reaction products of 2-Propenoic acid (2 moles) and Neopentylglycol hydroxypivalate (1 mole)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined Repeated dose toxicity study with the Reproduction/Developmental toxicity study is available on the registered substance. Based on the dose-range finding study, a maximal dose of 600 mg/kg/day was chosen for the main OECD 422 study. No adverse toxicity was observed in parental animal, on reproduction parameters and on foetal development at 600 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2017 - 15 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age: at the beginning of the treatment period, the males were approximately 10 weeks old and the females were approximately 11 weeks old
- Mean body weight: at the beginning of the treatment period, the males had a mean body weight of 425 g (range: 392 g to 449 g) and the females had a mean body weight of 262 g (range: 226 g to 296 g).
- Housing: F0 animals were individually housed, except during mating (males + females) and lactation (females + pups), in polycarbonate cages (Tecniplast 2154, 48 x 26.5 x 21 cm, 940 cm2) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Individual housing of F0 animals was chosen as group housing could adversely affect gestation and lactation, and to avoid aggressive behavior between males 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.
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the males were acclimated to the study conditions for 7 days before treatment and the females were acclimated to the study conditions for 5 days before the beginning of estrous cycle monitoring during the pre-treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 20 June 2017 to 15 August 2017.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING FORMULATIONS: solution in the vehicle
- Concentration in vehicle: 20, 60 and 120 mg/mL
- Amount of vehicle: 5 mL/kg/day.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred
- Proof of pregnancy: vaginal plug or sperm in the morning vaginal lavage referred to as day 0 post-coitum
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: Gas Chromatography with FID detection (GC-FID)
Test item concentrations: remained within an acceptable variation range (-3.4% to +5.4%) when compared to the nominal values.
Homogeneity: diluted analytical samples based on the marker peak prepared from 1.6 mg/mL and 240 mg/mL dose formulations in corn oil were found to be stable for 6 days when they were stored at room temperature and protected from light.

Duration of treatment / exposure:

In the males:
- 2 weeks before mating,
- during the mating period (until 1 week),
- until euthanasia (4 weeks in total) (until study Day 28),

In the females:
- 2 weeks before mating,
- during the mating period (until 1 week),
- during gestation,
- during lactation until Day 13 post-partum inclusive,
- until euthanasia for females which had no delivery.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected in agreement with the Sponsor, on the basis of the results of a previous study performed in the same species, in which the test item was administered daily by gavage to five males and five females at dose levels of 100, 300 or 1000 mg/kg/day for 2 weeks. In this study, the test item was clinically well tolerated at all dose levels. At necropsy, slightly higher liver weights were recorded in females treated at 1000 mg/kg/day. White discolorations in the forestomach was observed in both sexes treated at 1000 mg/kg/day associated with brown discolorations and/or yellow deposit in some males. These latter lesions correlated microscopically with erosion/ulcer and are suggestive of local intolerance to the test item. The erosion/ulcer being considered as adverse, the dose of 1000 mg/kg/day was considered to be higher than the Maximal Tolerable Dose (MTD).
Therefore 600 mg/kg/day was selected as the high-dose level in this study.
The low-dose and mid-dose were selected using a ratio representing approximately a 2 or 3 fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: stratification procedure.

Positive control:

no (not required)

Parental animals: Observations and examinations:

MORTALITY/MORBIDITY:
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then at least once a week until the end of the study.

CLINICAL SIGNS:
- Time schedule: from arrival, each animal was observed once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time of day, for the recording of clinical signs.

BODY WEIGHT:
- Time schedule: the body weight of each male was recorded on the first day of treatment (Day 1), then once a week until euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated and on Days 0, 7, 14 and 20 post-coitum (p.c.) (and on the day of euthanasia for females which did not deliver), and on Days 1, 4, 8 and 13 p.p.
All females prematurely sacrificed were weighed before sacrifice.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each male was measured once a week from the first day of treatment until the start of the mating period.
The quantity of food consumed by each female was measured once a week from the first day of treatment until the start of the mating period, during gestation for the intervals Days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval Days 1-4, 4-8 and 8-13 p.p.
During the mating period, food consumption was not measured for males or females.

HAEMATOLOGY:
- Time schedule: the parameters were determined from the first five males and lactating females from each group euthanized as scheduled, on the day of euthanasia.

