Esterification product of castor oil and tetrahydromethyl-1,3-isobenzofuranedione

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

REACH_NOAEL_300 mg/kg/bw | OECD 421 | #key study#

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-11 to 2018-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test

Version / remarks:

EPA 712-C-00-367, July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Justification for study design:

This test is performed on the rat. Although several mammalian species may be used, the rat is the preferred rodent species.
This study provides initial information concerning the effects of a test item on male and female reproductive performance such as gonadal function, mating behaviour,
conception, development of the conceptus and parturition. This screening test gives information at an early stage of assessing the toxicological properties of chemicals, or
on chemicals of concern.

Specific details on test material used for the study:

Active Components: 94 %, was taken into account in formulation preparation
Date of Analysis 26 June 2017

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
- Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Sex: male and female; the female animals were non-pregnant and nulliparous.
- Age at the start of the treatment period: approx. 14-15 weeks old
- Body weight at the allocation of the animals to the experimental groups:
males: 317 - 394 g (mean: 355.70 g, ± 20 % = 284.56 – 426.84 g)
females: 200- 255 g (mean: 222.90 g, ± 20 % = 178.32 – 267.48 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.
The animal study was authorized by local government under file no. 55.2-1-54-2532.0-31-2014.

HUSBANDRY
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 5 animals / sex / cage (type IV, polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Makrolon tunnels were provided for all males and for females until GD 18
- Nesting material were provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

PREPARATION OF ANIMALS
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. Only healthy animals were used for the study.
Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.
Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2 software).
Each animal was marked with its identification number by individual ear tattoo or tail marking.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400 (manufacturer: Merck KGaA, batch no.: S7378385727, physical state: liquid, storage conditions: at room temperature, expiry date: 09 March 2018)

Details on exposure:

PREPARATION OF TEST ITEM FORMULATIONS
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred and subjected to ultrasonic bath for approx. 30 min at approx. 60 °C until visual homogeneity was achieved. 
Based on the results of stability testing (Eurofins Munich Study No. 175740), the test item formulations were prepared at least once every 14 days (within stability time frame as given by Eurofins Munich Study No. 175740). The prepared formulation was stored at 2-8 °C. Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.
 
EXPERIMENTAL GROUPS AND DOSES
According to the results of a previous dose range finding study (BSL Munich Study No. 175736, non GLP) and in consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.

C 0 mg/kg bw/ day (Male No.:1-10/ Female No.:41-50)
LD 100* mg/kg bw/ day (Male No.:11-20/ Female No.:51-60)
MD 300* mg/kg bw/ day (Male No.:21-30/ Female No.:61-70)
HD 1000* mg/kg bw/ day (Male No.:31-40/ Female No.:71-80)
*= dose formulation were prepared based on active ingredient content within the test item (94 %)

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the high dose group.

ADMINISTRATION
The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

CONCENTRATIONS
C: 0 mg/mL;
LD: 26.6 mg/mL (corresponding to 25 mg/mL of active ingredient in the dosing formulation);
MD: 79.8 mg/mL (corresponding to 75 mg/mL of active ingredient in the dosing formulation);
HD: 266 mg/mL (corresponding to 250 mg/mL of active ingredient in the dosing formulation).
Dose formulations were prepared based on active ingredient content within the test item (94 %).

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 175740).
Study pre start stability analysis was included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day -15 to -35 °C.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was not shown to be homogenous according to Eurofins Study No. 175740, samples were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (40 samples).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 175742) and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle for a maximum period of 63 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days is completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Experimental Completion Date: 25 July 2016
Completion Date of Delegated Phase (Histopathology): 31 May 2017
Completion Date of Delegated Phase (Formulation Analysis): 29 March 2017
Arrival of the Test Item: 08 August 2017
Study Initiation Date: 11 December 2017
Amendment to Study Plan: 15 December 2017
2nd Amendment to Study Plan: 27 April 2018
Delivery of Animals: 19 December 2017
Acclimatisation Period: 19 December 2017 to 25 December 2017
Experimental Starting Date: 26 December 2017
Treatment Period: 09 January 2018 to 03 March 2018
Necropsies: 30 January 2018, 02 February 2018, 06 - 07 February 2018, 27 - 28 February 2018, 01 – 04 March 2018
Experimental Completion Date: 05 March 2018
Completion Date of Delegated Phase (Histopathology): 24 July 2018
Completion Date of Delegated Phase (Formulation Analysis): 25 July 2018

Dose / conc.:

0 mg/kg bw/day

Remarks:

control group: vehicle

Dose / conc.:

100 mg/kg bw/day

Remarks:

dose formulations were prepared based on active ingredient content within the test item (94 %)

Dose / conc.:

300 mg/kg bw/day

Remarks:

dose formulations were prepared based on active ingredient content within the test item (94 %)

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

dose formulations were prepared based on active ingredient content within the test item (94 %)

No. of animals per sex per dose:

100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.

