Santicizer P1400

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-SEP-17 to 2021-NOV-30

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 414 (Prenatal Developmental Toxicity Study)

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.31 (Prenatal Developmental Toxicity Study)

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 870.3700 (Prenatal Developmental Toxicity Study)

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, Japan, 12 - Nousan No. 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 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: 9-10 weeks old
- Weight at study initiation: Females: 177 - 248 grams
- Fasting period before study: Not specified
- Housing: individually in solid-floor cages
- Diet (e.g. ad libitum): pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited ad libitum
- Water (e.g. ad libitum): mains tap water (in bottles) ad libitum
- Acclimation period: at least 2 days

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-17 To: 2020-OCT-05

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on 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)
- Concentration in vehicle: 0, 25, 75, or 250 mg/mL for the control, 100, 300, and 1000 mg/kg/day dose groups, respectively.
- Amount of vehicle (if gavage): 4 mL/kg

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)).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

Day 6 to Day 19 of gestation, inclusive

Frequency of treatment:

Once daily

Duration of test:

Day 6 to Day 19 of gestation, inclusive

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control - corn oill)

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:

22/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; a 28 Day Oral (Gavage) Toxicity Study in the Rat with a 15 Day Treatment-Free Period. Sequani Study Number: JSK0016; and a 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 a stratified randomisation procedure based on individual body weights recorded on Day 0 of gestation by the supplier (ensuring that females mated with the same male were spread across the groups).

- Fasting period before blood sampling for (rat) dam thyroid hormones: No, animals were not fasted prior to blood sampling.

- Time of day for (rat) dam blood sampling: Day 20 of gestation between 08.00 and 09.00 hours

- Other:
- Justification for route of administration: P1400 is an industrial chemical and the route of administration corresponds to a possible route of human exposure during manufacture, use or handling.

- Justification for species selected: The rat is a suitable rodent species, acceptable to regulatory authorities and for which extensive background data are available.

Animals were dosed once daily from Day 6 to Day 19 of gestation, inclusive, by gavage, using a rubber catheter and disposable syringe at a constant dose volume of 4 mL/kg body weight. Individual doses were adjusted according to the most recently recorded body weight.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: examined twice daily for mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examination daily. From the start of dosing, animals were observed approximately.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on Day 0 of gestation by the supplier. At the CRO, body weights were recorded daily from Day 5 to Day 20 of gestation, inclusive.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The amount of food consumed by each animal was recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: Major organs were examined at necropsy. Gravid uterus and placenta weights were recorded and organs or tissues showing any macroscopic abnormalities were removed and retained in fixative. Liver and thyroids were weighed after trimming of fat and other contiguous tissue and the thyroids were weighed together after fixation.

HISTOPATHOLOGY
For all animals, the liver and thyroids were preserved in 10 % buffered formalin, wax embedded, cut at a nominal thickness of 4 μm to 5 μm, stained with haematoxylin and eosin
and examined microscopically.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

For all pregnant females, the number of corpora lutea and the number and distribution of implantations in each uterine horn were recorded. Implantations were classified as early
intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix. The foetuses and their placentae were removed and the uterus and ovaries were retained in 10 % buffered formalin.

Blood sampling:

- Serum: Yes
- Volume collected : 0.2 mL

Blood samples (0.2 mL) were taken from the tail vein into gel separator tubes and allowed to clot for at least 30 minutes at room temperature. All animals were sampled on Day 20 of gestation between 08.00 and 09.00 hours. 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, where possible (Aliquot 1 contained 50 μL and Aliquot 2 contained all remaining serum) and stored frozen (< -70°C) until analysis.

Aliquot 1 samples were analysed for thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH), using a commercially available analytical kit and analysed on the
Luminex MAGPIX. Aliquot 2 samples were retained frozen (< -70 °C) until satisfactory results were obtained.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes, anogenital distance was measured using digital callipers, measuring between the anus and caudal end of the genital tubercle

On Day 20 of gestation, live foetuses were killed by rapid cooling, weighed, sexed and examined for external abnormalities. Anogenital distance was measured using digital callipers, measuring between the anus and caudal end of the genital tubercle.

Approximately 50 % of the live foetuses were allocated to the fixed head examination. All foetuses were briefly placed in 70 % IDA and subjected to micro-dissection, where the viscera were examined and the foetuses eviscerated. The foetuses allocated to the fixed head examination were decapitated and the heads fixed in Bouin's fluid and examined by serial sectioning.