CLINICAL CHEMISTRY:
- Time schedule: the parameters were determined from the first five males and lactating females from each group euthanized as scheduled, on the day of euthanasia.

THYROID HORMONES:
- Time schedule:
- at termination on Day 4 p.p. from 2 pups/litter culled,
- at termination on Day 13 p.p. from 2 pups/litter,
- at termination on Day 14 p.p. from all F0 females,
- at termination from all F0 males.

REPRODUCTION (apart from indices):
- Pre-coital time and duration of gestation were recorded

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning (between 8 and 10 a.m.):
- during the 2 weeks of the pre-treatment period (including the 2 supplementary females per group),
- from the beginning of the treatment period during the pre-mating and mating periods, until the females were mated,
- on Day 14 p.p. before euthanasia, to allow correlation with reproductive organs histopathology.

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- weighing and microscopic examination: see Tissue Procedure Table below
- special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS:
On Day 4 p.p., the size of each litter was adjusted by randomly culling extra pups to obtain as nearly as possible four males and four females per litter. No cross-fostering was performed.

PARAMETERS EXAMINED:
- number and sex of pups,
- number of live, dead and cannibalized pups,
- presence of gross anomalies, weight gain, clinical signs,
- anogenital distance (AGD),
- presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination.
- males: after the end of the mating period (4 weeks of treatment in total),
- females: on Day 14 p.p.

The following F0 females were euthanized by the same way without overnight fasting:
- females which did not deliver: on Day 25 or 26 p.c. (after a body weight recording to check for a possible un-noticed delivery),
- female with total litter loss.

ORGAN WEIGHTS:
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males, or on Day 14 p.p. for lactating females) was recorded before euthanasia. For these animals, the organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection, or after fixation for thyroids with parathyroids.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all F0 animals including the prematurely sacrificed females.

PRESERVATION OF TISSUES:
The tissues of F0 animals specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the eyes with optic nerves and Harderian glands, and the testes and epididymides, which were fixed in Modified Davidson's fixative).
Thyroids with parathyroids of the selected pup/sex/litter euthanized on Day 13 p.p. were preserved in 10% buffered formalin.

PREPARATION OF HISTOLOGICAL SLIDES:
All tissues required for microscopic examination were trimmed based on the RITA guidelines, when applicable (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).
This tissue processing was performed at CiToxLAB France.

MICROSCOPIC EXAMINATION:
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table from the first five euthanized as scheduled males and lactating females of the control and high-dose groups (groups 1 and 4),
- all macroscopic lesions of all groups.
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Postmortem examinations (offspring):

SACRIFICE:
Pups were euthanized by an intraperitoneal injection of sodium pentobarbital, followed by exsanguination when the thyroids were sampled:
- pups not selected on Day 4 p.p.: on Day 4 p.p.,
- surviving pups: on Day 13 p.p.

GROSS NECROPSY: Pups euthanized on Day 13 p.p. were submitted to a detailed external examination (including orifices and buccal cavity) after euthanasia. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination, or after sampling of thyroids with parathyroids for the selected pups.

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

Statistics:

Body weight, food consumption and reproductive data
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Hematology, blood biochemistry, hormones, anogenital distance and post-implantation loss
CITOX software was used to perform the statistical analyses.

Organ weight
PATHDATA software was used to perform the statistical analysis of organ weight data.

Reproductive indices:

Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
Post-implantation loss = 100 * (Number of implantation sites - Number of live pups) / Number of implantations
Mating index = 100 * (Number of mated animals / Number of paired animals)
Fertility index = 100 * (Number of pregnant female partners / Number of mated pairs)
Gestation index = 100 * (Number of females with live born pups / Number of pregnant females)

Offspring viability indices:

Live birth index = 100 * (Number of live born pups / Number of delivered pups)
Viability index on Day 4 p.p. = 100 * (Number of surviving pups on Day 4 p.p. / Number of live born pups)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Ptyalism was observed with a dose-related incidence in test item-treated males and females during the premating, pregnancy and lactation periods. This sign, commonly noted when a test item is administered by gavage, was not considered to represent an adverse effect.
The other clinical signs recorded during the study, i.e. cutaneous observations on various parts of the body (areas of hair loss, cutaneous lesions, scabs), reflux at dosing, chromodacryorrhea, dacryorrhea, round back and/or emaciated appaearance, were considered to be unrelated to the test item treatment as they were present both in control and test item-treated animals, and/or were reported sporadically in only a few animals and/or were attributed to the gavage procedure.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

See table 1.
Males
No unscheduled death occurred in males at any dose level during the study.