Control animals:

yes, concurrent vehicle

Details on study design:

The test item is orally administered daily, i.e. 7 days per week in graduated doses to several groups of test animals (male and female), one dose level per group with a maximum exposure of 63 days in total in females (at least 14 days pre-mating, up to 14 days mating, approximately 22 days of gestation and up to post natal day 12).
Males are dosed for a minimum of four weeks until up to one day before the scheduled sacrifice (this includes a minimum of two weeks prior to mating, during the mating period and approximately two weeks post-mating).
During the administration period the animals are observed closely each day for signs of toxicity. Animals which die or are sacrificed during the test period are necropsied and, at the conclusion of the test, surviving animals are sacrificed and necropsied.
The litters are examined as soon as possible after birth. On day 13 of the lactation period, the pups are euthanised and externally examined for abnormalities.

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT AND FOOD CONSUMPTION
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups. All animals were weighed at termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

as part of Histopathology:
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Litter observations:

The duration of gestation was recorded and is calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. 
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.
Inadvertently, pup survival data of all pups from animals no. 79 (HD) were not recorded on day 3. Inadvertently, pups of dam no. 80 were weighed on PND 5 instead of PND 4.

Blood samples were collected from the defined site in serum separator tubes From 2 female pups (in dams with no enough females, males were used) wherever possible/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13 and from all adult males at termination. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary as no effect observed on T4 hormone levels of males and day 13 pups. Pup blood was pooled by litter for thyroid hormone analysis.
Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations, if possible. No pups were eliminated as litter size dropped below 8 pups. As there was only one pup available above a litter size of 8, only one pup was sacrificed. Except for the litters of dam no. 46 and 49 were no pups were sacrificed on PND 4 for thyroid hormone determination.

Postmortem examinations (parental animals):

PATHOLOGY
All male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29, while female animals were sacrificed on post-natal day 13 along using an anaesthesia (ketamine/xylazin). All surviving pups were killed by cervical dislocation on PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.  
Special attention was paid to the organs of the reproductive system. The following tissues from all male and female animals were preserved in 4 % neutral-buffered formaldehyde except for testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol - and subsequently examined histopathologically: all gross lesions, epididymides, ovaries, prostate and seminal vesicles with coagulating glands as a whole, testes, uterus with cervix, vagina and thyroid/parathyroid glands. The latter were not examined histopathologically.
All animals intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination: adrenal glands, all gross lesions, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), ovaries, oviducts, prostate and seminal vesicles with coagulating glands as a whole, rectum, spleen, stomach, testes, thymus, thyroid/parathyroid glands, trachea, urinary bladder, uterus with cervix and vagina.

ORGAN WEIGHTS
The wet weight of the reproductive organs [epididymides, testes, ovaries, uterus with cervix, prostate, seminal vesicles and coagulating glands, thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females), were weighed after fixation] of all sacrificed adult males and females from each group were recorded as soon as possible. Paired organs were weighed together. Organ weights of animals euthanised for animal welfare reasons were not recorded.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

HISTOPATHOLOGY
A full histopathology was carried out on the preserved organs and tissues (see above) of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid gland from pups and from the adult animals was not evaluated as it was not considered necessary as there was no test item related effect observed on thyroid weights in parental animals and T4 hormone level in parental males and pups sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which were euthanised due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon completion of the study.

Postmortem examinations (offspring):

All surviving pups were killed by cervical dislocation on PND 13.
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data were performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Reproductive indices:

P0: copulation, fertility, delivery and viability indices

Offspring viability indices:

