Alkanol reaction products with bis(aminoalkyl(C=1~6))carbomonocycle, alkoxy(C=3~8)alkanol(C=2~7) and 2,4-TDI

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April 2016 - 07 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

28 July 2015

Deviations:

yes

Remarks:

: see chapter 'any other information on materials and methods'

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Name of test material (as cited in study report): 20231250
- Physical state: white powder
- Storage condition of test material: room temperature (ca. 20°C) in the dark

Test animals

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 431 g (range: 393 g to 455 g) and the females had a mean body weight of 285 g (range: 250 g to 310 g).
- Housing: F0 animals were individually housed, except during mating and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids containing autoclaved sawdust.
- 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 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 10 May 2016 to 07 July 2016.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle.
The test item dose formulations were prepared daily and were delivered to the study room at room temperature and protected from light.
This preparation process was validated for a range of concentrations covering the lowest and highest concentrations used in this study.
.

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

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 as day 0 post-coitum
- After successful mating each pregnant female was caged individually again

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection (HPLC-UV)
Test item concentrations: Actual concentrations of the test item in the intermediate and high dose formulations analyzed on Day 1 were outside the acceptable range compared to the nominal concentrations (- 62.5 and +18.0%, respectively). After adjustement of the dose formulations preparation procedure on Day 3, the test item concentrations were re-checked in week 1and then once in weeks 2, 3 and 7. The test item concentrations remained within an acceptable range of variation (± 15%) throughout the study when compared to the nominal values (-12.2% to +8.6%) .
Homogeneity and stability of the dosage forms: homogeneity of the test substance in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 20 mg/mL and 200 mg/mL. Stability was not assessed as the test item was an UVCB, dose formulations were prepared daily.

Duration of treatment / exposure:

- in the males:
2 weeks plus 5 days (1) before mating,
during the mating period (1 week),
until euthanasia (at least 4 weeks in total, i.e. 31 days according to the necropsy schedule),

- in the females:
2 weeks plus 5 days (1) before mating,
during the mating period (1 week),
during gestation,
during lactation until Day 13 p.p. inclusive,
until euthanasia for females with no delivery.

(1) The pre-mating period was extended to 5 additional days further to the test item concentrations outside the acceptable range on Day 1.

Frequency of treatment:

7 days/week

Details on study schedule:

- No F1 parents (only one generation mated)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 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 and did not induce findings at macroscopic post-mortem examination.
Based on these results, the dose-levels of 100, 300 and 1000 mg/kg/day were selected.

- Rationale for animal assignment: stratified procedure.

Positive control:

no

Examinations

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.

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 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 at least once a week until euthanasia including on the day before euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mating, on Days 0, 7, 14 and 20 p.c. (post-coitum) and on Days 1, 4, 8 and 13 p.p.

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.

REPRODUCTION (apart from indices):
- The pre-coital time was calculated for each female.

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Parameters examined in males of parental generation:
- testis weight (all groups) + microscopic evaluation (control and high-dose groups)
- epididymis weight (all groups) + microscopic evaluation (control and high-dose groups)
- microscopic evaluation of stages of the spermatogenic cycle and testicular interstitial cells (control and high-dose groups)

Litter observations:

STANDARDISATION OF LITTERS: Yes

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

GROSS EXAMINATION OF DEAD AND SURVIVING PUPS:
- external and internal abnormalities

HORMONES:
- The levels of the thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) were determined for pups sampled on Day 14 p.p ( 2 pups/litter with blood pooled by litter)

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: all surviving animals after the end of the mating period
- Female animals: all surviving animals = Day 14 post-partum or, for females which had not delivered yet, on Day 25 post-coitum (after a body weight recording to check for a possible unnoticed delivery).

GROSS NECROPSY
A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all F0 animals including females that died during the study or were euthanized prematurely. Special attention was paid to the reproductive organs.
For females, the numbers of implantation sites in each uterine horn was counted. For females failing to produce a viable litter, the number of uterine implantation sites was re-checked after staining with ammonium sulphide (modification of the Salewski staining technique).

HISTOPATHOLOGY
A microscopic examination was performed on:
- Epididymides, testes and ovaries in animals of the control- and high-dose groups sacrificed at the end of the treatment period,
- all macroscopic lesions of all the animals sacrificed on completion of the treatment period,
Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

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 females) was recorded before euthanasia and liver, kidney,epididymides (Land R), prostate, testes (L and R), seminla vesicles and ovaries (Land R) were weighted (wet) as soon as possible after dissection.The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
- The body weight of 1 pup/sex/litter euthanized on Day 14 p.p. was recorded before euthanasia.

