Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity (OECD TG 422): NOAEL > 7200 ppm (corresponding to an actual intake of 246 mg/kg bw/day (highest dose tested)

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016- 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated Dose 28-day oral toxicity study in rodents
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
no
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Details on species / strain selection:
Rat is one of the rodent species acceptable to the regulatory authorities. Historical control data for the
strain are available at the Test Facility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately 11 weeks, Females Approximately 13 weeks
- Weight at study initiation: Males: 345 to 401 g; Females: 196 to 247 g
- Fasting period before study: no
- Housing: One air-conditioned room in a barrier protected unit. Animal were housed in plastic cages meeting European directive 2010/63/EU requirements. Cellulose bedding (Serlab, Montataire, France) or dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood. Animals received a small amount (handful) of shredded paper (SDS/Dietex) and the isolated animals had free access to a wooden gnaw block (Aspen Bricks, Dietex France).
- Diet (e.g. ad libitum): Rat powdered complete diet ad libitum (Diet reference A04C10 P2.5) sterilised by irradiation. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water (e.g. ad libitum): Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system or bottles).
- Acclimation period: 8 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3 °C (target range)
- Humidity (%): > 35 % (target)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 January 2017 To:27 February 2017
Route of administration:
oral: feed
Vehicle:
other: Basic powdered diet (Diet reference A04C10 P2.5)
Details on exposure:
DIET PREPARATION
A pre-mix preparation was performed: the test item and the vehicle were weighed. The test item was added slowly to the powder diet and mixed using a spatula. Then the mix preparation was performed: a part of the vehicle was placed into a mixer. The pre-mix preparation was added and the remaining powder diet was added. The formulation was mixed for at least 12 minutes (until a homogeneous mix was obtained).
Test item/diet mixes were formulated at least once weekly and aliquoted into daily aliquots stored frozen (between -15 and -25 °C).Daily aliquots were removed from the freezer just before delivery to the animal unit and were placed at refrigerated temperature (between +2 and +8 °C) and/or placed at
room temperature (between +15 and +25 °C) just before hopper filling. The temperature was outside the targets on 1 occasion (8.8 °C instead of 2-8 °C) during the study. This deviation did not noticeably affect the storage condition of the test item
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of gestation (G0)
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity of the test item in the diet mix was confirmed between 250 to 10000 ppm (validation study).
Stability of the test item in the diet mix (250 to 10000 ppm): 48 hours stored refrigerated (2 to 4 °C) and 8 days stored frozen (-15 to -25 °C), in bags. However, the test item formulation was not stable for 24 hours at room temperature (15 to 25°C) in the food hopper (decrease in achieved concentration more than 10% of time 0 concentration) (validation study).
Analysis of preparations:The diets for each dose group including the control prepared to be distributed during the first week of the study were analysed within the stability period for achieved
concentrations. Duplicate (2x 30 g) samples were taken from each formulation from the middle of the bulk diets on the day of preparation.The first set of samples was analysed at the Test Facility using a validated method. The transfer and validation of the analytical method was the subject of a separate study plan. Samples were stored frozen (-15 to -25 °C) up to analysis.
Duration of treatment / exposure:
males:14 days before mating, throughout the mating period and up to the day before necropsy (i.e. 31 days for males)
females:14 days before mating, throughout the mating period and up to the day before necropsy. During gestation (the first day of gestation was designated as G0) and 14 days after parturition up to and including the day before necropsy (the first day of birth is designated as L0). Apparently unmated females for 26 days after the last day of the mating period.
Frequency of treatment:
Continuously
Dose / conc.:
450 other: ppm; nominal 37.5 mg/kg bw
Dose / conc.:
1 800 other: ppm; nominal 150 mg/kg bw
Dose / conc.:
7 200 other: ppm; nominal 600 mg/kg bw
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Details on study design
- Dose selection rationale: in the preliminary study, test item given in diet at 9000 ppm induced body weight loss during the first days and a decreased body weight gain (associated with the decreased food consumption) over the 14 days of dosing, when compared with the control group. Therefore, the dose of 9000 ppm was considered a maximum tolerated dose for the preliminary 14-day study but was considered too high for a study of longer duration and with pregnant and lactating females.
Therefore, for this study, the mid dose of 1800 ppm used in the preliminary study was kept and a factor of 4 was used between each dose (instead of 5 in the preliminary study). Thus, the doses of pinyl isobutyraldehyde in this study were 450, 1800 and 7200 ppm.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of the males were recorded weekly from the first day of exposure (prior to the first exposure) up to necropsy.
Individual body weights for females were recorded weekly during pre-mating and mating periods (only pre-mating data are reported), on Days 0, 4, 7, 11, 14, 17 and 20 of gestation, and on Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of premating period (study Day 14)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (for at least 16 hours)
- How many animals: 5/sex/group (randomly selected)
- Parameters examined: according to guidelines

