Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January 2016 to 11 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted in accordance with OECD test guidelines in a GLP accredited laboratory

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

Deviations:

yes

Remarks:

See methods

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

Study design was based on recognised test guidelines

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

EC Number:

412-540-8

EC Name:

Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Cas Number:

22471-55-2

Molecular formula:

C12 H22 O2

IUPAC Name:

ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Constituent 2

Reference substance name:

Ethyl cis-2,2,6-trimethylcyclohexane-1-carboxylate

Molecular formula:

C12H22O2

IUPAC Name:

Ethyl cis-2,2,6-trimethylcyclohexane-1-carboxylate

impurity 1

Reference substance name:

Unidentified impurities

Molecular formula:

Not specified.

IUPAC Name:

Unidentified impurities

Test material form:

liquid: viscous

Details on test material:

Sample name: ET-344 SP

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study.The Sprague-Dawley strain was selected since background data from previous studies are available at CiToxLAB France.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals Number: 82 Sprague-Dawley, RjHan: SD (Rats CD®, 41 males and 41 females) were received at CiToxLAB France on 14 January 2016.
Breeder: Janvier, Le Genest-Saint-Isle, France.
Age/Weight on the first day of treatment: Males were approximately 10 weeks old and had a mean body weight of 414 g (range: 385 g to 439 g). Females were approximately 9 weeks old had a mean body weight of 253 g (range: 236 g to 275 g) The females were virgin.
The males and the females were sexually mature and were not siblings. Receipt: on arrival, the animals were given a clinical examination to ensure that they were in good health condition. Acclimation: Animals were acclimated to study conditions for 6 days prior to dosing. An additional animal/sex was acclimated to permit selection and/or replace individuals.Allocation to groups: during the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a computerized stratification procedure based on body weight (these data are not presented in this report), so that the average body weight of each group was similar. The weight variation did not exceed ± 20% of the mean weight for each sex. Identification: individual ear tattoo. At the beginning of the study, each animal received a unique CiToxLAB France identity number.Environmental conditions From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit. The animal room conditions were set as follows: temperature : 22 ± 2°C, relative humidity : 50 ± 20%, light/dark cycle : 12h/12h, ventilation : about 12 cycles/hour of filtered, non-recycled air. The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are recorded continuously (recording devices equipped with alarm systems). The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.Housing The animals were individually housed, except during mating and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing was chosen since it is preferable for pregnant animals and to avoid aggressive behavior around mating. Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material, a few days before delivery and during lactation period. The sawdust was replaced twice weekly. Rat hut and nylabone were given as environmental enrichment. The cages were placed in numerical order on the racks.Food: All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 3044117 and 1324730 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.Water: Bottles containing tap water (filtered with a 0.22 μm filter)Access to food and water: ad libitumContaminant analyses The batches of diet, sawdust and wood shavings were analyzed by the Suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Batch No: MKBS3571V

Details on exposure:

The oral route was selected since it is a route of administration which is recommended by the Regulatory Authorities for this type of study.

Details on mating procedure:

Females were paired with males from the same dose-level group. One female was placed with one male, in the latter's cage, during the night.
Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage. The day of confirmed mating was designated Day 0 p.c.Each female was placed with the same male until mating occurred or 7 days had elapsed. Pair with no evidence of mating after 7 days was separated and the female was placed for a further 7 days with a different male from the same dose-level group who had already mated. The pre-coital time was calculated for each female. Female with no evidence of mating after 7 days with the second male was sacrificed 24 days after the end of the mating period.The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the females were mated.

Analytical verification of doses or concentrations:

yes

Remarks:

Weeks 01, 04 and 07

Details on analytical verification of doses or concentrations:

The test item concentrations in the administered dose formulations analyzed in Weeks 01, 04 and 07 remained within an acceptable range of variations (-1.6% to +4.1%) when compared to the nominal values (± 10% of the nominal concentrations). No test item was observed in the control dose formulation.
Analysis was done using Gas Chromatography with Flame Ionization Detection

Duration of treatment / exposure:

The dose formulations were administered daily according to the following schedule:
In the males: - 2 weeks before mating, - during the mating period (up to 2 weeks), - until euthanasia (4 weeks in total).
In the females: - 2 weeks before mating, - during the mating period (up to 2 weeks), - during gestation, - during lactation until Day 4 p.p. inclusive, - until euthanasia for females with no evidence of mating or with no delivery. Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

Administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time.
Males (day 1 to day 28) Females (day 1 to day 34, including during mating, gestation and lactation)