see Results F1 section

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The female no. 67 (MD, sacrificed on GD 9) showed slight piloerection, abnormal breathing and red nasal discharge. The female no. 76 was euthanized on GD 5 with moderate piloerection, kyphosis and sunken flanks.
Animal no. 56 regurgitated test item on PMD 10 and was seen with abnormal breathing afterwards but recovered and was sacrificed at the scheduled end of study.
From premating day 8 onwards, all male animals of the HD and some female animals of the LD, MD and HD group were observed moving the bedding with the nose immediately after administration. One male HD (no. 38) and 9/10 HD females showed moreover salivation on different study days starting earliest on PM day 9 till terminal sacrifice. The clinical signs salivation and moving the bedding in males and females were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.
The clinical symptom like alopecia was noted in total in 3 animals (LD male no. 20, control female no. 49, MD female no. 70). This is not considered to be test item related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two animals were sacrificed in a moribund condition during the study: female animal no. 67 from the MD group on GD 9 and one female (no. 76) of the HD group on GD 5. No further mortalities were recorded at the other dose levels. Histolopathologically mortality was associated with a lung carcinoma and a suppurative necrotizing bronchopneumonia in female no. 67 and deemed to be incidential. Histopathologically cause of mortality or morbidity was not evident in female no. 76.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on body weight development in this study. Body weights of male and female animals were in the normal range of variation throughout the treatment of this study. However the female mean body weight gain of the LD and HD group was slightly attenuated without archiving significance during the 2nd part of the premating period, resulting in a slightly lower group mean body weight at the end of the study in LD and HD females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The test item had no effect on food consumption in this study. Mean daily food intake of male and female animals was in the normal range of variation throughout the treatment of this study. 
Slightly attenuated and significant (p< 0.05) mean food consumption of the female HD group between gestation days 0 to 7 and lactation days 4 to 9 is not considered to be treatment related. Slightly lower mean values for the food consumption of the female HD group can be attributed to one single female animal no. 71 which constantly has lower values than the other dams of this group.
Slightly attenuated food consumption in male animals from the MD and HD doses during the treatment period is not assumed to be toxicologically relevant as values were constantly within the normal range of variation for this gender, age and strain.

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:

no effects observed

Description (incidence and severity):

THYROID HORMONE (T4) ANALYSIS
No test item related effect of toxicological relevance or statistical significance was observed on male thyroxine hormone (T4) in the treatment groups when compared to the controls.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item induced histopathological changes.
All microscopic findings recorded in animal no. 67 and 76 were within the range of normal background lesions which may be recorded in animals of this strain and age and in this study type.
The test item did not produce any histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. Further, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.
The treatment with Cyclohexene-1,2-dicarboxylic acid, methyl-castor-oil alkyl esters did not induced histomorphological effects in the reproductive organs of the non-pregnant females from the high dose group (animal nos. 73 and 76) and its mating partner male no. 33 and 36 respectively.
All microscopic findings recorded in surviving animals were within the range of normal background lesions which may be recorded in animals of this strain and age and in this study type.
In conclusion, a histomorphological NOEL (no observed effect level) could be established at 1000 mg/kg bw/day.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see Mortality / Description

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The test item had no biologically significant effect on the estrous cycle analysed during the 2 weeks premating period and after the first administration in treatment groups when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the treatment groups and the control group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

see Histopathological findings: non-neoplastic / Description

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

PRECOITAL INTERVAL AND DURATION OF GESTATION
There were no test item related or statistically significant effects observed on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

REPRODUCTIVE INDICES
There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. However a lower viability index (PND 0-4) for dam no. 79 was recorded as 4 pups were found dead on PND 4. Slightly lower fertility and delivery indices for the HD group were recorded. These values are in the normal range of biological variation and not considered to be test item related.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Remarks:

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period as measured concentrations were within acceptance criterion of 20 %.

Sex:

male/female

Remarks on result:

other: No adverse effect was observed at the highest dose of treatment (1000 mg/kg bw/day). A histomorphological NOEL (no observed effect level) could be established at 1000 mg/kg bw/day.

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pup External Findings, see Details on Results F1

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see Details on results F1

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid Hormone (T4) Analysis: see Details on results F1

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

see Details on results F1

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

see Details on Results F1

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

see Details on Results F1

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

see Details on Results F1

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pup External Findings, see Details on Results F1

Histopathological findings:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Litter Data, see Details on Results F1

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

PUP SURVIVAL DATA
Pup mortality was recorded for 4 pups of dam no. 79 of the HD group between PND 2 – 4. Exact date of mortality cannot be determined as no data is available for PND 3 for this litter. No other mortality was recorded for the other dams of the other groups, except for the control dam no. 42, where one pup was found dead on PND 4. The mean total mortality for the HD group is therefore slightly higher than the control (3.85 %, compared to 0.77 % in the control). Since this slightly higher mean mortality value is due to only one dam and no other pup mortality in the HD group was recorded this is deemed to be incidental.
This resulted in a slightly lower mean viability index (Viability Index (%) = (No. of live offspring at day 4 / No. of live offspring at birth) X 100) during day 0-4 for the HD group (96.15 %) compared to the control group (99.23 %). The viability indices in all other groups during day 0-4 and 4-13 were 100 %.