Postmortem examinations (offspring):

SACRIFICE: Pups were anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination (or by decapitation on Day 4 p.p. when blood was sampled):
- pups not selected on Day 4 p.p.,
- surviving pups: on Day 14 p.p.

GROSS NECROPSY: Pups not selected on Day 4 p.p. were euthanized and discarded without further examination.
Pups sacrificed on Day 14 p.p. were submitted to a detailed external examination (including orifices and buccal cavity). This examination was done in addition to the daily observation for clinical signs, abnormal behavior and external abnormalities. Particular attention was paid to the external genital organs. Then, they were discarded without any further examination.

HISTOPATHOLOGY: No

ORGAN WEIGTHS: No

PRESERVATION OF TISSUES: Thyroids with parathyroids of one pup/sex/litter euthanized on Day 14 p.p. were preserved in 10% buffered formalin.

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).

Hormones, anogenital distance and post-implantation loss
Citox software was used to perform the statistical analysis of these data.

Organ weight
PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS (PARENTAL ANIMALS)
No test item-related clinical signs were observed during the pre-mating, pregnancy or lactation period in males or females at any dose-level.
All clinical signs recorded during the study, i.e. cutaneous observations on various parts of the body (areas of hair loss, cutaneous lesions, scabs), ptyalism, reflux at dosing, chromodacryorrhea and reddish vaginal discharge, 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 (PARENTAL ANIMALS) (See 7.8.1/2 in chapter 'any other information on results incl. tables')
There were no unscheduled deaths in males during the study while 7 females were sacrificed prematurely:

-> During the premating period, one female (G21146) given 1000 mg/kg/day was found dead on Day 22. Signs of poor clinical condition (round back, piloerection, pallor of extremities, coldness to the touch, dyspnea and abdominal breathing) and prolonged diestrus were observed prior to death. Necropsy findings (white discoloration on the external surface of the esophagus and lungs adherent to the pericardium) correlated microscopically with inflammation of the epicardium/pericardium/pleura and of tissues adjacent to the esophagus.

-> During the gestation period, four females were prematurely sacrificed due to difficulties in pup delivery: 1 out of 10 at 1000 mg/kg/day and 3 out of 10 at 300 mg/kg/day. At necropsy, these females were found to be pregnant. The female dosed at 1000 mg/kg/day (G21147) was sacrificed on Day 23 p.c. Signs of poor clinical condition (pallor of extremities/eyes, coldness to the touch and hypoactivity) were observed on the day of sacrifice, accompanied by reddish vaginal discharge and the presence of blood in the bedding.Necropsy findings (thymus adherent to the heart) correlated microscopically with inflammation in the thymus and of the epicardium/pericardium, associated with the presence of a grayish amorphous material in these inflammatory areas; it was considered likely that this material was test item. At 300 mg/kg/day, females G21131, G21132 and G21134 were sacrificed on Days 23, 24 and 23 p.c., respectively. Signs of poor clinical condition (piloerection, pallor of extremities/eyes, coldness to the touch, hypoactivity, half-closed eyes and/or abdominal breathing) were observed on the day of sacrifice. They were accompanied by reddish vaginal discharge in female G21131. No specific pathologic changes were observed for female G21131. The enlarged and gelatinous thymus in female G21132, with red content in the thoracic cavity, correlated microscopically with edema in the thymus and minimal inflammation in adjacent tissues.The thymus adherent to the heart in female G21134 correlated microscopically with inflammation in the thymus and of the epicardium/pericardium, associated with the presence of grayish amorphous material in these inflammatory areas; it was considered likely that this material was test item.

For all females mentioned above (except female G21131 given 300 mg/kg/day), pathological findings were observed mainly in the organs of the thoracic cavity (heart, thymus, lungs), and additionally in the esophagus of one female. They were consistent with pronounced sequels to trauma induced by the treatment procedure (gavage). The difficulties to deliver, associated with the deteriorating clinical condition leading to death or requiring premature sacrifice, were considered to have resulted from these inflammatory lesions, and were therefore considered to be unrelated to the test item administration. Although the cause of the poor clinical condition of female G21131 was undetermined from the gross and microscopic examinations, it was considered to be unrelated to the test item as it was an isolated case at the intermediate dose-level.