Females
Gestation period:
At 600 mg/kg/day, one female was prematurely sacrificed on Day 24 p.c. due to difficulties to deliver. Signs of poor clinical condition (piloerection and round back) were observed on the day of sacrifice, accompanied by reddish vaginal discharge and soiled urogenital area. Ptyalism was noted from Day 7. Three alive, five dead and one cannibalized pups were found in the bedding.
At necropsy, the thymus and spleen were reduced in size and the adrenals were enlarged and had a diffuse tan discoloration. In addition, a diffuse yellow discoloration of the mammary gland area was noticed. In the absence of microscopic examination, these observations were interpreted to be non specific, possibly related to stress. Six dead fetuses and placentae were found in the uterine horns and one placenta in the lumen. A relationship to administration of the test item was considered as dubious.

Lactation period:
At 300 mg/kg/day, one female was prematurely sacrificed on Day 2 p.p. due to the death of its litter (10 pups at birth). No clinical signs were noted prior to sacrifice. At necropsy, tan discoloration was noted on liver and adrenal glands. A relationship to administration of the test item was considered as dubious.

Due to no delivery, the following females were sacrificed on Day 25 or 26 p.c. without clinical signs prior to death:
- At 100 mg/kg/day: one female,
- At 300 mg/kg/day: two females.
At necropsy, these females were found to be non-pregnant.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

See table 2.
No effects were observed on mean body weights or mean body weight changes in males or females at any dose level.
The few statistically significant differences noted between control- and test item-treated animals in mean body weight change during the study (i.e. in males on Days 15-22 at 300 mg/kg/day, on Days 22-29 at 600 mg/kg/day, on Days 15-29 at 100 or 300 mg/kg/day and in females on Days 14-20 of the lactation period at 100 mg/kg/day) were not attributed to the test item treatment as they were isolated and of small magnitude and/or not dose-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

See table 3.
No effects on mean food consumption were observed in males and females at any dose level.
The only statistically significant difference between control and test item-treated animals, namely higher mean food consumption in females given 600 mg/kg/day on Days 0-7 p.c., was isolated and considered to be of no toxicological importance.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematology parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.
The only statistically significant differences from controls (i.e. higher large unstained cells in males at 600 mg/kg/day, lower erythrocyte counts in females at 300 or 600 mg/kg/day and lower hemoglobin concentration in females at 600 mg/kg/day) were considered to be of no toxicological significance as they were of small magnitude and/or not dose-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The blood biochemistry parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.
The only statistically significant differences from controls (i.e. higher glucose and urea levels in males at 100 or 600 mg/kg/day, respectively) were considered to be of no toxicological significance as they were of small magnitude and/or not dose-related.

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):

All examined high-dose males (5/5 rats) had a minimal or slight increase in renal hyaline droplets (versus 1/5 controls only affected, minimal severity). Hyaline droplets in renal tubules are caused by accumulation of alpha 2u-globulin. This is specific to male rats and irrelevant to human risk assessment. It was not considered as adverse in the absence of other associated changes.
All other changes were spontaneous in nature and bore no relationship to the test item treatment. In particular, there were no test item treatment-related changes in the thyroids or in any of the reproductive organs.

See table 6.
Vaginal mucification is still mostly observed as expected for lactating females (pups starting to eat solid food from Day 14 p.p.). Similarly, episodes of proestrus and metestrus stages are also expected to be observed during this transitory period (PND = 14).

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones
No effects on T4 or TSH levels were observed in pups sacrificed on Day 13 p.p. or in the adult males sacrificed at the end of the treatment period at any dose level.
The only statistically significant difference from controls, i.e. lower mean TSH level in males given 300 mg/kg/day, was not attributed to the test item treatment as it was reported without any dose relationship.

Hematology
The hematology parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.
The only statistically significant differences from controls (i.e. higher large unstained cells in males at 600 mg/kg/day, lower erythrocyte counts in females at 300 or 600 mg/kg/day and lower hemoglobin concentration in females at 600 mg/kg/day) were considered to be of no toxicological significance as they were of small magnitude and/or not dose-related.