PUP EXTERNAL FINDINGS (PND 0 and at Death)
No test item related gross external abnormalities of toxicological relevance on PND 0-12 were observed in the pups of any of the groups. On the day of necropsy slight alopecia on forelimbs, flanks and the back of all pups of dam no. 49 (control group) was noted. These findings are not considered to be test item related.

LITTER WEIGHT DATA
The pup mean weight on PND 0, PND 4 and PND 13 was lower without achieving statistical significance in the MD and the HD group when compared with the control (MD: 3 %, 8 % and 7 % lower than control; HD: 6 %, 12 %, 14 % lower than control) However, the values for the PND 13 mean pup weight were statistically significantly lower in HD group (p<0.05) than the control group.
Furthermore, total mean litter weights were significantly lower on PND 0, 4 and 13 in HD group although statistical significance was achieved only on PND 13 (-20 %, p<0.01) when compared with the control.
There was also statistically significant effect on group mean male litter weight on PND 0 (p<0.05), PND 4 (p<0.01) and PND 13 (p<0.01) in HD group when compared to the control. This effect on male litter was attributed to low number of male pups born in HD group as reflected with the statistically significantly low mean number of male pups on PND 4 and therefore this effect on group mean male litter weight was considered to be biological variation without any toxicological significance. 
The effect on group mean pup weight and total mean litter weights in HD group on PND 0, 4 and 13 was considered to be test item related.
The values for the LD and MD dose groups were in the range of biological variation and not deemed to be test item related.
 
ANOGENITAL DISTANCE AND NIPPLE RETENTION
No statistically significant effect of toxicological relevance was observed on mean pup weight on PND 0 for male and female, cube root of pup weight, anogenital distance, relative anogenital distance or on nipple retention of the male pups on PND 12 of any of the groups when compared with the controls.

THYROID HORMONE (T4) ANALYSIS
No test item related effect of toxicological relevance or statistical significance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls. However mean values for pup- thyroxine (T4) in the MD and the HD group were slightly but significantly (p<0.05) lower than seen in the control group without following dose dependency. Mean and single values are in the normal range of biological variation and therefor the effect is not considered to be test item related.

PRE- AND POSTNATAL DATA
No considerable test item related effects were noted for mean values of corpora lutea, implantations sites, live pups on PND0, 4 and 13 as well as pre implantation loss and post implantation loss in all dose groups.

LITTER DATA
The total number of pups on PND 0,4 and 13, the number of males and females on PND 0, 4 and 13, as well as sex ratio in the HD group was slightly lower than seen in the control. There were no considerable differences in the number of still births, runts on PND 0 noted in all dose groups compared to the control. The number of males on PND 4 before interim sacrifice was marginally and significantly (p<0.05) lower than seen in the control. This effect on lower male pups was considered as biological variation as confirmed test article related shift in sex ratio is very rarely observed. Although there is a possibility of alterations arising from direct effects on the male germ cell populations that would favor the production or destruction of Y or X chromosome-bearing sperm. However, in the light of absence of histopathological findings and hormonal alterations, this possibility was excluded and hence observed effect has no toxicological relevance.
All values for the other dose groups were comparable with the control.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

act. ingr.

Remarks:

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period as measured concentrations were within acceptance criterion of 20 %.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

The test item had an effect on group mean pup weight and total mean litter weights in HD group on PND 0, 4 and 13.

Key result

Critical effects observed:

no

Reproductive effects observed:

not specified

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:
The NOAEL of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in this study for general toxicity screening is considered to be 1000 mg/kg bw/d.
The NOAEL of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in this study for reproductive toxicity screening is considered to be 300 mg/kg bw/d.

Executive summary:

Summary

The aim of this study was to assess the possible effects of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters on male and female fertility and embryofetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a maximum treatment period up to 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but received PEG 400, the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:                        0        mg/kg bw/day

Low Dose:                    100*    mg/kg bw/day

Medium Dose:              300*    mg/kg bw/day

High Dose:                   1000*  mg/kg bw/day

*dose formulation was prepared based on active ingredient content within the test item (94%)

The test item formulation was prepared with PEG 400 within the stability range of 14 days (storage of formulation 2-8 °C). Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 4 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment days 29 and the females along with their pups were sacrificed on post natal day 13. Non-pregnant females were sacrificed on day 26.

 

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natal day 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues was performed on high dose and control animals, dead animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation ofadditional hematoxylin-PAS (Periodic Acid Schiff) stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.