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of premating period (study Day 14)
- Animals fasted: Yes (for at least 16 hours)
- How many animals: 5/sex/group (randomly selected)
- Parameters examined: according to guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: YES
Functional tests. 5/sex/group (randomly selected) shortly before kill (study Day 31 for males and on Day 13 of lactation for females). The tests included auditory reflex, pupillary reflex, surface righting reflex, forelimb grip strength, and locomotor activity in an open field test.

IMMUNOLOGY: No


Sacrifice and pathology
GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guidelines

Other examinations
Thyroid hormone analysis
Estrous cyclicity (parental animals):
Vaginal smears were taken daily and used to determine the stage of estrous for each female from two weeks before treatment, the first 14 days of treatment and during mating until identification of mating or separation from the male.
On the day of necropsy, a vaginal smear was taken to determine the stage of the estrous cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in P male parental generation:
testis weight, epididymis weight. For the testes from the 5 selected adult males of groups 1 (control) and 4 (high dose) and all males suspected to be infertile or which died before mating, in addition to the haematoxylin and eosin-stained slide, an additional slide were prepared and examined, stained with PAS. A detailed, qualitative evaluation of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The cell- or stage-specificity of any testicular findings were recorded, where appropriate.
Litter observations:
For each F1 litter the following data were recorded:
 number of pups born (live and dead)
 external abnormalities of the pups
 number, weight and sex of pups on Days 1, 4, 7 and 13 post-partum
 Anogenital distance (AGD) was measured on PND1. The AGD was normalized to the cube root of body weight.
 All males in each litter were examined for the number of areola/nipples on PND13.
On Day 4 post-partum, the size of each litter was adjusted (culling) to 8 pups, where possible, by eliminating extra pups by random selection to yield where possible 4 males and 4 females per litter. The extra pups were killed by an intraperitoneal injection of sodium pentobarbitone (CEVA santé animale).
Justification of the culling procedure: the culling of litters to a standard size is given as an optional procedure in the ICH regulatory guidelines. It was therefore decided to use culling in this study in order to be consistent with the historical data compiled by the Test Facility.
Blood samples were collected from two surplus pups where possible, pooled and used for determination of serum T4 levels.
No pups were eliminated when litter size dropped below the culling target (8 pups/litter). When there is only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible serum T4 assessments.
Postmortem examinations (parental animals):
SACRIFICE
All adult males and females were weighed before necropsy and killed by carbon dioxide inhalation followed by exsanguination and necropsied according to the following schedule:
Males: after completion of the mating period on study Day 32 (31 days of dose administration)
Females: on Day 14 of lactation.
On Day 26 post-coitum for mated females that failed to produce a viable litter.
On study Day 56 (27 days after the last day of the mating period) for apparently unmated female.
Pups: at culling (PND 4) or at terminal sacrifice (PND 13)


GROSS NECROPSY
- Gross necropsy was performed according to Guidelines

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues according to Guidelines were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
For each F1 litter the following data were recorded:
 number of pups born (live and dead)
 external abnormalities of the pups
 number, weight and sex of pups on Days 1, 4, 7 and 13 post-partum
 Anogenital distance (AGD) was measured on PND1. The AGD was normalized to the cube root of body weight.
 All males in each litter were examined for the number of areola/nipples on PND13.
Blood samples were collected from two surplus pups where possible, pooled and used for determination of serum T4 levels
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon
 the kurtosis of the data
 the probability of the Bartlett's test for homogeneity of the variances and
 an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
The locomotor activity in an open field and the estrous cycle, pre-coital interval and anogenital distance data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Reproductive indices:
Pre-coital interval (in days): sum of days until successful insemination/ number of inseminated females