Details on study schedule:

The dose formulations were administered by gavage, using a plastic syringe fitted with a plastic gavage tube, once a day, at approximately the same time. The quantity of the dose formulation administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 2 mL/kg/day was used. Control animals (group 1) received the vehicle only. The dose formulations were maintained under delivery conditions (at room temperature and protected from light) throughout the dosing procedure. The control and test item dose formulations were stirred for 15 minutes before administration.The formulations were maintained under continuous magnetic stirring throughout the dosing procedure.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)10 males (F21791 to F21800)10 females (F21871 to F21880)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 210 males (F21801 to F21810)10 females (F21881 to F21890)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 310 males (F21811 to F21820)10 females (F21891 to F21900)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 410 males (F21821 to F21830)10 females (F21901 to F21910)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

In this previous study, the test item was administered for 28 days by gavage to Sprague-Dawley rats at 15, 150 and 1000 mg/kg/day (5 rats/sex/groups). A control group received the vehicle (Arachis oil B.P.). Two satellite groups were treated at 1000 mg/kg/day or the vehicle and then maintained without treatment for a further 14 days. There were no unscheduled deaths. High-dose animals showed clinical signs (increased salivation immediately after dosing together with associated signs of wet and stained fur) from Day 2 onwards. No clinically observable signs were detected in satellite high-dose animals during the recovery period or in the low- or intermediate-dose groups. There were no effects on body weight gain, no adverse effects on food consumption but an increase in water intake in the high-dose group from Day 8 which recovered completely during the treatment free period. High-dose females had increases in total protein and albumin, and a reduction in albumin/globulin ratio. Increases in alanine aminotransferase and bilirubin were detected in high-dose females and gamma glutamyl transpeptidase was increased in both sexes treated at the high dose-level. High-dose females had increased reducing substances in the urine. No abnormalities were detected in the remaining dose groups or in satellite high-dose animals after 14 days without treatment. High-dose animals had dark and/or enlarged livers (males and females) and patchy pallor of the kidneys (males). Intermediate dose males had similar kidney changes to those of the high-dose group. No treatment-related macroscopic abnormalities were detected in low-dose animals.Increases in absolute and relative liver weight were seen in high-dose animals of either sex. Intermediate-dose males also had increased relative liver weights and relative liver weights were increased in satellite high-dose males after 14 days without treatment. Absolute and relative kidney weights increased in high- and intermediate-dose males. Hepatocellular enlargement and increased hepatocytoplasmic density were observed in males dosed at 1000 mg/kg/day. The condition was observed to have regressed in satellite 1000 mg/kg/day rats after a 14-day treatment free period. In kidneys, eosinophilic tubular degeneration was observed in all male rats (females were unaffected). Such renal changes are typical of hydrocarbon induced nephropathy observed in the renal tubules of male rats. Toxicologically significant effects were observed at 1000 and 150 mg/kg/day and the "No Observed Effect Level" (NOEL) was considered to be 15 mg/kg/day. However, this evaluation was mainly based on HBU and histopathology of liver/kidneys: mild clinical signs were recorded at 1000 mg/kg/day only with no effect on mean body weight change and no adverse effects on mean food consumption. Therefore to demonstrate that an enough high dose-level had been tested, 1000 mg/kg/day was selected as a high dose-level. The low-dose and mid dose-levels were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300 mg/kg/day).

Positive control:

Positive control was not used in this study.

Examinations

Parental animals: Observations and examinations:

Morbidity and mortality: Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.Clinical signs: Each animal was observed once a day at approximately the same time.
Body weight: Males were recorded on the day 1, then once a week until sacrifice. Females were measured on day 1 then once a week until mated (or sacrificed, if there were no evidence of mating). The on days 0, 7, 14 and 20 post-coitum (p.c.) and Days 1 and 5 p.p.Food consumption: Consumption by males were measured once a week from day 1 until mating period. Female consumption was measured once a week from day 1 until mating period Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-5 p.p.
Mating: Females were paired with males from the same dose-level group. One female was placed with one male, in the latter's cage, during the night. Confirmation of mating was made in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage. The day of confirmed mating was designated Day 0 p.c.. Each female was placed with the same male until mating occurred or 7 days had elapsed. Pair with no evidence of mating after 7 days was separated and the female was placed for a further 7 days with a different male from the same dose-level group who had already mated. The pre-coital time was calculated for each female. Female with no evidence of mating after 7 days with the second male was sacrificed 24 days after the end of the mating period.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the females were mated.