All carcasses were transferred to 95 % IDA. The carcasses of the intact foetuses were cleared in potassium hydroxide, stained with Alizarin red S and Alcian blue to visualise the ossified skeleton and cartilage and examined. The headless carcasses were discarded without further examination, following confirmation from the Sponsor, after examination of the intact skeletons.

Structural congenital abnormalities that impair, or potentially impair, the survival or constitution of the foetus were classified as major abnormalities. Abnormalities that in isolation, do not impair the survival or constitution of the foetus but could potentially interfere with normal bodily function were classified as minor abnormalities. Alternative structures occurring regularly in the control population, which may be permanent or transient, were classified as variants.

Foetuses with major external or visceral abnormalities were photographed.

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.

The percentage of foetuses affected were treated as continuous non-parametric data, without trend analysis, using Kruskal-Wallis and Wilcoxon tests. The number of litters affected were analysed, without trend analysis, using Chi-Squared and Fisher Exact test. 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 can be found in Table no. 2.

Indices:

1) Pre-implantation loss (%) = ((no. of corpora lutea – no. of implantation sites) / (no. of corpora lutea)) x 100

2) Post-implantation loss (%) = ((no. of implantation sites – no. of live foetuses) / (no. of implantation sites) x 100

3) Anogenital distance index (ACGI) = (Anogenital distance (mm) / cubed root body weight (g))

Historical control data:

Historical Control Data (HCD) presented in Appendix 13 of the final study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed at doses up to 1000 mg/kg/day. Clinical signs recorded, such as hair staining and/or loss and scabbing, were considered not to be toxicologically significant as they were present for only a few days and were also noted in the control animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality was observed through the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight, body weight gain, terminal body weight adjusted for the weight of the gravid uterus was observed to be similar in all groups and unaffected by treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption remained unaffected by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

A slight, non-statistically significant increase in group mean total T3 concentration was observed at 1000 mg/kg/day when compared with the corresponding controls. However, most of the individual T3 concentrations were within, or only slightly outside the historical control data range. No effect of treatment was observed on T4 or TSH concentrations.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of treatment on thyroid gland weight. Mean liver weight relative to body weight was statistically significantly higher than corresponding controls in female rats given 300 mg/kg/day and 1000 mg/kg/day (p≤0.05 and p≤0.001, respectively). At 300 mg/kg/day, individual values remained within the limits of the historical control range but at 1000 mg/kg/day, there were four animals with liver weight marginally above the upper limit of the range. However, mean liver weight adjusted for body weight was only 9 % higher than controls, and since there were no pathological changes in the liver, these increases were considered not to be an adverse treatment-related effect but represented an adaptive response.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross necropsy did not reveal any remarkable treatment-related findings. A variety of spontaneous findings was observed in 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 strain and age kept under laboratory conditions.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No microscopic findings in the liver or thyroid considered to be related to the test material. A variety of spontaneous findings was observed in 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 strain and age kept under laboratory conditions.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions observed.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the incidence of pre- implantations loss and post-implantation loss.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No resorptions observed.

Early or late resorptions:

no effects observed

Description (incidence and severity):

No resorptions observed.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead feotuses

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

On Day 20 of gestation there were 20, 20, 21, and 20 females with live foetuses in the groups given 0, 100, 300 or 1000 mg/kg/day, respectively.

Other effects:

no effects observed

Description (incidence and severity):

There was no effect on the numbers of corpora lutea or number of live foetuses.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: Systemic toxicity