Irregularity index: Mean standard deviation of length of the oestrous cycle/ √ (length of the oestrous cycle)

Days in estrus: (number of estrus days/ number of smears) x100

Copulation index (%): (number of inseminated females/ number of paired animals) x100

Fertility index (%): (number of pregnant females/ number of inseminated females) x100

Pre-birth loss (%): (number of implantations - number of offspring born/ number of implantations) x100
Offspring viability indices:
Live birth index (in %): (number of pups born alive/ number of pups born) x 100

Viability index (in %): (number of pups alive on PND 4 (before culling)/ number of pups alive at birth) x 100

Lactation index (%): (number of pups alive on PND 13/ number of pups alive on PND 4 (after culling)) x 100

Sex ratio (proportion of male) (in %): (number of males/ number of pups) x 100

Clinical signs:
no effects observed
Description (incidence and severity):
Torn claw, localized hairloss, sores, scabs or nodule on the tail were considered incidental.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths among F0 rats.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
males:
There was a dose-related lower mean body weight gain for males given 1800 and 7200 ppm throug
hout the pre-mating period (-18 and -35 %, respectively) compared with the control group, resulting in
lower final mean body weight on Day 29 (-3 and -7 %, respectively).
females:
During the pre-mating period, there was a lower mean body weight gain for all treated female groups
during the first 8 days of dosing when compared with the control group. Changes were not dose-rela
ted.
During the gestation period (G0 to G20), there was a dose-related lower mean body weight gain at
1800 ppm (mainly between G0 and G14) and at 7200 ppm, when compared with the control group.
During the lactation period, the mean body weight change between L1 and L13 was similar for all
groups, although a mean body weight gain at 1800 and 7200 ppm slightly lower than controls betwe
en L1 and L4.
These decreased in body weight gains induced minor effects on body weight at 1800 ppm (-4 % at the
end of the pre-mating period for males (Day 29) and -4 % and -2% at the end of the gestation (G20)
and lactation (L13) periods, respectively, for females).
At 7200 ppm, these changes induced relevant effect on body weight at the end of the gestation perio
d (-9 % on G20) and, at a lower severity, at the end of the pre-mating period for males (-7 % on Day 29), whereas the effect on body weight was minor at the end of the lactation period (-4% on L13).
(see tabel mean body weight changes of females in section any other results including tabels)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males
There was a dose-related lower mean food consumption for males treated at 1800 and 7200 ppm between Days 1 and 15 (-7% and -10%, respectively) compared with the control group. The decrease
was more pronounced between Days 1 and 3 (up to -28 % at 7200 ppm).
Females
There was a lower mean food consumption for females treated at 7200 ppm between Days 1 and 4 during the pre-mating period (up to -44 %) and a dose-related lower mean food consumption for females treated at 1800 and 7200 ppm between Gestation Days 1 and 20 (-11 and -21 %, respectively), compared with the control group.
During the lactation period, there was no test item effect in food consumption in any group. At 450 and 1800 ppm, the mean food consumption was even higher than in controls (+22% and +14 %,
respectively) and at 7200 ppm, it was comparable with controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects in haematology and coagulation parameters in any group.
The slightly lower mean fibrinogen concentration for females treated at 1800 and 7200 ppm was considered of no toxicological relevance in view of the low magnitude of the change and since mean values were within the background control range.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no relevant test item-related effects in serum clinical chemistry parameters in any group.
The following statistically significant differences compared with controls, were considered of no
toxicological significance in view of their low magnitude or since mean values remained close or w
ithin the normal historical control range:
- slightly higher mean total protein and albumin concentration for males treated at 1800 and 7200 ppm
- increased individual creatinine levels for 3 males at 7200 ppm
- increased individual phosphorus levels in 2 females at 1800 and 3 females at 7200 ppm.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There was a dose-related lower mean grip strength in all treated groups for both sexes compared with the control groups (-7%, -30% and -27% in males dosed 450, 1800 and 7200 ppm, respectively, and
-16%, -27% and -38% in females dosed 450, 1800 and 7200 ppm, respectively). Lower body weights in treated groups might be the cause of lower strength in treated groups. This effect was therefore
considered most probably incidental.
There were no test item-related changes in auditory, pupillary or righting reflexes.
There were no test item-related changes in open field data for males and females.
The mean total time spent in lateral regions was higher for all treated males compared with controls but values were within the historical control range. The mean control value for males was moreover low compared with historical control data. Although statistically significant, these differences were therefore considered as unrelated to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher mean absolute and relative liver weights were noted in males (+24%) and females (+26%) at 7200 ppm, as were thyroid with parathyroid glands (+25%) and kidney (+18%) weights in males at the same dose. Lower mean absolute and relative spleen (-18%), ovaries (-18%) and uterus (-24%) weights were seen in females at 7200 ppm. Of these changes, only the kidney weight variation could be accounted for histologically by tubular degeneration and basophilia, noted in all males at 7200 ppm only. Although the liver and thyroid weight variations had no histological correlate, they are likely to be attributable to an adaptive hepatocellular hypertrophic change with secondary thyroid follicular cell hypertrophy.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic observations that were considered to be associated with administration of the test item for treated males and females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the kidneys of males at 7200 ppm. Degeneration of tubules of the cortex and medulla was seen at a slight grade in all males at 7200 ppm, in association with tubular basophilia. Tubular degeneration was more pronounced at the corticomedullary junction, where tubules were dilated and filled with hyaline casts. Because of its unusual localisation and severity, the degenerative change could be considered to be potentially adverse and, because it is confined to males, it could be related to the protein metabolism of male rat kidneys.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly higher total T4 levels were noted for F0-males given 450 and 1800 ppm and for male PND 13 pups in the 450 ppm dose group, compared with concurrent control animals. These differences reached statistical significance, but were not considered test item-related in the absence of a dose-related distribution, because no increase in total T4 levels were observed in the animals in the 7200 ppm group.
Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment in length and regularity of the estrous cycle in any group.
There was an acyclic female in the 7200 ppm group, before mating during the treatment period. This isolated finding was considered incidental.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
There was no test item-related effect on mating performance in any group. All animals mated with the exception of one pair in the 450 ppm group. All mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was therefore normal (less than 4 days) in all groups.