Blood biochemistry
The blood biochemistry parameters were considered to be unaffected by the test item treatment in males and females at the end of the treatment period.
The only statistically significant differences from controls (i.e. higher glucose and urea levels in males at 100 or 600 mg/kg/day, respectively) were considered to be of no toxicological significance as they were of small magnitude and/or not dose-related.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were observed on the estrus cycle at any dose level during the treatment period.
The statistically significant lower mean number of estrus days at 300 mg/kg/day (3.7 vs. 4.3 days) was not attributed to the test item treatment as this difference was not dose-related.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

See table 4.
Pairing, mating and fertility
No test item-related effects were observed on the mating or fertility data. All females mated within a similar mean number of days.
All females were pregnant, except one female given 100 mg/kg/day and two females given 300 mg/kg/day, which accounts for the slightly lower mean fertility index recorded at these dose levels. These isolated cases, reported without any dose-relationship, were considered to be incidental and unrelated to the test item treatment.

Delivery data
See table 5.
All pregnant females delivered, except one female given 600 mg/kg/day which was sacrificed due to difficulty to deliver. This isolated occurrence was considered to be unrelated to the test item treatment.

No effects were observed on the number of corpora lutea, pre- and post-implantation losses or pups delivered, or on the duration of gestation at any dose level.

Table 1:

The incidence of prematurely sacrificed females is shown in the table below:

Sex Female
Dose level (mg/kg/day) 0 100 300 600
Number of females 10 10 10 10
Pre-mating/mating 0 0 0 0
Gestation
Difficulties to deliver 0 0 0 1
No delivery (non-pregnant) 0 1 2 0
Lactation 0 0 1 0
Total surviving females at
scheduled sacrifice 10 9 7 9

Table 2:
Mean body weights/Mean body weight changes (g)

Sex Males Females
Dose level (mg/kg/day) 0 100 300 600 0 100 300 600
Pre-mating(males and females) or whole study
Day 1 424 423 427 425 264 262 253 269
Day 8 470 468 477 476 274 270 264 282
Day 15 509 504 514 510 283 281 274 290
Day 22 525 518 523 527 / / / /
Day 29 532 517 523 525 / / / /
Days 1 - 29 108 95 96 101 / / / /
Gestation
Day 0 p.c. / / / / 286 284 272 292
Day 20 p.c. / / / / 438 423 429 450
Day 0-20 p.c. / / / / 151 138 157 159
Lactation
Day 1 p.p. 339 338 333 349
Day 13 p.p. 374 382 369 386
Days 1 - 13 p.p. 35 44 35 37
/: not applicable.

Table 3:
Mean food consumption (g/animal/day)

Sex Males Females
Dose level (mg/kg/day) 0 100 300 600 0 100 300 600
Pre-mating or whole study
Days 1 - 8 33 32 33 33 20 19 17 23
Days 8 - 15 32 32 31 33 19 19 16 23
Gestation
Days 0 - 7 / / / / 20 19 19 26*
Days 7 - 14 / / / / 22 22 22 24
Days 14 - 20 / / / / 28 26 27 28
Lactation
Days 1 - 4 / / / / 37 36 35 36
Days 4 - 8 / / / / 50 50 50 48
Days 8 - 13 / / / / 61 65 65 61
/: not applicable, statistically significance: *: p<0.05.

Table 4:
Pairing, mating and fertility data

Dose level (mg/kg/day) 0 100 300 600
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 10 10
Mean number of days taken to mate 2.5 2.3 3.0 2.9
Number of pregnant females 10 9 8 10
Number of females with live born pup 10 9 8 9
Mating index (female, %) 100 100 100 100
Fertility index (%) 100 90 80 100
M: males; F: females.