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, mean foetal weight was slightly, but statistically significantly lower, than control (p≤0.05). Individual values were within, or only marginally lower than the lower limit of the historical control data range and therefore, this slight decrease was considered not to be an adverse effect of treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect on the number of live foetuses.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect on the foetal sex ratio.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Foetal anogenital distance was not affected by treatment with the test material.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Major foetal abnormalities were noted in one foetus in each of the groups given 100 mg/kg/day (Interventricular septum, incomplete) and 300 mg/kg/day (Cervical vertebra; one or more neural arch, malformed), and in two foetuses from two litters in the group given 1000 mg/kg/day (Interventricular septum, incomplete; Head, dome shaped). There were no major foetal abnormalities noted in the control group. The nature, incidence, and intergroup distribution of these major foetal abnormalities were considered to be incidental and not treatment-related as they are commonly observed in the historical control data and were only seen in single litters and single foetuses.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, there were statistically significantly higher numbers of litters with foetuses displaying minor or variant abnormalities of the skull that included incomplete ossification of the squamosal, nasal, jugal and/or occipital (p≤0.05, p≤0.01). However, the incidences of these findings were within, or only slightly outside, the historical control ranges. Additionally, there were increased incidences of foetuses showing the minor abnormality of incomplete ossification of one or more neural arches of the sacral vertebrae (p≤0.001) and the variant findings of non-ossification of the 5th and 6th sternebrae (p≤0.01). The incidences of these findings were within, or only slightly higher than the historical control ranges. These incidences of slightly lower foetal skeletal ossification were considered to be associated to the slightly lower foetal weight observed in this group, and therefore, were considered to be non-adverse.

Visceral malformations:

effects observed, non-treatment-related

Effect levels (fetuses)

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: Developmental toxicity

Overall developmental toxicity

Key result

Developmental 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 5% of nominal concentrations which was within the acceptance criteria, confirming accurate formulation. The coefficient of variation values were less than 5 %, which was within the applied limits, confirming the precision of analysis.No test material was detected in control formulations.

Table 3. 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	25.4	25.0	24.9	25.1	+0.4	1.05	100.0
	3	75	77.8	72.0	73.5	74.4	-0.8	4.02	98.6
	4	250	245	245	243	244	-2.4	0.41	98.4
Last occasion	1	0	-	ND, ND	-	-	-	-	-
	2	25	26.1	25.5	25.7	25.8	+3.2	1.15	104.3
	3	75	78.1	77.8	78.3	78.1	+4.1	0.37	106.3
	4	250	261	263	263	262	+4.8	0.45	104.1

RME Relative mean error, representing the deviation from nominal

ND Not detected

CV Coefficient of variation

Table 4. Thyroid hormone assessment (Group mean values)
Group		Mean Total T3 (ng/mL)	Mean Total T4 (ng/mL)	Mean Total TSH (ng/mL)
		(#)	(#)	(# 1)
Group 1 (Control – 0 mg/kg/day)	Mean	18.693	373.439	1.610
	SD	8.464	23.998	0.822
	N	20	20	19
	Trend	↑	↓	↓
Group 2 (P1400 100 mg/kg/day)	Mean	18.941	391.337	1.337
	SD	7.235	40.834	1.030
	N	20	20	19
Group 3 (P1400 300 mg/kg/day)	Mean	17.140	365.278	1.813
	SD	7.838	34.829	0.917
	N	21	21	20
Group 4 (P1400 1000 mg/kg/day)	Mean	23.332	367.309	1.566
	SD	7.857	37.235	0.842
	N	20	20	19
	Trend	>0.05	>0.05	>0.05

(#) - Williams, Anova & Dunnett

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

Table 5. Organ weights: Absolute and Relative to Body Weight (Group mean values)
Group		Liver (g)	Adjusted Liver %	Liver Wt Body Weight
		(#)	(# 1)	(#)
Group 1 (Control – 0 mg/kg/day)	Mean	12.530	12.363	5.098
	SD	0.995	-	0.280
	N	20	20	20
	Trend	↑	↑	↑
Group 2 (P1400 100 mg/kg/day)	Mean	12.145	12.283	5.064
	SD	1.166	-	0.301
	N	20	20	20
	Trend	-	>0.05	>0.05
Group 3 (P1400 300 mg/kg/day)	Mean	12.661	12.774	5.267
	SD	1.429	-	0.276
	N	21	21	21
	Trend	>0.05	≤0.05*	≤0.05*
Group 4 (P1400 1000 mg/kg/day)	Mean	13.612	13.522	5.577
	SD	1.112	-	0.331
	N	20	20	20
	Trend	≤0.01**	≤0.001***	≤0.001***

(#) - 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): Adjusted Bodyweight [Rodent]}