Sperm parameters (parental animals):

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

Each live pup was identified individually on Day 1 p.p., by subcutaneous injection of Indian ink.
Litter size: The total litter size and sex of each pup were recorded as soon as possible after birth.
Any gross external malformations in pups were noted. The litters were observed daily in order to note the number of live, dead and cannibalized pups.
Clinical signs: The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities.
Body weight: The body weight of each pup was recorded on Days 1 and 5 p.p.

Postmortem examinations (parental animals):

Macroscopic examination:
Examination of the principal thoracic and abdominal organs was performed on all animals with special attention to reproductive organs. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on Day 5 p.p. and for the female sacrificed on Day 24 p.c. due to no delivery. The numbers of corpora lutea were also recorded for the female sacrificed 24 days after the end of the mating period with no evidence of mating. For the apparently non-pregnant female, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.
Preservation of tissues (F0 animals):
The tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in Modified Davidson's fixative).
Preparation of histological slides (F0 animals):
All tissues required for microscopic examination were trimmed based on the RITA guidelines (Ruehl Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).
One additional kidney slides of each control and high-dose males have been prepared and immunostained with antibodies for alpha-2-microglobulin protein (20 slides in total).

Microscopic examination:
A microscopic examination was performed on:
all tissues listed in the Tissue Procedure Table from all animals of the control and high-dose groups (groups 1 and 4) euthanized at the end of the treatment period (at the end of the mating period for males or on Day 5 p.p. for females),
all macroscopic lesions of all groups.,
immunostained kidneys from the control and high-dose males (groups 1 and 4) sacrificed at the end of the treatment period.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

No macroscopic post-mortem malformation was noted.
All pups (except moribund) were discarded without any examination.

Statistics:

Data are expressed as group mean values ± standard deviation (body weight, body weight change, foodconsumption, number of corpora lutea, number of implantations, number of pups and pup body weight,gestation length) or as proportions (pre-implantation loss, post-implantation loss, pup observations, mating index, fertility index, gestation index, live birth index, viability index). Whenever appropriate, the experimental unit of comparison was the litter.
Data of non-pregnant females are not included in group mean calculations (body weight, body weightchange, food consumption).

Reproductive indices:

Females were allowed to litter normally and rear their progeny until Day 5 p.p.. Any sign of a difficult or prolonged parturition was recorded.
The morning when the parturition was completed was designated Day 1 p.p.. The length of gestation was calculated.

Offspring viability indices:

Live birth index (number of live pups born/number of delivered pups) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Ptyalism (hypersalivation) was recorded in males receiving 100 mg/kg/day (2/10 males) and in nearly all males and females dosed from 300 mg/kg/day. This finding was considered to be related to the test item and a sign of discomfort but not an adverse effect.A test item treatment-related effect was considered to be unlikely for the few other clinical signs as they are commonly observed in this species and strain or observed with no dose-level relationship.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths in the control, 100, 300 and 1000 mg/kg/day groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males: When compared with controls, there were no effects on mean body weight at all dose-levels and on mean body weight change at 100 mg/kg/day. In the 300 mg/kg/day, males had a low mean body weight gain on study Days 8 to 15 (+28 g vs. +36 g in controls, p<0.05) which was considered to be fortuitous based on absence of effect at the high dose-level during the same treatment period. In the 1000 mg/kg/day group, males had a low mean body weight gain from study Days 1 to 29 (down to +36 g vs. +50 g in controls on Days 1 to 8, p<0.01). On initiation of the mating period (study Days 15 to 22), 2/10 males lost weight (-7 and -8 g males F21827 and F21830, respectively). Then body weight gain returned toward control values on study Days 22 to 29. Taking into account the amplitudes of the differences when compared with controls and the return toward controls values, this finding was considered to be test item treatment-related but non-adverse.
In females: There were no effects on mean body weights and mean body weight changes during the pre-mating, gestation and lactation periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no adverse effects on mean food consumption associated with test item treatment.
At 1000 mg/kg/day and when compared with controls, males and females had statistically significant higher mean food consumption (+10% in males on study Days 8 to 15, p<0.05 and up to +16% in females on Days 7 to 14 p.c., p<0.01).