Table 5: Delivery and litter data

Dose level (mg/kg/day) 0 100 300 600
Number of pregnant females 10 9 8 10
Number of females which delivered 10 9 8 9
Mean duration of gestation (days) 22.1 22.0 22.1 22.0
Mean number of corpora lutea 15.0 14.4 14.9 15.2
Mean number of implantation sites 14.6 13.9 14.5 15.0
Mean pre-implantation loss (%) 2.7 3.9 2.3 1.4
Mean number of pups delivered 12.6 12.4 12.3 13.2
Mean post-implantation loss (%) 13.2 10.1 15.7 11.7

Table 6: Microscopic examinations

Estrous cycle stage
Group Animal number Findings
1 K25723 Proestrus
K25726 Mucification
K25729 Metestrus
K25731 Mucification
K25732 Metestrus
4 K25757 Mucification
K25758 Mucification
K25760 Mucification
K25761 Mucification
K25763 Mucification

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: findings in the kidneys treatment related but non adverse

Dose descriptor:

NOEL

Effect level:

600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: mating and fertility

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

See table 8.
No test item-related clinical signs and no abnormal behaviour were observed at any dose level.
The few findings recorded during the lactation period were not attributed to the test item treatment as they were reported in isolated animals and/or are routinely observed in rat pups of this strain and age and/or were not dose-related.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

See tables 7 and 9.
No effects were observed on the incidence of pups found dead or cannibalized at any dose level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects were observed on mean body weight or mean body weight change at any dose level during the lactation period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

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:

effects observed, non-treatment-related

Description (incidence and severity):

See table 10.
Pup sex ratio
No significant effects were observed on the sex ratio (percentage of males) at any dose level.
The lower % of males at 100 mg/kg/day was considered to be unrelated to the test item treatment as this difference was reported with no statistical significance and was not noted at the two higher dose levels.

Pup development
See table 11.
Anogenital distance
No test-item related effects on the anogenital distance were observed in males and females at any dose level.
The statistically significant higher mean anogenital distance measured in female pups at 300 mg/kg/day was not attributed to the test item treatment as the difference was not dose-related.

Nipples and areolae numbers
No nipples were noted in any male pups.

Areolae were observed in the following pups on Day 12 p.p.:
- one pup at 300 mg/kg/day (2 areolae),
- five pups at 600 mg/kg/day, from two litters (2, 6 or 8 areolae per pup).

A relationship to the test item treatment could not be excluded. However, as this finding was isolated with low incidence and was no longer observed the day after in two pups at 600 mg/kg/day (observation made just before sacrifice, documented in the raw data), this was considered to be non-adverse.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Table 7:

Incidence of mortality

 

Dose level (mg/kg/day)	0	100	300	600
Number of litter	10	9	8	9
Number of pups found dead	8	1	8	12
Number of pups cannibalized	2	0	4	1
Number of litter affected	3	1	2	3

 

Table 8:

Incidence of clinical signs in lactating pups

 

Dose level (mg/kg/day)	0	100	300	600
Generalized pallor, n (L)	1 (1)	-	-	1 (1)
Alopecia (head), n (L)	1 (1)	-	-	-
Hematoma (head/hindlimb), n (L)	-	2 (2)	-	-
Necrose (tail), n (L)	-	1 (1)	-	-
Swollen abdomen, n (L)	-	-	-	2 (1)
Abnormal color/blackish abdomen, n (L)	-	-	-	3 (2)

n: number of pups, L: number of litter affected.

-: no clinical signs.

Table 9:

Pup viability

 

Dose-level (mg/kg/day)	0	100	300	600
Viability index on Day 4p.p.(%)	92.1	99.1	87.8	89.1
Lactation index on Day 13p.p.(%)	100	100	100	100

 

Table 10:

Sex ratio on Day 0 p.p. (% of males)

 

Dose level (mg/kg/day)	0	100	300	600
Sex ratio on Day 0 p.p. (% of males)	50.8	38.4	54.2	58.0

Table 11:

Anogenital distance corrected for body weight (AGD mm/BW g^1/3)

 

Sex	Males				Females
Dose level (mg/kg/day)	0	100	300	600	0	100	300	600
AGD/BW^1/3	2.57	2.53	2.48	2.53	1.35	1.42	1.57**	1.40

Statistically significance:**: p<0.01.

 

Conclusions:

The test item 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 13 post partum, at dose levels of 100, 300 or 600 mg/kg/day.

Based on the experimental conditions of this study:
- the No Observed Effect Level (NOEL) for parental toxicity was considered to be 600 mg/kg/day in females based on the absence of findings at this dose level; in males, given the non-adverse findings in the kidneys, the No Observed Adverse Effect Level (NOAEL) was considered to be 600 mg/kg/day,
- the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 600 mg/kg/day based on the absence of effects on mating or fertility at this dose level,
- the NOAEL for toxic effects on progeny was considered to be 600 mg/kg/day based on the absence of adverse findings on pups at this dose level.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until Day 13 post-partum (p.p.).

