day) were sacrificed for animal welfare reasons due to difficulties during parturition. As females were sacrificed, the corresponding litters were also killed and examined macroscopically:

- In the case of female no. 109, the cause for the poor condition was hematoma/hemorrhage, which most likely explains the sudden pallor displayed by that animal. Hysterectomy was performed and the foetuses (one male and one female in left uterine horn) that were found deadwere examined macroscopically, showingadvanced autolysis in abdominal and thoracic cavities.

- In female no. 161, 4 males and 4 females were alive when the hysterectomy was performed and 5 males and 2 females were found dead. No alterations were observed in their necropsy.

Females no. 102 (0 mg/Kg/day) and 149 (600 mg/Kg/day) were sacrificed on days 2 and 3 of lactation, respectively, due to total litter loss.

On day 24 of gestation, female no. 106 (0 mg/Kg/day) was sacrificed as no signs of parturition were observed. At necropsy, 1 implantation was observed in left uterine horn and consequently, this female was considered to be pregnant.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect on body weights during the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was no indication of an effect on food consumption during the treatment period.

The statistically significant increases noted at 300 and 600 mg/kg/day in males (postnatal days 8 and 15) or in females at 600 mg/kg/day on gestation day 14 were not considered to have been toxicologically relevantgiven theirmagnitude (no more than 1.2 times the Control mean values), as it was transient and as there were no effects in body weights that could be associated with it.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no changes of toxicological relevance.

Males receiving 600 mg/kg/day showed significantly higher mean neutrophil and basophil values compared to the Control group. This tendency was also observed in females receiving 600 mg/kg/day but it was not statistically significant. There was no dose-effect relationship. Females on day 21 of lactation showed no other differences from the Control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry examination performed on the day of sacrifice in males revealed a dose-related increase in protein and globulin values in males. These differences were statistically significant at 300 and 600 mg/kg/day when compared to the Control group. However, this increase in protein and globulin values was not observed in females.

No other differences were observed when comparing the test item administered groups with the Control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Motor activity was considered not to be affected by the test item. Statistically significant increases observed in females at 600 mg/Kg/day (compared to Control) at 5 minutes were not considered relevantor test material-related, as they were occasional and there was no similar difference observed in males.

There was no indication of any adverse effects of the test item on sensory reactivity or grip strength assessments.

Given the magnitude (from 9 to 11 % versus Control) and the lack of a dose-effect relationship, the statistically significant differences observed at 300 and 600 mg/kg/day in the mean forelimb grip strength of femalesand at 600 mg/kg/day in the mean forelimb and hind limbs (11%) were not considered to be toxicologically relevant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A dose-related increase was recorded in kidney weights in all male treatment groups (maximum effect was observed in adjusted mean values at 600 mg/kg/day and was 17% higher versus Control). In addition, mean adjusted liver and spleen weights in males at 600 mg/kg/day were 40 and 22% higher, respectively, than those recorded in the Control group. In males, mean adjusted thymus weight was 18% lower at 600 mg/kg/day when compared to the Control group. All these differences were statistically significant.

Dose-related decreases in uterus, cervix and oviduct weights were observed (maximum effect 23% in adjusted mean values). Statistically significant differences were observed at 600 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed on day 21 postpartum in females and after at least 28 days of treatment in males revealed no test-item-related lesions. All findings were considered to be incidental and unrelated to treatment with the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no histopathological correlates for the increased liver weight in F0 males treated at the high dose.

In the kidneys of some F0 males treated with the high dose, increased amounts of hyaline droplets were seen. This correlates with the increased kidney weight in this group. However, the level of hyaline droplet accumulation observed was within the normal limits in this species and strain and was therefore not considered to be related to treatment.

In the testes, no cell or stage specific abnormalities were noted in males treated at 600 mg/kg/day. Interstitial cells were also assessed qualitatively, and no alterations were seen in treated males.

In the thyroid glands, minimal hypertrophy of the follicular epithelium was observed in some males of all treated groups, with a dose-effect relationship. It was also observed in isolated females of all treated groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There was a minimal or slight hypertrophy of the follicular epithelium of the thyroid glands in males and femalesof all treated groups (150, 300 and 600 mg/kg/day). This finding could be considered to be within the normal variation in females at 150 and 300 mg/kg/day due to the low incidence recorded (2/12 at 150 mg/kg/day and 1/11 at 300 mg/kg/day), and the lack of effects in the T4 determinations. The decrease observed in T4 in F0 males at 600 mg/kg/day also correlates with the histopathological findings.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 600 mg/kg/day histological findings observed in males and females (minimal to slight microscopic changes in the thyroid glands) were considered non-adverse in the absence of consistent changes in hormone levels.
Key result
Critical effects observed:
no

Table 6. F0 Food consumption - group mean values (g/animal/day): Males

Dose Group

 

Pre-treatment Period

Treatment Period

Postnatal Period

Day 8

Day 12

Day 8

Day 15

Day 8

Day 15

Statistical Test

 

Av

Av

Wi

Wi

Wi

Wi

Males

Control

Mean

26

26

21

20

18

19

SD

3.7

3.2

0.5

0.3

1.2

1.1

N

3

3

3

3

12

12

 

150 mg/Kg/day

Mean

25

25

21

20

18

18

SD

2.4

3.3

0.7

0.4

1.9

1.4

N

3

3

3

3

12

12

 

300 mg/Kg/day

Mean

25

25

22

22

20**

21*

SD

3.0

3.2

1.0

0.8

1.8

1.7

N

3

3

3

3

12

12

 

