Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2016 - 15 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
There were no prostate and seminal vesicles weighing following a confusion between guideline versions 2015 and 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3,5-trimethylcyclohexyl acrylate
EC Number:
289-200-9
EC Name:
3,3,5-trimethylcyclohexyl acrylate
Cas Number:
86178-38-3
Molecular formula:
C12H20O2
IUPAC Name:
3,3,5-trimethylcyclohexyl prop-2-enoate
Test material form:
liquid

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 study initiation: the males were approximately 10 weeks old and the females were approximately 11 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 459 g (range: 417 g to 500 g) and the females had a mean body weight of 274 g (range: 236 g to 304 g)
- Housing: F0 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 of F0 animals was chosen in order not to jeopardize gestations and lactations 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 anf the females were acclimated to the study conditions for 7 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: 11 October 2016 to 15 December 2016.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation (mating period): until mating occurred or 14 days have elapsed
- 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: GC-FID (Gas Chromatography with FID detection)
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Preparation process validated at 2 and 200 mg/mL
Duration of treatment / exposure:
In the males:
- 2 weeks before mating,
- during the pairing period,
- until sacrifice (4 weeks in total),

In the females:
- 2 weeks before mating,
- during the mating period,
- during gestation,
- during lactation until Day 14 post-partum inclusive,
- until sacrifice for females with no evidence of mating.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
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.:
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 2 week study where groups of five Sprague Dawley rats per sex received the test item in corn oil at 100, 300 or 1000 mg/kg/day by gavage.
There were no premature deaths. Test item-treated animals had ptyalism (all animals at 300 and 1000 mg/kg/day) and sometimes chromodacryorrhea. Mean food consumption was in high-dose males slightly lower than controls (-17%) in the last days of treatment. At necropsy, mean liver and kidneys weights were in males higher than controls (up to about +17% at the high-dose). Accentuated lobular pattern was seen in the liver of a few animals of the high-dose mainly.
Therefore, 1000 mg/kg/day was selected as the high dose-level and was expected to induce some toxicity. The low-dose and mid dose were selected using a ratio representing approximately a 3-fold interval (i.e. 300 and 100 mg/kg/day) and the low-dose was expected to be devoid of adverse effects.

- Rationale for animal assignment: stratification procedure.
Positive control:
no (not required)

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.

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

CLINICAL SIGNS:
- Time schedule: From arrival, each animal was observed at least 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.

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 including 1 or 2 days before euthanasia.
The body weight of each female was recorded on the first day of treatment (Day 1), then once a week until mated (or until euthanasia for the female with no evidence of mating) and on Days 0, 7, 14 and 20 post coitum (p.c.) and 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.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: The first five males and the first five females euthanized on Day 15 p.p. from each group were evaluated with a Functional Observation Battery once at the end of the treatment period. For females, this was performed on Day 14 p.p. after euthanasia of the pups.

HAEMATOLOGY:
The parameters were determined from the first five males and from five to seven females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

CLINICAL CHEMISTRY:
The parameters were determined from the first five males and the first five females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

URINALYSIS:
The parameters were determined from the first five males and the first five females euthanized on Day 15 p.p. from each group on the day of euthanasia, after food fasting.

THYROID HORMONES:
Blood samples were taken, in the first half of the morning (generally before 10 a.m.), at termination from all F0 animals, after food fasting.

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 two 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 15 p.p. before euthanasia.
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:
- Performed on Day 4 post-partum: yes
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed.

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 PUPS:
- external abnormalities

HORMONES
- blood samples were taken, in the first half of the morning (generally before 10 a.m.), on Day 4 post-partum from up to 2 pups culled /litter (pooled) and on Day 14 post-partum from 2 pups/litter (pooled).
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 15 p.p.

The following females were euthanized by the same way without overnight fasting:
- female with no evidence of mating: 25 days after the end of the mating period.

ORGAN WEIGHTS:
The body weight of each F0 animal euthanized as scheduled (after the end of the mating period for males or on Day 15 p.p. for females) was recorded before euthanasia. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.

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

HISTOPATHOLOGY:
A microscopic examination was performed on:
- all tissues listed in the study protocol from the first five euthanized as scheduled males and the first five females euthanized on Day 15 p.p. of the control and high-dose groups (groups 1 and 4),
- adrenal glands (males only), liver and kidneys (both sexes) from the first five euthanized as scheduled males and the first five females euthanized on Day 15 p.p. of the low- and intermediate-dose groups (groups 2 and 3),
- all macroscopic lesions of all groups,
- reproductive organs from the pair that did not mate, to investigate possible causes.
Postmortem examinations (offspring):
SACRIFICE: on Day 14 post-partum

GROSS NECROPSY: Pups euthanized on Day 14 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.