There was no effect of treatment on fertility in any group. There were 3, 1 and 2 mated females in the control, 1800 ppm and 7200 ppm groups respectively that did not become pregnant. These isolated cases, including controls, were considered incidental.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
1 800 other: ppm; actual intake 83 mg/kg bw
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction: fertility
Effect level:
7 200 other: ppm; actual intake 246 mg/kg bw
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
7 200 other: ppm; actual intake 246 mg/kg bw
System:
other: Body weight; liver, thyroid and kidney weight increase and specifically the histopathological effects in the kidneys in males
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no pup observations that suggested any association with maternal treatment.
Incidental occasional signs such as incomplete hair growth were noted in all groups including controls.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean live litter size and live birth index were comparable in all groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean pup body weight was lower for pups at 7200 ppm than the mean pup control body weight. The difference increased during the lactation period. The mean body weight was approximately 14 % lower than controls for both sexes on PND 13. This difference may be related to the lower mean body weight of dams treated at 7200 ppm, during the gestation and lactation periods.
parturition: There were 7, 9, 9 and 8 females that completed delivery in the control, 450, 1800 and 7200 ppm groups, respectively. The mean duration of gestation was comparable (approximately 22 days) in all groups. There was no effect of treatment in the mean number of implantation sites in any group.