600 mg/Kg/day

Mean

38

39

22

22

22**

21**

SD

20.0

20.5

1.2

1.6

2.0

2.5

N

2

2

3

3

11

11

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests where appropriate

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

Table 7. F0 Food consumption - group mean values (g/animal/day): Females

Dose Group

 

Pre-treatment Period

Treatment Period

Gestation Period

Lactation Period

Day 8

Day 12

Day 8

Day 15

Day 7

Day 14

Day 20

Day 7

Day 14

Statistical Test

Av

Av

Wi

Wi

Wi

Wi

Wi

Sh

Sh

Females

Control

Mean

22

24

16

17

18

21

21

27

48

SD

4.3

5.5

0.8

0.5

2.0

2.0

2.4

6.7

10.9

N

3

3

3

3

12

12

12

9

9

 

150 mg/Kg/day

Mean

19

19

16

16

19

21

20

30

52

SD

3.0

2.9

0.2

0.1

2.3

1.6

2.3

4.7

4.2

N

3

3

3

3

12

12

12

12

12

 

300 mg/Kg/day

Mean

24

24

16

16

19

20

22

42

51

SD

5.2

5.5

0.2

0.3

2.3

2.2

2.8

31.3

4.9

N

3

3

3

3

12

12

12

12

11

 

600 mg/Kg/day

Mean

17

18

16

16

20

22*

23

27

45

SD

0.5

1.0

0.3

0.6

2.1

1.9

1.8

16.5

13.1

N

2

2

3

3

3

12

12

11

11

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests where appropriate

Sh: Treated groups compared with Control using Shirley’s test

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

Table 8. F0 Body weight - group mean values - Males

Period

Day

(Statistical Test)

Dose group:

Control

150 mg/Kg/day

300 mg/Kg/day

600 mg/Kg/day

Pre-treatment Period

Day 2 (Av)

Mean

344.6

343.6

344.3

346.7

SD

9.36

7.72

11.98

11.54

N

12

12

12

12

% of control

-

100

100

101

Day 12 (Av)

Mean

369.5

369.8

368.0

370.6

SD

10.59

11.03

12.42

10.85

N

12

12

12

12

% of control

-

100

100

100

 

Treatment Period

Day 1 (Wi)

Mean

371.7

372.8

371.9

373.8

SD

11.09

12.56

12.82

11.41

N

12

12

12

12

% of control

-

100

100

101

Day 4 (Wi)

Mean

374.2

378.3

375.7

377.3

SD

12.75

12.97

13.18

12.74

N

12

12

12

12

% of control

-

101

100

101

Day 8 (Wi)

Mean

382.2

384.6

381.7

385.3

SD

14.08

14.81

13.76

12.09

N

12

12

12

12

% of control

-

101

100

101

Day 11 (Wi)

Mean

384.7

383.4

385.0

381.9

SD

16.26

15.87

12.25

10.93

N

12

12

12

12

% of control

-

100

100

99

Day 14 (Wi)

Mean

390.0

391.0

390.4

391.9

SD

16.78

16.35

13.85

13.06

N

12

12

12

12

% of control

-

100

100

100

 

Postnatal Period

Day 1 (Wi)

Mean

387.1

386.2

386.2

387.5

SD

17.00

17.15

13.88

12.92

N

12

12

12

11

% of control

-

100

100

100

Day 3 (Wi)

Mean

389.4

389.8

391.2

392.9

SD

13.95

16.33

13.35

13.17

N

12

12

12

11

% of control

-

100

100

101

Day 7 (Wi)

Mean

391.8

391.7

394.3

396.1

SD

16.14

17.20

14.00

10.92

N

12

12

12

11

% of control

-

100

101

101

Day 10 (Wi)

Mean

395.6

394.0

397.9

399.6

SD

18.07

17.24

14.59

10.74

N

12

12

12

11

% of control

-

100

101

101

Day 14 (Wi)

Mean

398.4

396.5

399.8

399.5

SD

18.75

17.84

14.60

16.48

N

12

12

12

11

% of control

-

100

100

100

Day 17 (Wi)

Mean

398.8

404.3

402.7

396.0

SD

19.51

23.90

15.19

19.54

N

7

6

7

6

% of control

-

101

101

99

Day 21 (Wi)

Mean

395.8

405.6

400.6

400.1

SD

24.03

22.68

17.35

20.0

N

6

6

6

6

% of control

-

102

101

101

Day 24 (Wi)

Mean

398.8

407.3

402.0

400.1

SD

24.72

21.76

18.01

20.62

N

6

6

6

6

% of control

-

102

101

100

Day 28 (Tt)

Mean

396.7

-

399.8

-

SD

38.47

-

-

-

N

2

-

1

-

% of control

-

-

101

-

Table 9. F0 Body weight - group mean values - Females

Period

Day

(Statistical Test)

Dose group:

Control

150 mg/Kg/day

300 mg/Kg/day

600 mg/Kg/day

Pre-treatment Period

Day 2 (Av)

Mean

228.3

227.2

226.4

225.3

SD

10.21

8.61

9.79

10.01

N

12

12

12

12

% of control

-

100

99

99

Day 12 (Av)

Mean

232.2

232.4

231.5

230.7

SD

8.83

8.17

7.75

9.33

N

12

12

12

12

% of control

-

100

100

99

 

Treatment Period

Day 1 (Wi)

Mean

237.1

237.6

235.6

235.2

SD

12.22

9.52

9.69

10.95

N

12

12

12

12

% of control

-

100

99

99

Day 4 (Wi)

Mean

236.4

239.3

237.8

235.2

SD

11.89

7.04

13.00

11.85

N

12

12

12

12

% of control

-

101

101

99

Day 8 (Wi)