Results

Two mortalities were recorded during the study: one female (no. 67) of the MD group was sacrificed moribund on GD 9 and one HD female (no. 76) was sacrificed moribund on GD 5. In female no. 67 mortality was associated with a lung carcinoma and a suppurative necrotizing bronchopneumonia. This finding is deemed to be incidental and not test item related. No microscopic finding could be associated with the morbidity of dam no. 76, but was not deemed to be test item related. No further mortalities were recorded.

No test item related clinical findings were recorded during the study. However prematurely sacrificed female no. 67 showed slight piloerection, abnormal breathing and red nasal discharge and female no. 76 was euthanized showing moderate piloerection, kyphosis and sunken flanks.

The test item had no effect on body weight or body weight development. All values were in the normal range of variation for this strain and age.

The food consumption of the animals was not affected by the test item. Slightly attenuated food consumption in the female HD group were directly correlated to one single dam and therefore not considered to be test item related.

The test item had no biologically significant effect on estrous cycle anaylsed during the 2 weeks premating period.

The total number of pups on PND 0, 4 and 13, the number of males and females on PND 0, 4 and 13 as well as the sex ratio was slightly lower in the HD group when compared to the control. There were no considerable differences in the number of still births, runts on PND 0 noted in all dose groups compared to the control. The number of males on PND 4 before interim sacrifice was marginally and significantly (p<0.05) lower than seen in the control. This effect on lower male pups was considered as biological variation and of no toxicological significance.

The test item had an effect on group mean pup weight and total mean litter weights in HD group on PND 0, 4 and 13. 

There was no test item related effect observed on the duration of precoital interval and the duration of the gestation in all dose groups compared with the control.

No test item related effect was observed for mean values of corpora lutea, implantations sites, live pups on PND0, 4 and 13 as well as preimplantation loss and post implantation loss in all dose groups.

There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group.

The pup mortality was not affected by the test item during the study period.

No effect of the test item of toxicological relevance was recorded for the anogenital distance, nipple retention, thyroid hormone or external examination of the pups.

The organ weights showed no considerable differences between the dose groups and the control groups and variations were in the normal range of biological variation.Under the conditions of this study, the test item did not produce any histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, oviducts, uterus and cervix, and vagina. Further, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.The treatment with Cyclohexene-1,2-dicarboxylic acid, methyl-castor-oil alkyl esters did not induce histomorphological effects in the reproductive organs of the non-pregnant females from the high dose group (animal nos. 73 and 76) and their mating partners male nos. 33 and 36 respectively. In conclusion, a histomorphological NOEL (no observed effect level) could be established at 1000 mg/kg bw/day.

Dose formulation analysis for nominal concentration revealed that nominal concentrations for all formulations were confirmed throughout the study period as measured concentrations were within acceptance criterion of 20%.

Conclusion

On the basis of this reproduction/developmental toxicity screening test with Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in male and female Wistar rats with dose levels of 100, 300, and 1000 mg/kg body weight day the following conclusions can be made:

There was one mortality observed in the study (female no. 67 of MD group) on gestation day 9 and one female (no. 76 of HD group) was sacrificed in moribund condition on gestation day 5 due to animal welfare reasons. Histopathologically mortality was associated with a lung carcinoma and a suppurative necrotizing bronchopneumonia in female no. 67 and deemed to be incidential. Histopathologically cause of mortality or morbidity was not evident in female no. 76.

There was a test item related effect observed on pup group mean weight and total group mean litter weights on post natal day 0, 4 and 13 in HD group.

No adverse effects of test item were found on male and female clinical observations, body weight development, food consumption, estrous cyclicity, litter data, precoital interval and duration of gestation, pre and post-natal data, reproductive indices, pup survival data, anogenital distance and nipple retention, pup thyroid weight and thyroid hormone analysis in parental males and pups sacrificed on PND 13, pup external findings, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups.

The NOAEL of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in this study for general toxicity screening is considered to be 1000 mg/kg body weight/day.

The NOAEL of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in this study for reproductive toxicity screening is considered to be 300 mg/kg body weight/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No study available

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

The NOAEL of Cyclohexene-1,2-dicarboxylic acid, methyl-, castor-oil alkyl esters in this study for reproductive toxicity screening is considered to be 300 mg/kg body weight/day. Because there was a test item related effect observed on pup group mean weight and total group mean litter weights on post natal day 0, 4 and 13 in the High Dose group (1000 mg/kg body weight/day). These effects are not considered severe enough to warrant classification for toxicity to reproduction according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.