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, urinalysis, hormones, anogenital distance and post-implantation loss data
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)
Lactation index on day 14 p.p. = 100 * (Number of surviving pups on day 14 p.p. / Number of surviving pups on day 4 p.p.)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only test item-related clinical sign was ptyalism noted at all dose-levels in a dose-related manner (incidence and duration).
The other clinical signs observed in test item groups (chromodacryorrhea, area of hair loss, cutaneous lesion, scab, desquamation) were considered to be incidental (noted in isolated animals and commonly observed in this species and strain at this age of breeding).
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths in test item-treated groups.
In the control group, one male was prematurely sacrificed on Day 14. Prior to sacrifice, piloerection, dyspnea, swollen and hard abdomen were observed. This animal had the highest body weight gain in the first week of treatment which may also be related to its prostatic sarcoma.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 1.
There were no adverse effects in any sex.

In males at 1000 mg/kg/day and compared with controls, there was a slightly statistically significantly lower mean body weight gain over the treatment period which was considered to be test item-related but of limited toxicological significance as mean body weights were comparable with controls.

In females at 1000 mg/kg/day and compared with controls, there was a statistically significantly higher mean body weight change in the first days of the lactation period which was considered to be test item related but of limited toxicological significance as mean body weights during lactation were comparable with controls. There were no effects on mean body weight or mean body weight change during premating and gestation periods at any dose-levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See table 2.
There were no adverse effects in any sex.

In males at 1000 mg/kg/day and compared with controls, there was a slightly statistically significantly higher mean food consumption in the second week of treatment which was considered to be test item-related but of limited toxicological significance as the magnitude of difference from controls was minimal.

In females at 1000 mg/kg/day and compared with controls, there was a statistically significantly higher mean food consumption in the last days of gestation and in the first week of lactation which was considered to be test item-related but of minor toxicological significance in view of the magnitude of difference from controls.

There were no relevant effects on mean food consumption at 100 and 300 mg/kg/day.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details in the section 7.5 Repeated dose toxicity (OECD 422)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details in the section 7.5 Repeated dose toxicity (OECD 422)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
See details in the section 7.5 Repeated dose toxicity (OECD 422)
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See details in the section 7.5 Repeated dose toxicity (OECD 422)
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details in the section 7.5 Repeated dose toxicity (OECD 422).

Adrenal glands : Minimal cortical cell hypertrophy was noted in 3/5 males treated at 1000 mg/kg/day and correlated with the increased mean weights.
This finding was considered to be non-adverse in view of its low magnitude and of the absence of associated degeneration/necrosis.

No finding was observed in the other reproductive organs.

Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
See details in the section 7.5 Repeated dose toxicity (OECD 422).

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on estrus cycles during the first 2 weeks of treatment.
The statistically significantly lower mean number of cycles at 300 mg/kg/day (2.7 vs. 3.0 in controls, p<0.05) was considered to be incidental [no dose-relationship and partly due to one female in diestrus for 6 days].

At the end of the treatment period, females were in diestrus (sometimes listed as metestrous) as expected at mid lactation which generally correlated with diestrus stages seen at microscopy when examined in control and high-dose groups, with the exception of one control female and one female treated at 100 mg/kg/day which were respectively seen in proestrus or estrus on Day 15 p.p. (there were no microscopic examination for these females).
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
See tables 3 and 4.
Mating and fertility data
There were no test item-related effects on mating and fertility data.

There were 1, 1, 1 and 2 pairs that mated in 13, 13, 7 and 12/14 days at 0, 100, 300 and 1000 mg/kg/day, respectively. The females of those pairs were mostly in diestrus (pseudo-pregnancy), with the exception of the female at 300 mg/kg/day which was for 5 days in estrus (listed as proestrus) and one female treated at 1000 mg/kg/day which was cycling. As the incidences were comparable between groups, a relationship with the test item treatment was considered unlikely.

Besides, there was one pair with no signs of mating (no spermatozoid and no sperm plug) during the allocated mating period (14 days) at 1000 mg/kg/day, which may be related to an almost complete testicular atrophy and a strongly reduced sperm content in epididymides from one male. This event was not considered to be due to the test item treatment.