Mean

239.6

240.7

239.0

237.1

SD

11.28

8.12

12.85

12.44

N

12

12

12

12

% of control

-

100

100

99

Day 11 (Wi)

Mean

235.8

239.4

238.3

235.8

SD

10.65

7.98

12.20

12.51

N

12

12

12

12

% of control

-

101

101

100

Day 14 (Wi)

Mean

238.2

239.2

237.5

237.1

SD

11.52

7.23

10.32

12.16

N

12

12

12

12

% of control

-

100

100

100

 

Gestation Period

Day 0 (Wi)

Mean

240.7

243.4

241.3

239.2

SD

12.20

8.49

10.58

12.48

N

12

12

12

12

% of control

-

101

100

99

Day 4 (Wi)

Mean

255.3

255.5

255.5

253.1

SD

11.05

9.38

13.72

12.71

N

12

12

12

12

% of control

-

100

100

99

Day 7 (Wi)

Mean

263.0

264.3

263.3

261.6

SD

12.05

10.18

14.80

14.02

N

12

12

12

12

% of control

-

101

100

99

Day 11 (Wi)

Mean

277.4

278.4

275.6

275.4

SD

12.96

10.73

15.85

15.05

N

12

12

12

12

% of control

-

100

99

99

Day 14 (Wi)

Mean

286.8

288.9

287.9

289.5

SD

17.91

11.15

16.76

15.64

N

12

12

12

12

% of control

-

101

100

101

Day 17 (Wi)

Mean

305.6

312.8

310.3

313.5

SD

23.87

11.48

17.4

19.21

N

12

12

12

12

% of control

-

102

102

103

Day 20 (Wi)

Mean

328.0

339.9

339.7

338.2

SD

29.46

15.08

18.70

23.98

N

12

12

12

12

% of control

-

104

104

103

 

Lactation Period

Day 1 (Wi)

Mean

253.5

249.0

250.2

254.7

SD

15.27

15.58

15.94

17.73

N

10

12

12

11

% of control

-

98

99

100

Day 4 (Wi)

Mean

269.2

268.9

270.6

276.8

SD

10.74

14.68

14.62

16.43

N

9

12

11

11

% of control

-

100

101

103

Day 7 (Wi)

Mean

278.8

275.4

277.4

283.3

SD

8.11

15.07

12.72

18.41

N

9

12

11

11

% of control

-

99

100

102

Day 14 (Wi)

Mean

288.8

295.4

294.4

297.0

SD

12.62

18.49

18.41

22.64

N

9

12

11

11

% of control

-

102

102

103

Day 17 (Wi)

Mean

296.3

293.8

297.4

295.4

SD

15.36

14.64

16.52

22.23

N

9

12

11

11

% of control

-

99

100

100

Table 10. F0 FOB: Motor Activity - group meanabsolute values (beam counts)

Dose Group

Motor Activity

5 (min)

10 (min)

15 (min)

20 (min)

25 (min)

30 (min)

Total (min)

Males

Statistics Test

 

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Control

Mean

179.4

138.4

86.2

126.0

82.8

46.8

659.6

SD

30.36

58.54

58.26

53.83

48.85

43.07

139.68

N

5

5

5

5

5

5

5

 

150 mg/Kg/day

Mean

201.8

76.2

41.0

109.4

69.4

22.2

520.0

SD

100.08

75.41

26.33

74.33

75.93

16.35

260.34

N

5

5

5

5

5

5

5

% of control

112

55

48

87

84

47

79

 

300 mg/Kg/day

Mean

199.4

141.2

124.8

95.4

64.6

101.2

726.6

SD

70.22

77.39

85.46

60.39

58.05

72.79

220.35

N

5

5

5

5

5

5

5

% of control

111

102

145

76

78

216

110

 

600 mg/Kg/day

Mean

276.4

199.4

134.8

136.6

88.2

18.8

854.2

SD

126.82

77.68

51.69

54.48

65.72

21.79

288.94

N

5

5

5

5

5

5

5

% of control

154

144

156

108

107

40

130

Females

Control

Mean

160.2

78.8

73.8

44.0

53.0

48.8

458.6

SD

19.32

50.26

57.80

46.82

47.75

80.42

205.16

N

5

5

5

5

5

5

5

 

150 mg/Kg/day

Mean

111.6

69.2

22.4

39.8

44.8

27.6

315.4

SD

80.62

55.93

32.35

38.00

39.47

40.83

195.63

N

5

5

5

5

5

5

5

% of control

70

88

30

90

85

57

69

 

300 mg/Kg/day

Mean

147.4

124.2

33.4

8.8

22.6

26.2

362.6

SD

26.23

97.49

58.06

8.61

29.11

43.38

154.14

N

5

5

5

5

5

5

5

% of control

92

158

45

20

43

54

79

 

600 mg/Kg/day

Mean

262.8**

152.2

116.2

55.4

24.0

25.0

635.6

SD

37.25

43.96

62.35

43.66

38.52

30.28

174.61

N

5

5

5

5

5

5

5

% of control

164

193

157

126

45

51

139

* p≤0.05

** p≤0.01

Table 11. F0 FOB: Grip Strength - group mean absolute values (g)

Dose Group

 

Forelimb Mean (g)

Hindlimb Mean (g)

Statistics Test

 

Wi

Wi

Males

Control

Mean

880.01

777.97

SD

104.052

175.758

N

5

5

 

150 mg/Kg/day

Mean

930.38

891.20

SD

104.071

230.406

N

5

5

% of control

106

115

 