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

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination of Kidneys:
Only the kidneys with gross findings were examined. Numerous cytoplasmic hyaline droplets were observed in many renal tubules from males treated at 300 or 1000 mg/kg/day with Thesaron. This was associated with pelvis dilation and tubular basophilia in some animals. Immunohistochemistry confirmed that these hyaline droplets were consistent with alpha-2-microglobulin protein. These findings are typical of hydrocarbon induced nephropathy observed in the renal tubules exclusively found in adult male rats (Hildebrand et al., 1997) and were considered to be adverse in this animal species (based on pelvis dilation and tubular basophilia). They are not considered relevant to humans.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no indications of any Thesaron-related changes in the ovaries in treated rats.
The numbers of corpora lutea, of growing follicles and atretic follicles were similar in treated and control females.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

There were no indications of any Thesaron-related changes in the epididymides/testes in treated rats.
There were no signs of degeneration in germ cells or interstitial cells in the testes.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item treatment-related effects on time taken to mate and, on mating, fertility and gestation indexes.In the 300 mg/kg/day, one female (F21891) did not mate and one pregnant female (F21899) did not deliver.

Details on results (P0)

No other information available

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on the renal changes in males at 300 mg/kg/day (these findings are typical of hydrocarbon induced nephropathy observed in the renal tubules exclusively found in adult male rats which are considered not relevant to humans).

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: The No Observed Adverse Effect Level (NOAEL) for female toxicity was considered to be 1000 mg/kg/day based on the absence of adverse effects in females at this dose level.

3

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In all groups, there were no morphological abnormalities at external examination of the pups.Dam F21892 from the 300 mg/kg/day group had a pup with clinical signs on Day 1 p.p. which was cannibalized on Day 2 p.p..Dam F21901 from the 1000 mg/kg/day group had 2 pups with clinical signs, one being found dead on Day 4 p.p..

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no test item treatment-related effects on the distribution of found dead and/or cannibalized pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, there was a tendency towards a decrease in mean pup body weight and mean body weight changes. This finding was considered to be test item treatment-related but of minor toxicological significance as the difference was of low magnitude and remained within the range of 2009-2012 Historical Control Data (6.5-18.0 g and 6.4-17.0 g in male and female pups, respectively).

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:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on live birth, viability and lactation indexes.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects on reproductive/developmental toxicity at highest dose tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Clinical signs F0

Sex	Male
Dose-level (mg/kg/day)	0	100	300	1000
Ptyalism		2 (from study Day 16 up to 18)	10 (from study Day 9 up to 31)	10 (from study Day 6 up to 31)
Reflux at dosing			1 (study Day 7)
Dacryorrhea		1 (study Day 11)
Chromodacryorrhea			1 (study Days 17 to 19)
Cutaneous lesion		1 (study Days 15 to 31)	1 (study Days 18 to 31)
Sex	Female
Dose-level (mg/kg/day)	0	100	300	1000
Tremors	1 (P) (study Days 6 to 7)
Ptyalism		1 (G and L) (study Days 12 to 21 p.c. and Day 0 to 1 p.p.)	10 (P) (from study Day 9 up to 51 (a)) 9 (G) (from Day 0 up to 21 p.c.) 8 (L) (study Days 0 to 4 p.p.)	10 (P, G and L) (from study Day 6 up to 18, Days 0 to 21 p.c. and Days 0 to 4 p.p.)
Reflux at dosing			1 (P) (study Day 17)	1 (P) (study Day 17) 2 (G) (from Day 4 up to 8 p.c.)
Chromodacryorrhea	1 (P and G) (study Days 15 to 17 and Days 0 to 11 p.c.)
Chromorhinorrhea	1 (G) (study Days 12 to 17 p.c.)
Cutaneous lesion	2 (G) (from Day 0 to 20 p.c.)	1 (P and G) (study Days 15 to 18, Days 19 to 21 p.c.)	1 (G) (study Days 2 to 20 p.c.)	1 (P and G) (study Day 18 and Days 0 to 18 p.c.)

P: premating and mating periods, G: gestation period, L: lactation period.

p.c.:post-coitum, p.p.:post-partum.

(a): female F21891 did not mate.