Delivery data
There were no effects on mean delivery data.

Details on results (P0)

Table 1: Body weight

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Pre-mating or whole study
Day 1 461 457 460 459 276 270 268 282
Day 8 511 509 500 497 280 278 277 293
Day 15 541 548 537 530 291 286 287 299
Day 29 576 584 575 559 / / / /
Days 1-8 +50 +52 +40 +38* +4 +7 +9 +11
Days 1-15 +83 +92 +77 +70 +14 +15 +19 +17
Days 15-29 +35 +35 +38 +29 / / / /
Days 1-29 +118 +127 +115 +99* / / / /
Gestation
Day 0 p.c. / / / / 300 297 289 312
Day 20 p.c. / / / / 465 464 454 473
Days 0-20 p.c. / / / / +165 +167 +165 +161
Lactation
Day 1 p.p. / / / / 373 362 355 369
Day 4 p.p. / / / / 370 367 363 383
Day 13 p.p. / / / / 397 396 384 410
Days 1-4 p.p. / / / / -4 +5 +8 +14**
Days 1-13 p.p. / / / / +24 +34 +30 +40*
/: not applicable, statistical significance *: p<0.05, **: p<0.01.

Table 2: Food consumption

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Pre-mating or whole study
Days 1-8 32 32 31 33 18 18 18 19
Days 8-15 32 32 34 36* 20 19 20 21
0 +6 +13 -5 0 +5
Gestation
Days 0-7 p.c. / / / / 23 22 23 25
Days 7-14 p.c. / / / / 25 25 26 28
Days 14-20 p.c. / / / / 29 28 29 34**
-3 0 +17
Lactation
Days 1-4 p.p. / / / / 34 38 38 44#
+12 +12 +29
Days 4-8 p.p. / / / / 49 52 52 59**
+6 +6 +20
Days 8-13 p.p. / / / / 63 68 67 69
/: not applicable, statistical significance: *: p<0.05, **: p<0.01, #: p<0.001.
in italic: differences from controls (%).

Table 3: Mating and fertility data

Dose-level (mg/kg/day) 0 100 300 1000
Number of animals paired (M + F) 9 + 10 10 + 10 10 + 10 10 + 10
Number of males mated 9 10 10 9
Number of females mated 10 10 10 9
Mean number of days taken to mate 4.3 3.9 2.8 5.1
Number of pregnant females 10 10 10 9
Fertility index 100% 100% 100% 100%
Number of females with live born pup(s) 10 10 10 9
Gestation index 100% 100% 100% 100%
M: males; F: females.

Table 4: Delivery data

Dose-level (mg/kg/day) 0 100 300 1000
Number of females which delivered 10 10 10 9
Mean duration of gestation (days) 22.0 22.0 22.0 22.0
Mean number of corpora lutea 15.4 15.5 15.5 15.7
Mean number of implantations 14.4 14.8 14.6 15.2
Mean pre-implantation loss (%) 7.9 5.9 5.2 3.0
Mean number of pups delivered 12.3 13.6 12.7 13.1
Mean post-implantation loss (%) 16.7 7.1 12.8 12.2

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic and local toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOEL
Remarks:
reproductive performance
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
See table 2.
None of the findings observed were attributed to the test item treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
See table 1.
There were no test item-related effects on pup mortality and viability.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
See table 3.
There were no effects on mean pup body weight on Day 1 p.p.

Thereafter, in the absence of statistical significance and since there was one control litter with generally high body weights, a test item effect that could explain the minimally lower mean body weights and mean body weight changes was considered unlikely.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital distance (see table 5)
When compared with controls, AGD was statistically significantly lower at 100 and 300 mg/kg/day as indicated by the ratios AGD to cube root of body weight. However, taking into account the absence of dose relationship and the standard deviations, a test item treatment-related effect was considered unlikely.

Nipples and areolae numbers
There were no test item-related effects on number of nipples and of areolae in male pups.

Thyroid hormones (see table 6)
There were no efefcts on mean T4 and TSH levels in Day 14 p.p. litters.