300 mg/Kg/day

Mean

914.08

901.83

SD

127.802

104.196

N

5

5

% of control

104

116

 

600 mg/Kg/day

Mean

897.09

877.35

SD

72.559

165.634

N

5

5

% of control

102

113

Females

Control

Mean

860.65

765.07

SD

34.236

53.565

N

5

5

 

 150 mg/Kg/day

Mean

896.43

760.92

SD

34.236

25.793

N

5

5

% of control

104

99

 

 300 mg/Kg/day

Mean

957.66*

724.79

SD

58.709

82.191

N

5

5

% of control

111

95

 

 600 mg/Kg/day

Mean

940.01*

677.67*

SD

53.845

22.658

N

5

5

% of control

109

89

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

Table 12. F0 Hematology and Coagulation - Group mean value

Dose Group

 

Neutrophils (x 109/L)

Basophils (x 109/L)

LRT (x 1012/L)

Statistics Test

 

Wi

Wi

Wi

Males

Control

Mean

0.54

0.01

0.097

SD

0.148

0.004

0.0179

N

5

5

5

 

150 mg/Kg/day

Mean

0.49

0.01

0.088

SD

0.085

0.004

0.0160

N

5

5

5

 

300 mg/Kg/day

Mean

0.57

0.01

0.089

SD

0.149

0.004

0.0133

N

5

5

5

 

600 mg/Kg/day

Mean

0.88*

0.02**

0.090

SD

0.373

0.011

0.0111

N

5

5

5

Females

Control

Mean

0.089

0.01

0.0115

SD

0.313

0.007

0.0133

N

5

5

5

 

150 mg/Kg/day

Mean

0.82

0.01

0.106

SD

0.125

0.004

0.0172

N

5

5

5

% of control

91

120

92

 

300 mg/Kg/day

Mean

0.66

0.01

0.113

SD

0.078

0.000

0.0130

N

5

5

5

% of control

74

100

98

 

600 mg/Kg/day

Mean

1.27

0.02

0.093*

SD

0.433

0.009

0.0159

N

5

5

5

% of control

142

160

93

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

LRT: Reticulocyte maturity index

Table 13. F0 Clinical Biochemistry - Group mean values post mating

Dose Group

 

Protein (g/L)

Globulin (g/L)

Statistics Test

 

Wi

Wi

Males

Control

Mean

62.14

22.48

SD

1.80

1.70

N

5

5

 

150 mg/Kg/day

Mean

63.84

24.66

SD

2.02

2.06

N

5

5

 

300 mg/Kg/day

Mean

65.08*

25.12*

SD

2.64

2.59

N

5

5

 

600 mg/Kg/day

Mean

68.82**

26.96**

SD

2.13

1.09

N

5

5

Females

Control

Mean

53.64

17.62

SD

1.849

2.364

N

5

5

 

150 mg/Kg/day

Mean

53.62

17.38

SD

1.743

2.009

N

5

5

% of control

100

99

 

300 mg/Kg/day

Mean

55.66

17.72

SD

1.716

1.571

N

5

5

% of control

104

101

 

600 mg/Kg/day

Mean

55.20

19.02

SD

2.758

2.123

N

5

5

% of control

103

108

Wi: Treated groups compared with Control using Williams’ test

* p≤0.05

** p≤0.01

Table 14. F0 Organ weights - group mean absolute and adjusted values (g)

Dose Group

 

Organ

Kidneys

Liver

Spleen

Thymus

Uterus and Cervix and Oviducts

Statistics Test

 

Wi

Wi

Wi

Wi

Sh

Males

Control

Mean

2.237

10.908

0.602

0.370

-

SD

0.194

0.866

0.027

0.044

-

N

5

5

5

5

-

Adjusted Mean

2.252

11.137

0.621

0.378

-

 

150 mg/Kg/day

Mean

2.313

13.045

0.668

0.295

-

SD

0.231

2.330

0.138

0.067

-

N

5

5

5

5

-

% of control

103

120

111

80

-

Adjusted Mean

2.312

13.021

0.666

0.294

-

 

300 mg/Kg/day

Mean

2.431

12.628

0.626

0.278

-

SD

0.172

2.633

0.114

0.078

-

N

5

5

5

5

-

% of control

109

116

104

75

-

Adjusted Mean

2.416

12.387

0.605

0.270

-

 

600 mg/Kg/day

Mean

2.612

15.260

0.736

0.304

-

SD

0.205

1.395

0.083

0.068

-

N

5

5

5

5

-

% of control

117

140

122

82

-

Adjusted Mean

2.615*

15.296**

0.739*

0.306*

-

Females

Control

Mean

1.808

10.381

0.680

0.219

0.730

SD

0.131

0.802

0.089

0.039

0.229

N

5

5

5

5

9

Adjusted Mean

1.810

10.394

0.681

0.219

0.731

 

150 mg/Kg/day

Mean

1.783

10.650

0.699

0.239

0.664

SD

0.104

0.398

0.101

0.061

0.129

N

5

5

5

5

12

% of control

99

103

103

109

91

Adjusted Mean

1.795

10.723

0.702

0.239

0.667

 

300 mg/Kg/day

Mean

1.931

10.859

0.705

0.232

0.618

SD

0.074

0.496

0.095

0.040

0.117

N

5

5

5

5

11

% of control

107

105

104

106

85

Adjusted Mean

1.943

10.927

0.708

0.232

0.619

 

600 mg/Kg/day

Mean

1.801

11.624

0.673

0.213

0.563

SD

0.230

1.887

0.101

0.038

0.146

N

5

5

5

5

11

% of control

100

112

99

97

77

Adjusted Mean

1.776

11.471

0.666

0.214

0.559*

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

* p≤0.05

** p≤0.01

- Not applicable

Table 15. Mean plasma TSH concentrations (pg/mL)