This study provides information:

. on the possible health hazards likely to arise from repeated exposure over a relative limited period of time,

. on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item.

The test item was administered daily by the oral route (gavage) at dose levels of 100, 300 or 600 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of 4 weeks: two weeks before mating, during the mating period (up to one week) until the day before sacrifice. Females were treated for an overall period of 7 to 8 weeks: two weeks before mating, throughout mating (up to one week) and gestation (3 weeks) until Day 13 post-partum (p.p.) inclusive. Another group of 10 males and 10 females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group.

The actual test item concentrationsin the dose formulations were determined in Weeks 1 and 6 using a validated GC-FID analytical method.

Animals were checked daily for clinical signs and mortality. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stage was determined each morning from two weeks before mating until the females had mated and on Day 14 p.p. before euthanasia.

The animals were paired for mating after two weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13 p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4 p.p. by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups not selected on Day 4 p.p. were sacrificed and discarded without further examination.

The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8 and 13 p.p. The physical development of pups was assessed by measuring the anogenital distance on Day 1 p.p. and by counting the number of nipples and areolae in male pups on Day 12 p.p.

Hematology and blood biochemistry investigations were performed on at least five males and females in each group at scheduled sacrifice. Thyroid hormones (TSH and T4) were determined in males at sacrifice and in pups sacrificed on Day 13 p.p.

Males were sacrificed after completion of the mating period. Dams were sacrificed on Day 14 p.p.

A full macroscopic post-mortem examination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups.

Pups were sacrificed on Day 13 p.p. and submitted to a detailed external examination with a particular attention to the external genital organs.

Results

Actual concentrations of the test item in the dose formulations analyzed during the study remained within an acceptable range of variation (-3.4% to +5.4%) when compared to the nominal concentrations.

 

F0 animals:

No test item-related deaths occurred in males or females during the study.

Ptyalism was observed with a dose-related incidence at all dose levels of the test item. This sign, commonly noted when a test item is administered by gavage, was not considered as an adverse effect.

Body weight and food consumption were unaffected by the test item treatment.

The estrous cycle was not impacted by the test item treatment.

The mating, fertility and delivery data were unaffected by the test item treatment.

Hematology and blood biochemistry parameters were not impacted by the test item treatment.

Thyroid hormone analyses did not reveal any disturbance in F0 males at sacrifice.

At pathology examination, no effects on organ weights and no test item-related macroscopic or microscopic findings were observed in females. In male rats, an increase in renal tubular hyaline droplets was observed. This finding is specific to male rats and has no relevance for human risk assessment.

 

Pups:

Observations of the pups from birth to Day 13 p.p. did not show any effects on mortality, viability, clinical signs, body weight evolution, sex ratio or anogenital distance. The only observation consisted of the presence of areolae in one and five pups at 300 and 600 mg/kg/day, respectively. As this finding was isolated with low incidence and was no longer observed the day after in two pups at 600 mg/kg/day, this was considered to be non-adverse.

Thyroid hormone analyses did not reveal any disturbance in pups on Day 13 p.p.

 

Conclusion

The test item 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 13 post-partum, at dose levels of 100, 300 or 600 mg/kg/day.

 

Based on the experimental conditions of this study:

. the No Observed Effect Level (NOEL) for parental toxicity was considered to be 600 mg/kg/day in females based on the absence of findings at this dose level; in males, given the non-adverse findings in the kidneys, the No Observed Adverse Effect Level (NOAEL) was considered to be 600 mg/kg/day,

. the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 600 mg/kg/day based on the absence of effects on mating or fertility at this dose level,

. the NOAEL for toxic effects on progeny was considered to be 600 mg/kg/day based on the absence of adverse findings on pups at this dose level.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is considered to be reliable with a klimish score fo 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A combined Repeated dose toxicity study with the Reproduction/Developmental toxicity study (Haag 2018) = OECD 422

The objective of this study was to evaluate the potential toxic effects of the test item following daily oral administration (gavage) to male and female rats from before mating, during mating and, for the females, throughout gestation until Day 13 post-partum (p.p.).