Sex ratio (see table 4)
There were no test item-related effects on the percentgae of male pups at birth which was comparable among groups and close to 50%.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Table 1: Mortality

Dose-level (mg/kg/day) 0 100 300 1000
Viability index on Day 4 p.p. (%) 91.1 99.3 93.7 97.5
Lactation index on Day 14 p.p. (%) 98.6 98.7 100 100

The distribution of prematurely dead pups was the following:

Dose-level (mg/kg/day) 0 100 300 1000
Number of cannibalized pups (litters affected) 3 (3) 1 (1) 3 (2) 3 (3)
Number of found dead pups (litters affected) 9 (4) 1 (1) 5 (1) 0 (0)
Total of prematurely dead pups (litters affected/total of litters) 12 (5/10) 2 (2/10) 8 (2/10) 3 (3/9)

Table 2: Clinical signs and external examination

Dose-level (mg/kg/day) 0 100 300 1000
Absence of milk in stomach 8 (3) 1 (1) 5 (1)
Thin appearance 1 (1)
Dehydration 1 (1) 1 (1) 1 (1)
Hematoma (back/ear/head/neck/tail) 1 (1) 3 (3)
Scab (abdomen) 1 (1)
Swollen (ear/abdomen) 1 (1) 1 (1)
Blackish/abdomen 1 (1)
Total of litters affected/total 4/10 1/10 1/10 3/9
Number of pups (litters) affected.

Table 3: Body weight

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
Day 1 p.p. 8.3 8.3 8.3 8.2 7.8 7.8 7.9 7.6
Day 4 p.p. (preculling) 12.7 12.1 12.5 12.0 12.2 11.5 11.9 11.5
Day 4 p.p. (postculling) 12.7 12.0 12.5 12.0 12.3 11.6 11.9 11.5
Day 8 p.p. 23.4 22.5 22.8 22.3 22.8 21.4 22.2 21.3
Day 13 p.p. 38.5 38.1 37.9 36.6 38.3 37.1 36.9 35.2
Days 1-4 p.p. 4.4 3.8 4.1 3.9 4.4 3.7 4.0 3.8
Days 4-8 p.p. 10.6 10.5 10.3 10.3 10.5 9.8 10.3 9.8
Days 8-13 p.p. 15.1 15.6 15.0 14.3 15.6 15.6 14.7 13.9

Table 4: Sex ratio

Dose-level (mg/kg/day) 0 100 300 1000
Sex ratio at birth (% of males) 44.3 49.3 52.8 48.3

Table 5: Development

Sex Male Female
Dose-level (mg/kg/day) 0 100 300 1000 0 100 300 1000
AGD (mm) 6.78
± 0.67 6.27**
± 0.69 6.44*
± 0.90 6.82
± 0.63 4.23
± 0.59 3.94
± 0.67 3.60**
± 0.74 4.18
± 0.42
Ratio AGD/3vbody weight (mm/g1/3) 2.97
± 0.30 2.75**
± 0.32 2.79**
± 0.36 3.00
± 0.27 1.87
± 0.25 1.76*
± 0.29 1.58**
± 0.32 1.86
± 0.20
-7 -6 +1 -6 -16 -1
Mean data based on individual pup data.
Statistical significance: *: p<0.05, **: p<0.01; in italic: differences from controls (%).

Table 6: Thyroid hormones

Dose-level (mg/kg/day) 0 100 300 1000
T4 (ng/mL) 34.2 35.9 36.0 36.4
TSH (pg/mL) 2269 1979 2062 1956

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 14 p.p., at dose levels of 100, 300 and 1000 mg/kg/day.

Based on the experimental conditions and results of this study:
- the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on the absence of adverse effects at this dose and the non adverse findings in clinical signs, body weight change, food consumption and hematology, blood biochemistry, urinalysis and pathology,
- the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 1000 mg/kg/day based on the absence of test item effects on mating, fertility and delivery data,
- the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day based on the absence of test item effects in pups.
Executive summary:

The objective of this GLP 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, through mating and, for females, through gestation until Day 14 post-partum (p.p.).

This study provides information:

.         on the possible health hazards (including neurological and immunological effects) 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 (F0 animals) received the test item daily, by oral administration (gavage), 2 weeks before mating, during mating and, for the males, until sacrifice, for the females, throughout gestation until Day 14 post-partum (p.p.) inclusive. Another group of ten males and ten females received the vehicle (corn oil) and acted as a control group. A constant dosage-volume of 5 mL/kg/day was used.

The concentration of the dose formulation was checked in study Weeks 1 and 6.

 

The F0 animals were checked daily during the dosing period for mortality, morbidity and clinical signs. Detailed clinical observations were performed once a week. Body weight and food consumption were recorded once a week during premating, mating (food consumption not during mating), gestation (0, 7, 14 and 20 p.c.) and lactation (Days 1, 4, 8 and 13 p.p.) periods.