Dose Group

 

F0 Males

F0 Female on Day 21 Post-partum

Statistics Test

Wi

Sh

Control

Mean

1560

1860

SD

1010

N/A

N

12

9

 

150 mg/Kg/day

Mean

1800

543

SD

749

134

N

12

12

 

300 mg/Kg/day

Mean

1550

607

SD

685

20

N

10

11

 

600 mg/Kg/day

Mean

1350

751

SD

571

472

N

10

10

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

* p≤0.05

** p≤0.01

 

Table 16. Mean serum T4 concentrations (pg/mL)

Dose Group

 

F0 Males

F0 Female on Day 21 Post-partum

Statistics Test

 

Wi

Sh

Control

Mean

47600

39578

SD

6512

5434

N

12

9

 

150 mg/Kg/day

Mean

49158

39333

SD

6331

6447

N

12

12

 

300 mg/Kg/day

Mean

44650

37209

SD

5207

3529

N

10

11

 

600 mg/Kg/day

Mean

40109**

39191

SD

3739

7049

N

11

11

Wi: Treated groups compared with Control using Williams’ test

Sh: Treated groups compared with Control using Shirley’s test

* p≤0.05

** p≤0.01

Conclusions:
Based on the results observed, the systemic toxicity No Observed Adverse Effect Level (NOAEL) in males and females rats was determined to be 600 mg/Kg/day. At this dose level, histological findings (minimal to slight microscopic changes in the thyroid glands) observed were considered non-adverse in the absence of consistent changes in hormone levels. At 600 mg/Kg/day, thyroid hormone changes were observed. However, given that the mechanism leading to these changes is unknown, and in the absence of effects on reproductive performance, their relevance to humans is uncertain and could be therefore, considered non-adverse.
Executive summary:

A key Guideline 422 study was conducted to evaluate the effects of repeated oral (gavage) administration of the test material (P1400: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2 (phenylmethyl) ester, CAS 1200806-67-2) to rats during a four-week period in F0 males two weeks before mating through a minimum of 28 days (after confirming female pregnancy), until day 20 postpartum for females including the day before sacrifice, and from birth to sexual maturation (PND 70) in F1 pups. 12 rats per sex per dose received the test material in corn oil once daily via oral gavage at does of 150, 300, or 600 mg/Kg/day while the control animals received corn oil only.

 

Animals were observed twice daily for mortality and for any clinical signs following administration of the test material. Parameters such as body weight, food consumption, behavioural assessments such as grip strength, motor activity, and sensory reactivity assessment were evaluated for. Additionally, blood samples were extracted from the retro-orbital plexus of all animals under light isoflurane anesthesia and clinical biochemistry as well as haematology assessments were undertaken. Blood samples were taken from all surviving adult males, at termination and from all surviving dams on day 21 postpartum for thyroid hormone analysis. At necropsy, post-mortem examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and their contents with emphasis on the uterus (females only), number of implantation sites (females only) was performed. Organ weights were recorded and all organ and tissue samples (except for the nose) of selected animals from the control and high dose groups (5 animals/sex) were processed, embedded, cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin. Reproductive organs of all control and high dose animals as well as of animals selected for sexual maturation were also examined. Histopathological analysis was undertaken and slides of organs and tissues collected at necropsies of all animals to be evaluated, except for the bone marrow smear for which no test was planned, were examined by the pathologist. Additionally, based on treatment-related morphological changes detected in thyroids in high-dose animals, this organ from the other treatment group animals were examined in all adult animals. A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups was also performed.

 

No treatment-related mortality, clinical signs (other than salivation) or effects on functional observation battery were observed in the parental generation. There was no effect on body weights during the study and there was no indication of an effect on food consumption during the treatment period. The statistically significant increases noted at 300 and 600 mg/Kg/day in males (postnatal days 8 and 15) or in females at 600 mg/Kg/day on gestation day 14 were not considered to have been toxicologically relevant given their magnitude (no more than 1.2 times the Control mean values), as it was transient and as there were no effects in body weights that could be associated with it. Haematology, coagulation and clinical biochemistry effects observed, in the absence of a dose-effect relationship (neutrophil and basophils) or given that the differences were only observed in one sex (protein and globulin values in males), were not considered to be toxicologically relevant. Gross necropsy did not reveal any remarkable findings and all findings were considered to be incidental and unrelated to treatment with the test material.

 

In parental males, a dose-related increase in mean kidney weights, correlating to an increase in the amount of hyaline droplets, was observed at histopathological examination. However, this was considered to be within the normal historical range for rats of this strain and age. At 600 mg/Kg/day in parental animals, there were no histopathological findings that correlate with the increased liver and spleen weights in males or in the decrease in uterus, cervix and oviducts in females. A minimal or slight hypertrophy of the follicular epithelium of the thyroid glands was observed in males and females of all treated groups (150, 300 and 600 mg/Kg/day). This finding could be considered to be within the normal variation in females at 150 and 300 mg/Kg/day due to the low incidence recorded (2/12 at 150 mg/Kg/day and 1/11 at 300 mg/Kg/day), and the lack of effects in the T4 determinations. The decrease observed in T4 in parental males at 600 mg/Kg/day also correlated with the histopathological findings.

