Santicizer P1400

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test guideline 3

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

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

benzyl butyl cis-cyclohexane-1,2-dicarboxylate

Cas Number:

1931129-39-3

Molecular formula:

C19H26O4

IUPAC Name:

benzyl butyl cis-cyclohexane-1,2-dicarboxylate

Test material form:

liquid

Details on test material:

Name of substance: 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester
Alternative names: benzyl butyl cis-cyclohexane-1,2-dicarboxylate; Santicizer® Platinum P1400
Batch Number: 5840
CAS Numbers: 1931129-39-3 (cis-isomer); 1200806-67-2
Purity: 99.609 % (GC)
Expiration date: 2021-MAR-04

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control - corn oil)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 (Low dose)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 (Intermediate dose)

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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, 231^1	244, 240^1	242, 254^1	241	-3.6	3.61	94.1^2, 95.5^2, 94.8^2

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

Applicant's summary and conclusion

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.