-> Due to no delivery,two females were sacrificed on Day 25 p.c. without clinical signs prior to death: female G21124 given 100 mg/kg/day ( At necropsy, four implant scars were observed) and female G21140 given 1000 mg/kg/day (At necropsy, this female was found to be non-pregnant).

BODY WEIGHT (PARENTAL ANIMALS) (See 7.8.1/3 in chapter 'any other information on results incl. tables')
No effects were observed on mean body weight or mean body weight change in males or females at any dose-level.
The only statistically significant difference between control and test item-treated animals, namely higher mean body weight gain in females given 1000 mg/kg/day on Days 7-14 p.c. (+41 g vs. +31 g in controls), was isolated and considered to be incidental.

FOOD CONSUMPTION (PARENTAL ANIMALS) (See 7.8.1/4 in chapter 'any other information on results incl. tables')
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 1000 mg/kg/day on Days 7-14 p.c. (+26 g vs. +22 g in controls), was isolated and considered to be incidental.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related changes in the mean organ weights. When compared to controls, the mean absolute epididymide weight was statistically lower in males treated at 300 mg/kg/day. Considering the low magnitude (-11%) and the absence of a similar trend at 1000 mg/kg/day, any relationship with the test item was excluded.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test item-related changes were observed at in animals sacrificed at the end of the treatment period. The right testis of one male treated at 300 mg/kg/day was enlarged, correlating with diffuse tubule dilation. In this animal, the left seminal vesicle was small, correlating with reduced secretion.The right testis and epididymidis of another male treated at 300 mg/kg/day were small, correlating histologically with marked degeneration/atrophy of tubules and azoospemia.
These isolated changes were considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In animals sacrificed at the end of the treatment period, minimal or slight tubular atrophy/degeneration was observed in the testes of 1 control males and 3 treated males at 1000 mg/kg/day. Marked atrophy/degeneration was also noted in one male treated at 300 mg/kg/day, along with azoospermia in the epididymidis and correlating with small size of the testes and epididymidis seen at necropsy.
As these changes were seen unilaterally and in the absence of a dose-related trend in the severity, and as they are commonly seen as a spontaneous change in rats, any relationship to the test item was excluded. In addition, following careful examination of the different stages of spermatogenesis in tubules, no test item related changes were observed.

OTHER EFFECTS:
No effects on T4 or TSH levels were observed in pups sacrificed on Day 14 p.p. or in the adult males sacrificed at the end of the treatment period at any dose-level.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No test item-related effects were observed on the estrous cycle at any dose-level during the treatment period. The statistically significant lower mean number of metestrus days at 300 mg/kg/day (2.9 vs. 5.0 days) was not attributed to the test item treatment as this difference was not dose-related.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) (See 7.8.1/5 in chapter 'any other information on results incl. tables')
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 1000 mg/kg/day, which accounts for the slightly lower mean fertility index recorded at this dose-level. This event was considered to be incidental and unrelated to the test item treatment as the value was close to the historical control range (90-100%).
The number of females that delivered was 10/10, 9/10, 7/10 or 7/8 in the groups treated at 0, 100, 300 or 1000 mg/kg/day, respectively. No delivery was observed for female G21124 given 100 mg/kg/day; at necropsy, four implantation sites were recorded. Difficulty to deliver was noted for females G21131, G21132 and G21134 given 300 mg/kg/day and for female G21147 given 1000 mg/kg/day; at necropsy, between 14 and 16 implantation sites were recorded. These occurrences were considered to be unrelated to the test item treatment as they were noted with no dose-relationship (female G21124 at 100 mg/kg/day and female G21131 at 300 mg/kg/day) or were attributed to the pathological lesions induced by the treatment procedure (females G21132 and G21134 at 300 mg/kg/day and female G21147 at 1000 mg/kg/day, see MORTALITY (PARENTAL ANIMALS)).
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.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

GENERAL TOXICITY (F1) (See 7.8.1/6 in chapter 'any other information on results incl. tables')
No test item-related clinical signs and no abnormal behaviour were observed at any dose-level.

MORTALITY (F1) (See 7.8.1/7 in chapter 'any other information on results incl. tables')
No effects at birth were observed on the incidence of pups found dead or cannibalized at any dose-level.
No test item-related effects were observed on pup viability ( day 4 p.p.) at any dose-level.
The statistically significant lower mean number of surviving pups on Day 4 p.p. in the 300 mg/kg/day group (75 vs. 113 in controls) and the associated lower mean viability index % were due to 3/7 litters with mainly found-dead pups on Day 1 p.p. As this difference was not dose-related, it was not attributed to the test item treatment.