 

Based on the results observed, the systemic toxicity No Observed Adverse Effect Level (NOAEL) in males and females rats was determined to be 600 mg/Kg/day. At this dose level, histological findings (minimal to slight microscopic changes in the thyroid glands) observed were considered non-adverse in the absence of consistent changes in hormone levels. At 600 mg/Kg/day, thyroid hormone changes were observed. However, given that the mechanism leading to these changes is unknown, and in the absence of effects on reproductive performance, their relevance to humans is uncertain and could be therefore, considered non-adverse.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
One key sub-chronic and two short-term sustance specific OECD Guideline studies (oral route) available for assessment.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A key OECD Guideline 408 sub-chronic toxicity study (Sequani Limited, 2021) was conducted to assess the toxicity of the test material (1, 2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester), when administered by gavage to the rat once daily for at least 90 days.

 

The test material was administered to Crl:WI(Han) Wistar rats (5/sex/dose) once daily via oral gavage for a period of 90 days at dose levels of 0, 100, 300, or 1000 mg/kg/day in a corn oil vehicle. Animals were subsequently observed for the following parameters: clinical observations, ophthalmoscopy, body weight, food intake, behavioural observations and functional observation battery, vaginal smear on the day of necropsy, haematology, blood chemistry, thyroid hormone assessment, organ weights, macroscopic and microscopic pathology.

 

No mortality or signs of clinical toxicity were observed through the study period. There was no effect on body weight or body weight gain in female rats at any dose level or in male rats given 100 mg/kg/day or 300 mg/kg/day. A slight (7%), non-statistically significant lowering of overall body weight gain was observed in males given 1000 mg/kg/day, when compared with the corresponding controls. Food consumption was unaffected by treatment. No treatment-related ocular changes or abnormalities were observed after exposure to the test material.

 

At 1000 mg/kg/day, mean platelet volume (MPV) was statistically significantly higher than controls in males (p≤0.001) and females (p≤0.05), and platelet distribution width was higher than controls in males (p≤0.05). Mean cell volume (MCV) was statistically significantly lower than controls in male rats (p≤0.01) and female rats (p≤0.05) given 1000 mg/kg/day. Reticulocyte haemoglobin content (CHr) was lower than controls in males at all dose levels (p≤0.05 top≤0.001) and haemoglobin distribution width (HDW) in female rats was statistically significantly higher than controls at 300 or 1000 mg/kg/day (p≤0.01).These changes were marginal, with most or all individual values within the historical control range, and as they had no effect on the primary red cell parameters, were considered not to be adverse.Mean reticulocyte corpuscular volume (MCVr) in male rats given 1000 mg/kg/day was statistically significantly lower than the corresponding control mean. However, this was due to three highindividual control values (i.e. values above the upper limit of the historical control range) rather than an effect of the test material.

 

White blood cell count in males given 1000 mg/kg/day was statistically significantly higher than controls (p≤0.05), primarily due to slight increases in absolute lymphocyte and monocyte counts. However, all individual values were within the historical control range and since these changes were minor in nature, considered not to be adverse.Plasma cholesterol in both sexes was higher compared to controls at 300 (p≤0.05 top≤0.01) and 1000 mg/kg/day (p≤0.01). All individual values, with the exception of one female given 1000 mg/kg/day, and one male given 300 or 1000 mg/kg/day, were within the historical control background ranges.Triglyceride concentration was statistically significantly higher in males given 300 or 1000 mg/kg/day (p≤0.05 andp≤0.01, respectively) but most individual values for were within the historical control background ranges.Plasma calcium was statistically significantly higher than controls in males at 1000 mg/kg/day (p≤0.001). However, all individual values were within the historical control background range. Statistically significant (p≤0.05) increases in plasma sodium concentration at 300 and 1000 mg/kg/day, and lowering of plasma chloride concentrations (p≤0.01) at 1000 mg/kg/day were observed in male rats, however most or all individual values were within the historical controlrange.These changes in plasma cholesterol, triglyceride, calcium, sodium, and chloride levels wererelatively minor and, in the absence of corroborative pathology changes, were considered not tobe adverse.

 

In male rats given 1000 mg/kg/day, group mean TSH concentration was 2.6-fold higher than controls (p≤0.01), where 7 out 10 males were above the mean historical control range. In females at this dose level, there was a similar 2.5-fold increase in TSH concentration when compared with corresponding controls where 4 out of 10 females were above the mean historical control range although, this did not achieve statistical significance.In female rats, there was a marginal, but statistically significant (p≤0.05) increase in group mean T4 concentration at 1000 mg/kg/day where 9 out 10 females were above the mean historical control range. The increase in TSH concentration was considered to be associated with the rodent-specific adaptive epithelial hypertrophy in the thyroid gland and, in the absence of any marked changes in T3 or T4 concentrations, this finding was considered to be non-adverse.

 

Several instances of hyper- and hypoactivity in the arena were observed in all dose groups during the weekly functional observations throughout the study in both sexes. However, as the incidence was low and the pattern and frequency of these observations not dose-related, this observation was not considered to be treatment-related. At 100 mg/kg/day on Day 77, two males had a straub tail and one female excessively reared in the arena. On Day 91 of the study, at 300 mg/kg/day, one male displayed an excessive response to tail-pinching. These observations were considered not to be treatment-related due to their infrequent and transient nature. At 1000 mg/kg/day, in males and females, there were two and one instances of slight salivation on removal from the home cage, respectively, and one instance of salivation in the arena in males.Mean movement counts and distance travelled for all groups, including control rats, generallydecreased with time in both sexes. In males, there was a statistically significant increase in movement count and distance travelled during Period 1 at 300 and 1000 mg/kg/day (p≤0.05), but these differences were not dose-related. In female rats given 1000 mg/kg/day, during Period 1, there was a statistically significant increase in distance travelled (p≤0.05), and a decrease in time at rest (p≤0.05). Thereafter, there were no statistically significant intergroup differences in movement counts or distance travelled at any dose level, in either sex. As these inter-group differences were observed in Period 1 only and not at any other time point, they were considered unlikely to be treatment-related.Fore and hindlimb grip strength was statistically significantly increased in females at 1000 mg/kg/day (p≤0.05) compared with controls. However, as this reduction was not consistent between sexes, it was considered likely to be due to chance rather than any effect of the test material.