Table 6. Pregnancy Data (Summary)
Sex: Female		Group 1 (Control – 0 mg/kg/day)	Group 2 (P1400 100 mg/kg/day)	Group 3 (P1400 300 mg/kg/day)	Group 4 (P1400 1000 mg/kg/day)
Group Size		22	22	22	22
Not pregnant		2	2	1	2
Not pregnant %		9.1	9.1	4.5	9.1
Not pregnant Died/Killed	Sum	0	0	0	0
Not pregnant Schedule Kill	Sum	2	2	1	2
Pregnant		20	20	21	20
Pregnant %		90.9	90.9	95.5	90.9
Pregnant Died/Killed/Aborted	Sum	0	0	0	0
Pregnant with Total Resorption	Sum	0	0	0	0
Number with Live Foetuses	Sum	20	20	21	20

Table 7. Uterine and Implantation Data (Group mean values)
Sex: Female		Group 1 (Control – 0 mg/kg/day)	Group 2 (P1400 100 mg/kg/day)	Group 3 (P1400 300 mg/kg/day)	Group 4 (P1400 1000 mg/kg/day)
Number with Live Foetuses (GD 20)		20	20	21	20
Number of Corpora Lutea (#)	Sum	228	229	243	236
	Mean	11.4	11.5	11.6	11.8
	SD	1.3	2.1	1.5	1.0
	Trend	↑	-	-	>0.05
Number of Implantations (#1)	Sum	213	202	230	218
	Mean	10.7	10.1	11	10.9
	SD	1.0	2.9	1.4	1.9
	Trend	↑	-	-	>0.05
% Pre-implantation Loss (#1)	Mean	6.0	12.9	5.0	8.0
	Trend	↑	-	-	>0.05
Number of Early Deaths (#1)	Sum	15	13	12	20
	Mean	0.8	0.7	0.6	1.0
	SD	1.2	0.9	0.9	1.6
	Trend	↓	-	-	>0.05
Number of Late Deaths (#)	Sum	0	1	0	1
	Mean	0.0	0.1	0.0	0.1
	SD	0.0	0.2	0.0	0.2
	Trend	↑	-	-	>0.05
Number of Dead Foetuses	Sum	0	0	0	0
	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
Number of Live Foetuses (#)	Sum	198	188	218	197
	Mean	9.9	9.4	10.4	9.9
	SD	1.6	3.2	1.6	1.9
	Trend	↑	-	-	>0.05
% Post-implantation Loss (#)	Mean	7.2	8.2	5.1	8.8
	Trend	↑	-	-	>0.05
Live Foetuses as % of Implants	Mean	92.8	91.8	94.9	91.2

(#) - Williams, Anova & Dunnett(Log) (#1) - Shirley, Kruskal-Wallis & Steel

Table 8. Litter weights (g) / Foetal data (Group mean values)
Sex: Female		Group 1 (Control – 0 mg/kg/day)	Group 2 (P1400 100 mg/kg/day)	Group 3 (P1400 300 mg/kg/day)	Group 4 (P1400 1000 mg/kg/day)
Number with Live Foetuses		20	20	21	20
Number of Live Foetuses	Sum	198	188	218	197
No of Male Foetuses	Sum	100	111	104	98
No of Female Foetuses	Sum	98	77	114	99
% of Male Foetuses (#)	Mean	50.8	60.0	47.5	49.6
	Trend	↑	-	-	>0.05
Litter Weight (#1)	Mean	37.9	35.64	38.48	35.05
	Trend	↓	-	-	>0.05
Foetal Weight (M+F) (#2)	Mean	3.86	3.91	3.71	3.55
	Trend	↓	-	>0.05	≤0.05*
Foetal Weight (M)	Mean	3.97	4.00	3.82	3.63
Foetal Weight (F)	Mean	3.76	3.76	3.63	3.47
Placental Weight (#2)	Mean	0.50*	0.54	0.48	0.52
	Trend	↓	-	-	>0.05

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

(#1) - Williams, Anova & Dunnett

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

Table 9. Examination of foetuses (number of foetuses examined)
Sex: Female		Group 1 (Control – 0 mg/kg/day)	Group 2 (P1400 100 mg/kg/day)	Group 3 (P1400 300 mg/kg/day)	Group 4 (P1400 1000 mg/kg/day)
Litters Examined	N	20	20	21	20
External Foetuses Examined	Sum	198	188	218	197
Visceral Foetuses Examined	Sum	198	188	218	197
Visc Head Foetuses Examined	Sum	98	95	105	97
Skeletal Foetuses Examined	Sum	98	95	105	97
Skel Skull Foetuses Examined	Sum	98	95	105	97
Bouins Head Foetuses Examined	Sum	100	93	113	100
Brain Foetuses Examined	Sum	98	95	105	97