BODY WEIGHT (F1)
No effects were observed on mean body weight or mean body weight change at any dose-level during the lactation period.

OTHER EFFECTS (F1) (See 7.8.1/8 in chapter 'any other information on results incl. tables')
No test item-related effects were observed on the sex ratio at any dose-level. The statistically significant lower mean number of male pups in the 300 mg/kg/day group (54 vs. 49 in controls) and the associated lower mean sex ratio % were not attributed to the test item treatment as this difference was of low magnitude and not dose-related.
The anogenital distance was considered to be unaffected by the test item treatment in both sexes at all dose-levels.
No nipples or areolae were observed in any male pups.
No effects on T4 or TSH levels were observed in pups sacrificed on Day 14 p.p.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

7.8.1 /2 Mortality (Parental animals):

Animal number	Group/dose-level	Status of death/ Day of death	Observations in female genital tract
G21131	Group 3: 300 mg/kg/day	Sacrificed due to poor condition on Day 46	Uterus: three dead fetuses in the right horn Vagina: one dead fetus
G21132	Group 3: 300 mg/kg/day	Sacrificed due to poor condition on Day 46	Uterus: one placenta in the right horn and one in the left horn Vagina: one dead fetus
G21134	Group 3: 300 mg/kg/day	Sacrificed due to poor condition on Day 46	Uterus: one placenta and one dead fetus
G21147	Group 4: 1000 mg/kg/day	Sacrificed due to poor condition on Day 46	Uterus: six living fetuses in the right horn and eight living fetuses in the left horn

 

7.8.1 /3 Body weight (Parental animals):

 

Sex	Males				Females
Dose-level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Pre-mating (females) or whole study (males)
Day 1	435	430	430	430	283	283	287	286
Day 8	482	473	475	480	288	288	290	290
Day 15	513	501	503	512	297	295	296	297
Day 22	524	516	510	528	/	/	/	/
Gestation
Day 0p.c.	/	/	/	/	305	302	305	305
Day 20p.c.	/	/	/	/	456	450	463	469
Days 0 - 20p.c.	/	/	/	/	+150	+148	+159	+164
Lactation
Day 1p.p.	/	/	/	/	364	351	362	366
Day 13p.p.	/	/	/	/	388	379	383	392
Days 1 - 13p.p.	/	/	/	/	+23	+27	+22	+26

/: not applicable.

7.8.1 /4 Food consumption (Parental animals):

 

Sex		Males				Females
Dose-level (mg/kg/day)		0	100	300	1000	0	100	300	1000
Pre-mating or whole study
Days 1 - 8		32	31	32	33	18	19	19	19
Days 8 - 15		32	31	30	34	19	18	17	20
Gestation
Days 0 - 7		/	/	/	/	21	19	21	22
Days 7 - 14		/	/	/	/	22	22	23	26*
Days 14 - 20		/	/	/	/	27	28	28	31
Lactation
Days 1 - 4	/		/	/	/	32	30	33	38
Days 4 - 8	/		/	/	/	44	42	48	51
Days 8 - 13	/		/	/	/	58	60	64	68

/: not applicable; *: p<0.05.

7.8.1 /5 Reproductive performance (Parental animals):

Pairing, mating and fertility data

 

Dose-level (mg/kg/day)	0	100	300	1000
Number of animals paired (M + F)	10 + 10	10 + 10	10 + 10	9 + 9
Number of males mated	10	10	10	9
Number of females mated	10	10	10	9
Mean number of days taken to mate	1.9	1.5	2.1	1.6
Number of pregnant females	10	10	10	8
Mating index (%)	100	100	100	100(a)
Fertility index (%)	100	100	100	89

M: male; F: female.

(a): excluding female G21146, which was not paired

Delivery data

 

Dose-level (mg/kg/day)	0	100	300	1000
Number of pregnant females	10	10	10	8
Number of females which delivered	10	9	7	7
Mean duration of gestation (days)	22.1	22.3	22.1	22.0
Mean number of corpora lutea	16.6	15.0	15.9	15.0
Mean number of implantation sites	14.3	14.1	14.9	14.6
Mean pre-implantation loss (%)	14.7	5.4	6.0	2.6
Mean number of pups delivered	11.7	11.2	12.1	12.6
Mean post-implantation loss (%)	22.5	20.5	19.1	13.5

 

7.8.1 /6 General toxicity (F1)

 

Dose-level (mg/kg/day)	0	100	300	1000
Tail necrotic, n (L)	1 (1)	-	-	-
Increase in size of right hindlimb, n (L)	-	1 (1)	-	-
Hematoma on tail, n (L)	-	-	1 (1)	-
Left hindlimb necrotic, n (L)	-	-	1 (1)	-
Cold to the touch, n (L)	1 (1)	9(1)^a	-	-

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

^a: this findings was limited to Days 1 and 2p.p.