 

Gross necropsy did not reveal any remarkable treatment-related findings.

Body weight-related liver weight in female rats given 300 or 1000 mg/kg/day and in male rats at all dose levels were statistically significantly higher than controls (p≤0.05 top≤0.001). Most individual body weight-related liver weights for male rats given 1000 mg/kg/day were above the upper limit of the historical background range, with group mean relative liver weight being 35% and 17% higher than corresponding controls, for males and females respectively, correlating to the adaptive centrilobular hypertrophy of the liver seen microscopically.In male rats, mean absolute and body weight-related thyroid weights were statistically significantly higher (p≤0.05 top≤0.01) than the control mean across all dose groups in a dose-related manner, by up to 2.3-fold, correlating with the adaptive follicular epithelial hypertrophy seen microscopically in the thyroid. Most individual values were, however, within the historical background range.Body weight-related kidney weights for males (p≤0.001) and females (p≤0.05) given 1000 mg/kg/day were statistically significantly higher than controls, by 12% and 5%, respectively. All individual values were within the historical background range, and there were no microscopic correlates. Therefore, this change was considered not to be of toxicological significance.In males, absolute and body weight-related spleen weight was higher than controls by up to 24% at 1000 mg/kg/day (p≤0.001). All individual body weight-related spleen weights were within the historical background range, and there were no microscopic correlates, therefore this change was considered not to be of toxicological significance.Absolute epididymis weight at 300 and 1000 mg/kg/day was statistically significantly lower than controls (p≤0.05). However, this was not strictly dose-related, and there were no microscopic correlates, therefore this intergroup difference was considered to be due to chance rather than any effect of the test material.

 

Histopathology revealed treatment-related effects in the liver (hypertrophy centrilobular), thyroid glands (hypertrophy of the follicular epithelium and increased basophilia of colloid), and urinary bladder (hyperplasia of the urothelium). These effects in the liver and thyroid were considered to be rodent specific adaptive responses. Hyperplasia of the urothelium of the urinary bladder was considered likely to be a local effect.

 

Based on the lack of adverse treatment-related effects observed through the study period, the sub-chronic oral toxicity No Observed Adverse Effect Level (NOAEL) for 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester was determined to be 1000 mg/kg/day.

A key short-term (28-day) OECD Guideline 407 study was also conducted to assess the toxicity of the test material (1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester), when administered by gavage to the rat once daily for 28 days and to assess the reversibility of any observed effects over a 15 day treatment-free period (Sequani Limited, 2020).

 

The test material was administered to Crl:WI(Han) Wistar rats (5/sex/dose) once daily via oral gavage for a period of 28 days at dose levels of 0, 100, 300, or 1000 mg/kg/day in a corn oil vehicle. An additional five males and five females were dosed with 0 (vehicle) or 1000 mg/kg/day for 28 days and were then allocated to the 15 day treatment-free period. Animals were subsequently observed for the following parameters: clinical observations, body weight, food intake, haematology, blood chemistry, urinalysis, behavioural observations and functional observation battery, organ weights, macroscopic and microscopic pathology.

 

No treatment-related mortality was observed through the study period although two females were euthanised as a result of trauma associated with the gavage dosing procedure. No treatment-related signs of clinical toxicity were observed in either sex following exposure to the test material at 100 and 300 mg/kg/day. Exposure to the test material at 1000 mg/kg/day resulted in sporadic clinical signs of ploughing, increased/decreased activity and prostration although these were seen notably in females. One clinical sign observed in females was excessive licking of other female genitals from Days 7 and 8 of the study at the 1000 mg/kg/day dose level. These clinical signs either ceased or were only observed sporadically throughout the study. Body weight gain was unaffected in males given 100 mg/kg/day or in female rats at any dose level. A slight decrease in body weight gain was observed at 300 or 1000 mg/kg/day in males compared with the control animals and at the end of the treatment-free period, body weight gain in male rats previously given 1000 mg/kg/day remained lower than the corresponding controls.

 

Food consumption remained unaffected by treatment and aside from isolated instances of excessive salivation observed in male rats given 1000 mg/kg/day (consistent with post-dose observations), there was no effect on functional observations following exposure to the test material. Mean locomotor movement counts and distance travelled for all groups, including controls, generally decreased with time in male rats. Time at rest generally increased with time across all of these groups. There was a dose-related increase in movement counts and distance travelled during Period 1 in treated females compared with control animals and, at 1000 mg/kg/day, these differences attained statistical significance (p≤0.01). Time at rest decreased compared with the controls (p≤0.05) in these animals at this time-point. Thereafter, there were no statistically significant intergroup differences in movement counts or distance travelled at any dose level. Mean locomotor movement counts and distance travelled were similar to the corresponding controls at the end of the treatment-free period.

 

Minor changes in haematology and blood chemistry parameters were observed which were either partially or fully reversible by the end of the treatment-free period. Slight increases in plasma globulin, total protein, and cholesterol concentrations observed were considered to be possibly related to the increase in liver weight seen at 1000 mg/kg/day. The haematology and blood chemistry effects observed, although treatment-related, were not considered to be adverse findings. Urine analysis revealed increased protein content in urine in males given 300 or 1000 mg/kg/day when compared with the control animals. Urine pH was decreased in males at all dose levels and in females given 1000 mg/kg/day. However, at the end of the treatment-free period, protein present in the urine and urine pH was observed to be similar to the corresponding control rats.