Body weight and body weight change (mean) F0

Sex	Male				Female
Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pre-mating
Day 1	413	417 (+1)	411 (0)	416 (+1)	253	252 (0)	250 (-1)	256 (+1)
Day 8	464	464 (0)	456 (-2)	453 (-2)	272	268 (-1)	267 (-2)	273 (0)
Day 15	500	496 (-1)	484 (-3)	483 (-3)	286	280 (-2)	282 (-1)	286 (0)
Day 22	512	506 (-1)	497 (-3)	488 (-5)	/	/	/	/
Day 29	536	531 (-1)	519 (-3)	510 (-5)	/	/	/	/
Days 1 to 8	+50	+47	+45	+36**	+19	+15	+17	+18
Days 8 to 15	+36	+33	+28*	+31	+14	+12	+15	+13
Days 15 to 22	+11	+10	+13	+5	/	/	/	/
Days 22 to 29	+25	+25	+22	+22	/	/	/	/
Days 1 to 15	+87	+80	+73	+67**	+33	+28	+32	+31
Days 1 to 29	+123	+115	+107	+94*	/	/	/	/
Gestation
Day 0p.c.	/	/	/	/	291	285 (-2)	286 (-2)	293 (+1)
Day 7p.c.	/	/	/	/	325	324 (0)	321 (-1)	320 (-2)
Day 14p.c.	/	/	/	/	368	365 (-1)	361 (-2)	364 (-1)
Day 20p.c.	/	/	/	/	459	459 (0)	447 (-3)	455 (-1)
Days 0 - 20p.c.	/	/	/	/	+168	+174	+161	+162
Lactation
Day 1p.p.	/	/	/	/	359	358 (0)	347 (-3)	349 (-3)
Day 5p.p.	/	/	/	/	372	378 (+2)	365 (-2)	369 (-1)
Days 1 to 5p.p.	/	/	/	/	+13	+20	+18	+19

p.c.:post-coitum, p.p.:post-partum,/: not applicable.

( ): in brackets percentage of changevs.controls.

Statistical significance: *: p<0.05, **: p<0.01

Mean food consumption (g/animal/day)F0

Sex	Male				Female
Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pre-mating
Days 1 to 8	30	31 (+3)	30 (0)	30 (0)	15	18 (+20)	18 (+20)	18 (+20)
Days 8 to 15	31	32 (+3)	32 (+3)	34* (+10)	20	19 (-5)	20 (0)	21* (+5)
Gestation
Days 0 to 7p.c.	/	/	/	/	23	23 (0)	23 (0)	25 (+9)
Days 7 to 14p.c.	/	/	/	/	25	26 (+4)	27 (+8)	29** (+16)
Days 14 to 20p.c.	/	/	/	/	30	32 (+7)	32 (+7)	34** (+13)
Lactation
Days 1 to 5p.p.	/	/	/	/	46	45 (-2)	44 (-4)	47 (+2)

p.c.:post-coitum, p.p.:post-partum,/: not applicable.

( ): in brackets percentage of changevs.controls.

Statistical significance: *: p<0.05, **: p<0.01.

Summary of mating and fertility dataF0

Dose-level (mg/kg/day)	0	100	300	1000
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	10 + 10
Number of males mated	10	10	10	10
Number of females mated	10	10	9	10
Mean number of days taken to mate	2.1	3.7	3.1	3.2
Number of pregnant females	10	10	9	10
Mating index (%)	100	100	90	100
Fertility index (%)	100	100	100	100
Gestation index (%)	100	100	88.9	100

 

Summary of delivery and litterF0

Dose-level (mg/kg/day)	0	100	300	1000
Number of pregnant females	10	10	9	10
Number of females which delivered	10	10	8	10
Mean duration of gestation (days)	21.9	22.0	22.0	22.0
Mean number ofcorpora lutea	15.8	15.6	16.4	16.4
Mean number of implantations	15.0	15.2	15.0	15.6
Mean pre-implantation loss (%)	4.9	2.3	8.1	3.7
Mean number of pups delivered	13.9	13.4	12.9	14.2
Mean post-implantation loss (%)^a	6.9	12.5	13.7	8.0
Viability index on Day 4p.p.(%)	98.6	98.5	99.0	98.6

 

Mortality F1

Dose-level (mg/kg/day)	0	100	300	1000
Number of pups delivered	139	134	103	142
Number of pups found dead	1	2	0	1
Number of pups cannibalized	3	0	1	2

 

Clinical signs and gross external examination F1

Dose-level (mg/kg/day)	0	100	300	1000
Number of pup examined	135	132	102	139
Few milk in the stomach				1
Generalized pallor				1
Cold to the touch				1
Dehydration			1
Hematoma (abdomen)			1
Number of pups with finding(s)	0	0	1	2

 

Pup viability F1

Dose-level (mg/kg/day)	0	100	300	1000
Live Birth Index (%)	100	100	100	100
Pups dying, missing and/or cannibalized (Days 1-4p.p., %)	1.4	1.5	1.0	1.4
Viability Index (Day 4p.p., %)	98.6	98.5	99.0	98.6
Lactation Index (Day 5p.p., %)	98.5	100.0	100.0	99.3