The F0 animals were paired for mating after 2 weeks of treatment and the females were allowed to litter and rear their progeny until Day 14 p.p.The total litter sizes and the sex of each pup were recorded after birth. The pups were observed daily for clinical signs, abnormal behaviour and external abnormalities and weighed on Days 1, 4, 8 and 13 p.p.

A Functional Observation Battery (FOB) including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed on five F0 animals per sex and group at the end of the treatment period. Analysis of hematology, blood biochemistry and urine parameters were performed from blood and urine samples taken prior to sacrifice from at least five F0 animals per sex and group. Thyroid hormones (T4 and TSH) plasma levels were determined from all F0 males and Day 14 p.p.pups.

The F0 males were sacrificed after 4 weeks of treatment and the F0 females on Day 15 p.p. Final body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was performed on long list of organs from five F0 animals per sex in the control and high-dose groups and on all macroscopic lesions.

Pups not selected on Day 4 p.p. were sacrificed and discarded without further examination.

Pups selected were sacrificed on Day 14 p.p. and submitted to a detailed external examination with particular attention to the external genital organs.


Results

Chemical analyses

The test item concentrations in the analyzed dose formulations were within an acceptable range of variations (± 10% of the nominal concentrations required) and no test item was observed in the control dose formulations analyzed.

 

F0 animals

There were no unscheduled deaths in test item-treated groups andthe only test item-related clinical sign was ptyalism noted at all dose-levels in a dose-related manner (incidence and duration). There were no test item-related functional effects at FOB.

 

There were no relevant effects on mean body weights. At 1000 mg/kg/day when compared with controls, mean body weight change was lower in males over the treatment period (+99 g vs.+118 g in controls, p<0.05; limited toxicological significance) or higher in females on Days 1-4 p.p.(+14 g vs.-4 g, p<0.01; limited toxicological significance). There were no effects on mean body weight change in females during premating and gestation periods.

 

At 1000 mg/kg/day when compared with controls, mean food consumption was slightly higher in males in the second week of treatment (+13%, p<0.05; limited toxicological significance) and higher in females from Day 14p.c.to Day 8 p.p.(up to +29% on Days 1 -4 p.p.; minor toxicologically significant).

There were no relevant effects on mean food consumption at 100 and 300 mg/kg/day.

 

At pathology, in animals treated at 1000 mg/kg/day, dose-related hepatocellular hypertrophy in the liver, hyaline droplets in males, tubular dilation and basophilia in the kidneys, and hypertrophy of cortical cells in the adrenals (males only) were observed and correlated with increased mean organ weights.

In animals treated at 300 mg/kg/day, dose-related hepatocellular hypertrophy in the liver, hyaline droplets, tubular dilation and basophilia in the kidneys from males were observed, and correlated with increased mean organ weights in males.

In animals treated at 100 mg/kg/day, dose-related hepatocellular hypertrophy in the liver from males only, hyaline droplets in males and tubular dilation in females.

There were no test item-related effects on estrous cycles during the first 2 weeks of treatment and on mean fertility, mating and delivery data.

 

These effects on clinical signs, mean body weight change and mean food consumption, and at laboratory investigations and pathology, were considered as test item-related and non-adverse.


Pups

There were no effects considered as test item-relatedon pup mortality/viability, clinical signs, mean body weight, anogenital distance on Day 4 p.p., development of nipples and areolae in male pups on Day 13 p.p., ormean T4 and TSH levels on Day 14 p.p.There were notest item-relatedeffects on the percentage of male pups at birth.

 

Conclusion

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, gestation and until Day 14 p.p., at dose-levels of 100, 300 and 1000 mg/kg/day.

 

Based on the experimental conditions and results of this study:

.         the No Observed Adverse Effect Level (NOAEL) for parental toxicity (systemic and local) was considered to be 1000 mg/kg/day based on the absence of adverse effects at this dose and the non-adverse findings in clinical signs, body weight change, food consumption and hematology, blood biochemistry, urinalysis and pathology,

.         the No Observed Effect Level (NOEL) for reproductive performance (mating, fertility and delivery) was considered to be 1000 mg/kg/day based on the absence of test item effects on mating, fertility and delivery data,

.         the NOEL for toxic effects on progeny was considered to be 1000 mg/kg/day based on the absence of test item effects in pups.

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