 

Gross necropsy did not reveal any remarkable treatment-related findings. Body weight-related liver weight for both sexes given 1000 mg/kg/day (p≤0.001) and males given 300 mg/kg/day (p≤0.01) were statistically significantly higher than controls. Most individual body weight-related liver weights for males given 1000 mg/kg/day were above the upper limit of the historical background range, with the group mean relative liver weight being 34% and 19% higher than controls for males and females, respectively, given 1000 mg/kg/day. However, in the absence of histopathological changes correlated with these liver findings, this effect was considered not adverse.

 

In males, absolute (p≤0.01) and body weight-related (p≤0.05) thyroid weights were increased across all dose groups compared with controls, however, this increase was not dose-related. In females given 1000 mg/kg/day, there was an increase in absolute (p≤0.05) and body weight-related (p≤0.01) thyroid weight; body weight-related thyroid weight was 51% higher than controls. There were microscopic correlates in the thyroid, however, all individual values were within the historical background range, therefore this change was considered to be adaptive. The apparent increases in both thyroid and liver weights and observed hypertrophy in the thyroid gland reflect a commonly observed adaptive response in the liver-thyroid axis which is associated with the metabolism of xenobiotics or their metabolites in the rat.

Thymus weight in males (absolute and body weight related) was statistically significantly lower in animals given 1000 mg/kg/day (p≤0.05) compared with controls. All individual values were within the historical background range and there were microscopic correlates in the thymus and these changes were considered to be related to stress and not adverse. Body weight-related kidney weights in males given 100, 300, and 1000 mg/kg/day (p≤0.05 top≤0.01) and in females given 1000 mg/kg/day (p ≤ 0.05) were statistically significantly higher than controls, although individual weights were within the historical background range for both males and females, and there were no microscopic correlates therefore this change was considered not to be adverse and to be due to chance. In females, absolute pituitary weight was lowered by 23% at 300 mg/kg/day and by 36% at 1000 mg/kg/day (p≤0.05 andp≤0.01 respectively), compared with controls. Body weight related pituitary weights were similarly lower than controls. All body weight-related pituitary weights were within the historical background data, and there were no microscopic correlates, therefore this change was considered not to be adverse and to be due to chance.

 

All changes in organ weights were partially or fully reversed at the end of the treatment-free period with the exception of absolute and body weight-related thyroid weight in males given 1000 mg/kg/day, which remained statistically significantly higher than controls (p≤0.01). However, the effect in the thyroid weight was considered to be adaptive and not treatment-related.

 

Treatment-related microscopic findings were seen in the thyroid gland (hypertrophy of the follicular epithelium) and thymus (atrophy). The changes in the thyroid gland are generally considered adaptive and rodent specific while the thymus atrophy was likely to be the result of non-specific toxicity.

 

Treatment-related changes were observed at the highest dose 1000 mg/kg/day (restricted to rat specific adaptive responses consisting of secondary thyroid follicular hypertrophy consistent with liver enzyme induction). However, these were considered to be adaptive changes and not adverse and based on the findings, the 28-day oral toxicity No Observed Adverse Effect Level (NOAEL) for 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester was determined to be 1000 mg/kg/day.

Additionally, a key Guideline 422 study was conducted to evaluate the effects of repeated oral (gavage) administration of the test material (P1400: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2 (phenylmethyl) ester, CAS 1200806-67-2) to rats during a four-week period in F0 males two weeks before mating through a minimum of 28 days (after confirming female pregnancy), until day 20 postpartum for females including the day before sacrifice, and from birth to sexual maturation (PND 70) in F1 pups. 12 rats per sex per dose received the test material in corn oil once daily via oral gavage at does of 150, 300, or 600 mg/Kg/day while the control animals received corn oil only (Envigo CRS, 2018a).

 

Animals were observed twice daily for mortality and for any clinical signs following administration of the test material. Parameters such as body weight, food consumption, behavioural assessments such as grip strength, motor activity, and sensory reactivity assessment were evaluated for. Additionally, blood samples were extracted from the retro-orbital plexus of all animals under light isoflurane anesthesia and clinical biochemistry as well as haematology assessments were undertaken. Blood samples were taken from all surviving adult males, at termination and from all surviving dams on day 21 postpartum for thyroid hormone analysis. At necropsy, post-mortem examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and their contents with emphasis on the uterus (females only), number of implantation sites (females only) was performed. Organ weights were recorded and all organ and tissue samples (except for the nose) of selected animals from the control and high dose groups (5 animals/sex) were processed, embedded, cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin. Reproductive organs of all control and high dose animals as well as of animals selected for sexual maturation were also examined. Histopathological analysis was undertaken and slides of organs and tissues collected at necropsies of all animals to be evaluated, except for the bone marrow smear for which no test was planned, were examined by the pathologist. Additionally, based on treatment-related morphological changes detected in thyroids in high-dose animals, this organ from the other treatment group animals were examined in all adult animals. A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups was also performed.

 

No treatment-related mortality, clinical signs (other than salivation) or effects on functional observation battery were observed in the parental generation. There was no effect on body weights during the study and there was no indication of an effect on food consumption during the treatment period. The statistically significant increases noted at 300 and 600 mg/Kg/day in males (postnatal days 8 and 15) or in females at 600 mg/Kg/day on gestation day 14 were