Three groups of ten male and ten female Sprague-Dawley rats received the test item. The test item was administered daily by the oral route (gavage) at dose levels of 100, 300 or 600 mg/kg/day, under a constant dosage volume of 5 mL/kg/day.

Males were treated for an overall period of 4 weeks: two weeks before mating, during the mating period (up to one week) until the day before sacrifice. Females were treated for an overall period of 7 to 8 weeks: two weeks before mating, throughout mating (up to one week) and gestation (3 weeks) until Day 13post-partum(p.p.) inclusive. Another group of 10 males and 10 females received the vehicle only (corn oil) under the same experimental conditions and acted as a control group.

The actual test item concentrationsin the dose formulations were determined in Weeks 1 and 6 using a validated GC-FID analytical method.

Animals were checked daily for clinical signs and mortality. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. Estrous cycle stage was determined each morning from two weeks before mating until the females had mated and on Day 14p.p.before euthanasia.

The animals were paired for mating after two weeks of treatment and the dams were allowed to litter and rear their progeny until Day 13p.p. The total litter sizes and numbers of pups of each sex were recorded after birth. The size of each litter was adjusted on Day 4p.p.by culling extra pups to obtain as nearly as possible four males and four females per litter. Pups not selected on Day 4p.p.were sacrificed and discarded without further examination.

The pups were observed daily for clinical signs or abnormal behaviour and were weighed on Days 1, 4, 8 and 13p.p.The physical development of pups was assessed by measuring the anogenital distance on Day 1p.p.and by counting the number of nipples and areolae in male pups on Day 12p.p.

Hematology and blood biochemistry investigations were performed on at least five males and females in each group at scheduled sacrifice. Thyroid hormones (TSH and T4) were determined in males at sacrifice and in pups sacrificed on Day 13p.p.

Males were sacrificed after completion of the mating period. Dams were sacrificed on Day 14p.p.

A full macroscopicpost-mortemexamination was performed, with a particular attention to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from five males and lactating females in the control and high-dose groups.

Pups were sacrificed on Day 13p.p.and submitted to a detailed external examination with a particular attention to the external genital organs.

No test item-related deaths occurred in F0 males or females during the study. Ptyalism was observed with a dose-related incidence at all dose levels of the test item. This sign, commonly noted when a test item is administered by gavage, was not considered as an adverse effect. Body weight and food consumption were unaffected by the test item treatment. The estrous cycle was not impacted by the test item treatment. The mating, fertility and delivery data were unaffected by the test item treatment.

Hematology and blood biochemistry parameters were not impacted by the test item treatment. Thyroid hormone analyses did not reveal any disturbance in F0 males at sacrifice. At pathology examination, no effects on organ weights and no test item-related macroscopic or microscopic findings were observed in females. In male rats, an increase in renal tubular hyaline droplets was observed. This finding is specific to male rats and has no relevance for human risk assessment.

 

Observations of the pups from birth to Day 13p.p.did not show any effects on mortality, viability, clinical signs, body weight evolution, sex ratio or anogenital distance. The only observation consisted of the presence of areolae in one and five pups at 300 and 600 mg/kg/day, respectively. As this finding was isolated with low incidence and was no longer observed the day after in two pups at 600 mg/kg/day, this was considered to be non-adverse.

Thyroid hormone analyses did not reveal any disturbance in pups on Day 13p.p.

 

Based on the experimental conditions of this study:

. the No Observed Effect Level (NOEL) for parental toxicity was considered to be 600 mg/kg/day in females based on the absence of findings at this dose level; in males, given the non-adverse findings in the kidneys, the No Observed Adverse Effect Level (NOAEL) was considered to be 600 mg/kg/day,

. the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 600 mg/kg/day based on the absence of effects on mating or fertility at this dose level,

. the NOAEL for toxic effects on progeny was considered to be 600 mg/kg/day based on the absence of adverse findings on pups at this dose level.

Effects on developmental toxicity

Description of key information

Based on the OECD 422 study, the NOAEL for toxic effects on progeny was considered to be 600 mg/kg/day based on the absence of adverse findings on pups at this dose level.

No specific study on fetal development was performed.

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

Based on the available data, no classification for reproduction toxicity is required for the registered substance according to the Regulation EC N°1272/2008.