-: no clinical signs.

 

7.8.1 /7 Mortality (F1)

Incidence of mortality at birth

Dose-level (mg/kg/day)	0	100	300	1000
Number of litter	10	9	7	7
Number of pups found dead	3	6	9	0
Number of pups cannibalized	1	4	1	0
Number of litter affected	2	4	3	0

Pup viability

 

Dose-level (mg/kg/day)	0	100	300	1000
Pups dying, missing and/or cannibalized (Days 1-4p.p. %)	3.4	9.9	11.8	0.0
Viability index (Day 4p.p. %)	96.6	90.1	88.2*(a)	100
Lactation index (Day 14p.p.%)	100	100	100	100

Statistically significant from controls: *: p<0.05.

(a): the statistical significance concerned the number of surviving pups on Day 4p.p.and not the %.

7.8.1 /8 Other effects (F1)

Sex ratio

Dose-level (mg/kg/day)	0	100	300	1000
Sex ratio	42.2	50.0	63.5**(a)	53.4

Statistically significant from controls: **: p<0.01.

(a): the statistical significance concerned the number of males and not the %.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the dose-level of 1000 mg/kg/day was considered to be the No Observed Effect Level (NOEL) for parental systemic toxicity, for reproductive performance (mating and fertility) and for toxic effects on progeny.

Executive summary:

In a screening study for reproductive / developmental effects performed according to OECD 421 guideline , the objective was to evaluate the potential toxic effects of the test substance following daily oral administration (gavage) to male and female rats from before mating, through mating and, for females, through gestation until day 13 post-partum (p.p.).

Three groups of 10 male and 10 female rats received the test substance by gavage at doses of 100, 300 or 1000 mg/kg/day. Males were treated for 15 days plus 5 additional days before pairing until the day before sacrifice after a minimum treatment period of 4 consecutive weeks. Females were treated daily for a minimum of 15 days plus 5 additional days before pairing, throughout mating and gestation until Day 13 post-partum (p.p.) inclusive.  The pre-mating period was extended to 5 additional days as test item concentrations for intermediate- and high-dose groups were outside the acceptable range in the first measurement in Week 1 of treatment. Another group of 10 males and 10 females received the vehicle corn oil under the same experimental conditions and acted as a control group.The actual test item concentrationsin the dosage formulations were determined using a validated HPLC/UV analytical method.

Animals were checked daily for clinical signs and mortality. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation.

The animals were paired for mating after 20 days of treatment and the dams were allowed to litter and rear their progeny until Day 14 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. The pups were observed daily for clinical signs and external abnormalities 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 4 p.p. (all pups before culling) and by counting the number of nipples and areolae in male pups on Day 13 p.p. Thyroid hormones (TSH and T4) were determined in males at sacrifice and in pups sacrificed on Day 14 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 particular attention accorded to the reproductive organs. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on epididymides, ovaries and testes from the control- and high-dose groups and on all macroscopic lesions. A macroscopic post-mortem examination was performed on pups, including those found dead before study termination.

Actual concentrations of the test item in the intermediate and high dose formulations analyzed on Day 1 were outside the acceptable range compared to the nominal concentrations (-62.5 and +18.0%, respectively). After adjustement of the dose formulation preparation procedure on Day 5, the test item concentrations remained within an acceptable range of variation throughout the study (-12.2% to +8.6% compared to nominal concentrations).

No test item-related deaths occurred and no test item-related clinical signs were observed during the study. 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.Thyroid hormone analyses did not reveal any disturbance in males at sacrifice. At pathology examination of parents, no effects on organ weights and no test item-related macroscopic or microscopic findings were observed.

Observations of the pups from birth to Day 14 p.p. did not show any effects on mortality, viability, clinical signs, body weight evolution, sex ratio or physical development. Thyroid hormone analyses did not reveal any disturbance in pups on Day 14 p.p.

Based on these results, the No Observed Effect Level (NOEL) for parental toxicity , reproductive performance and toxic effects on progeny was considered to be 1000 mg/kg/day