 

Pup body weight F1

Sex	Male				Female
Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
. Day 1p.p.	8.1	8.3 (+2)	8.0 (-1)	7.7 (-5)	7.7	7.9 (+3)	7.7 (0)	7.2 (-6)
. Day 5p.p.	13.5	13.5 (0)	13.4 (-1)	12.3 (-9)	12.9	13.1 (+2)	13.0 (+1)	11.5 (-11)
. Days 1 to 5p.p.	+5.4	+5.2	+5.4	+4.6	+5.2	+5.1	+5.3	+4.3

 

Applicant's summary and conclusion

Conclusions:

The test material was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and (for females) throughout gestation and until Day 4 post partum, at dose-levels of 100, 300 or 1000 mg/kg/day.At the dose-level of 1000 mg/kg/day (highest dose tested), ET-344 SP CAS: 22471-55-2 was not associated with any signs of reproductive or developmental toxicity.
Based on the experimental conditions of this study:
• the No Observed Adverse Effect Level (NOAEL) for male toxicity was considered to be 100 mg/kg/day based on the renal changes from 300 mg/kg/day (these findings are typical of hydrocarbon induced nephropathy observed in the renal tubules exclusively found in adult male rats which are considered not relevant to humans),
• the No Observed Adverse Effect Level (NOAEL) for female toxicity was considered to be 1000 mg/kg/day based on the absence in females at this dose level,
• the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 1000 mg/kg/day based on the absence of effects on mating and fertility at this dose level,
• the NOAEL for toxic effects on progeny was considered to be 1000 mg/kg/day based on the absence of adverse finding on pups at this dose-level.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, Thesaron, following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until Day 4 post partum (p.p.).

The study provided initial information on possible effects on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Three groups of ten male and ten female Sprague-Dawley rats received the test item, Thesaron (batch No. 5H0001), daily, by oral administration (gavage), before mating, during mating and, for the females, throughout gestation until Day 4p.p., at dose‑levels of 100, 300 or 1000 mg/kg/day. An additional group of ten males and ten females received the vehicle control, arachis oil, under the same experimental conditions. The dosing volume was 2 mL/kg/day.

The study was in accordance with OECD 421 Reproduction/Developmental Toxicity Screening Test Adopted by the Council on 27th July 1995 and conducted in a GLP accredited laboratory.

The test item concentrations in the administered dose formulations analyzed in Weeks 01, 04 and 07 remained within an acceptable range of variations (-1.6% to +4.1%) when compared to the nominal values (± 10% of the nominal concentrations). No test item was observed in the control dose formulation.

Mortality: there were no unscheduled deaths.

Clinical signs: ptyalism (hypersalivation) was recorded in males receiving 100 mg/kg/day (2/10 males) and in nearly all males and females dosed from 300 mg/kg/day. This finding was considered to be related to the test item and a sign of discomfort but not an adverse effect.

Body weight, body weight change and food consumption: there were no adverse effects.

Mating and fertility data: there were no treatment-related effects on mating and fertility data.

All animals mated within a comparable mean number of days.

Delivery data: there were no toxicologically significant findings.

Pup mortality: there were no treatment-related effects on the distribution of found dead and/or cannibalized pups.

Pup clinical signs and gross external examination: there were no test item treatment-related findings at pup examinations.

Pup viability: there were no toxicologically significant effects on live birth, viability and lactation indexes.

Pup body weight and body weight gain: there was a tendency towards a decrease in mean body weight and mean body weight changes in pups at 1000 mg/kg/day. This finding was considered to be test item treatment-related but non-adverse.

Pup sex ratio: there were no treatment-related effects on sex-ratios (% of male pups).

Pathology: there were no test item treatment-related organ weights changes. At macroscopic examination, gross findings were observed in the kidney and consisted of tan discoloration (1/10 and 3/10 male rats respectively treated at 300 or 1000 mg/kg/day) and dilation of pelvis (2/10 and 3/10 male rats respectively treated at 300 or 1000 mg/kg/day).

Hyaline droplets were observed in these kidneys from males treated at 300 mg/kg/day or 1000 mg/kg/day in association with tubular basophilia and pelvis dilatation. These droplets were consistent with alpha 2 microglobulin protein as demonstrated by immunohistochemistry. These treatment related changes correlated with gross observations seen in kidneys and were considered to be adverse in the rat (not considered relevant to humans).

Under the conditions of this study, Thesaron does not meet the classification criteria under Regulation 1272/2008.