Table 10. Examination of foetuses (Overall summary)
		Group 1 (Control – 0 mg/kg/day)	Group 2 (P1400 100 mg/kg/day)	Group 3 (P1400 300 mg/kg/day)	Group 4 (P1400 1000 mg/kg/day)
Number of Foetuses Examined:		198	188	218	197
Number of Litters Examined:		20	20	21	20
All classifications
Number of Foetuses		112	109	119	114
Group % of Foetuses		56.6	58.0	54.6	57.9
Number of Litters [#]	N+ve	20	20	21	20
	%	100.0	100.0	100.0	100.0
Litter % of Foetuses [#1]	Mean	56.37	59.72	54.82	58.14
Major
Number of Foetuses		0	1	1	2
Group % of Foetuses		0.0	0.5	0.5	1.0
Number of Litters [#]	N+ve	0	1	1	2
	%	0.0	5.0	4.8	10.0
Litter % of Foetuses [#1]	Mean	0.00	1.00	0.53	1.13
Minor
Number of Foetuses		24	48	37	45
Group % of Foetuses		12.1	25.5	17.0	22.8
Number of Litters [#]	N+ve	12*	18*	19*	18*
	%	60.0	90.0	90.5	90.0
Litter % of Foetuses [#1]	Mean	12.73*	26.39**	17.61*	23.23**

[#] - Chi-Squared & Fisher's Exact: * = p ≤ 0.05

[#1] - Kruskal-Wallis & Wilcoxon: * = p ≤ 0.05; ** = p ≤ 0.0

Applicant's summary and conclusion

Conclusions:

Based on the lack of adverse treatment-related effects observed at the highest dose tested, the maternal and developmental toxicity NOAEL for 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester in the rat was determined to be 1000 mg/kg/day.

Executive summary:

A key OECD Guideline 414 study in rats was conducted to evaluate the effects of the test material (1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester (alternative name: Santicizer® Platinum 1400)) on the embryonic and foetal development of the rat when administered from Day 6 to Day 19 of gestation, inclusive.

 

The test material was administered to groups of sexually mature, timed-mated, female Crl:WI(Han) rats (22/dose) once daily via oral gavage in a corn oil vehicle at 0, 100, 300, or 1000 mg/kg/day from Day 6 to Day 19 of gestation, inclusive. All females were observed daily from the start of dosing and body weight and food intake were recorded at regular intervals. Blood samples for thyroid hormone assessment were obtained on the day of necropsy. Animals were killed on Day 20 of gestation, a necropsy was performed and the internal organs were examined for macroscopic abnormalities, and selected organs were weighed and examined microscopically. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, sexed and examined for external, visceral, skeletal and cartilage abnormalities.

 

No mortality or clinical signs of treatment-related toxicity were observed through the study period and there was no effect of treatment on body weight or food intake at any dose level. Thyroid hormone (T3, T4, & TSH) concentrations were unaffected by exposure to the test material at any dose level.

 

Gross necropsy did not reveal any remarkable treatment-related findings and microscopic evaluation did not reveal any treatment-related findings in the liver or thyroid. At 300 and 1000 mg/kg/day, a non-adverse increase in mean liver weight compared with control animals was observed while there was no effect on thyroid weight.

 

There was no effect on the numbers of corpora lutea, implantations, the incidence of pre-implantations loss, post-implantation loss or on the number of live foetuses. Foetal weight was observed to be slightly lower than that of corresponding controls at 1000 mg/kg/day. However, individual values were within, or only marginally lower than the lower limit of the historical control data range and therefore, this slight decrease was considered not to be an adverse effect of treatment. There was no effect on foetal sex ratio, placental weight, or foetal anogenital distance.

 

Slight increases in minor and variant foetal abnormalities of incomplete or non-ossification of some skull bones, vertebral neural arches and sternebrae were observed at 1000 mg/kg/day. These were associated with the lower foetal weights in this group compared with control animals rather than a direct effect of treatment and considered to resolve postnatally.

 

Based on the results observed the maternal and developmental toxicity NOAEL for 1,2-Cyclohexanedicarboxylic Acid, 1-butyl 2-(phenylmethyl) ester in the rat was determined to be 1000 mg/kg/day.