pup viability and litter sizes: The percentage of pre-birth loss was slightly higher in the 7200 ppm group (11.86 %) compared with the concurrent control (7.16 %) but remained comparable with the historical control data range (1.2 to 12.9 %). Moreover, individual values did not indicate any test item-related trend. This change was therefore considered as unrelated to test item.
There were 2 litters in the 1800 ppm group (nos. 55 and 59) with 1 and 4 stillborn pups, respectively, compared with none in the concurrent control group. With no similar finding in the high dose group, this was considered incidental. The mean live litter size and live birth index were comparable in all groups.
The viability and lactation indices were comparable in all groups.
There was no test item-related effect on the sex ratio.

pup observations: There were no pup observations that suggested any association with maternal treatment.
Incidental occasional signs such as incomplete hair growth were noted in all groups including controls.

number of areola/nipples (male pups): There were no test item-related effects on areola/nipple retention. No nipples were observed in the examined male pups at PND 13.

pup anogenital distance: The mean anogenital distance (normalized for body weight) in male and female pups was comparable in all groups.


Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 7 200 other: ppm (actual intake 246 mg/kg bw)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Summary of mean achieved concentrations throughout the study period (deviations from theoretical concentrations in brackets)

Period

Target dose level in mg/kg BW/day (dose in ppm)

37.5 (450)

150 (1800)

600 (7200)

Pre-mating

D1-D8

Males

 

31

(- 18 %)

 113

(- 25 %)

445

(- 26 %)

Females

30

(- 19 %)

165

(+ 10 %)

413

(- 31 %)

Pre-mating

D8-D15

Males

 

27

(- 28 %)

 104

(- 25 %)

412

(- 31 %)

Females

28

(- 27 %)

115

(- 23 %)

465

(- 22 %)

Gestation

G0-G20

(Females)

Range

32 to 36

(- 3 to – 15 %)

114 to 136

(- 10 to – 24 %)

410 to 492

(- 18 to - 32 %)

Lactation

L1-L4

(Females)

Range

56 to 83

(+ 51 to + 121 %)

214 to 309

(+ 43 to + 106 %)

784 to 1167

 (+ 31 to + 95 %)

 Actual lowest dose   

 Female

 21

 83

 323

 Male

 21

 94

 246

Formulation analysis:All analyzed samples of fomulations prepared at nominal concentrations of 450, 1800 and 7200 ppm of Pinyl iso Butyraldehyde alpha in basic powder diet (A04C10 P2.5) taken from each preparation, including the control, one day during the first week of treatment were in agreement with acceptance criteria (± 20 %), except group 2 formulation at 450 ppm showing a deviation from nominal concentration of -20.2 % slightly above acceptance criteria of +/-20 %. The deviations from the nominal concentrations of formulation at 1800 and 7200 ppm were -18.0 % to -18.4 %. Investigation of the result outside acceptance criteria did not allowed identifying a root cause which may explain the low concentration observed. Duplicate sample from group 2 were not reanalyzed as stability of test item was limited to 5 days at room temperature (+15 to +25 °C) in open feeder and showed degradation for further duration storage.

No test item was present in the vehicle sample.

Achieved dose:Throughout the pre-mating period, the achieved intake of test item, calculated from the body weight and food consumption data, was lower, at all dose levels, than the theoretical concentrations, for males and females (-18 to -32 %), except at the mid-dose where there was a +10 % higher achieved dose during the first week for females only.

During the gestation period, the decreased achieved intake of test item was more slight at the low and mid dose level (-3 to – 24 %), than during the pre-mating period. At the high dose level, the decreased achieved concentration ranged from -18 to -32 %.

During the lactation period, due to higher food consumption for treated females (see section 11.7), the achieved intake of test item was markedly higher than the theoretical concentrations (+31 to + 121 %).

In addition, after 24-hour storage in the food hopper at room temperature, a decrease in test item formulation concentration of approximately -20 % was noted when compared with the nominal concentrations.

  Summary of salient mean body weight changes of females (in g - percent difference from concurrent controls in brackets)

Period

Dose level (ppm)

0

450

1800

7200

Pre-mating

D1-D8

7.26

2.62 *

(- 64 %)

4.77 *

(- 34 %)

1.04 ***

(- 86 %)

Gestation

G0-G20

110.34

-

99.83

(- 10 %)

78.88 ***

(- 29 %)

Lactation

L1-L4

14.33

-

10.83

(- 24 %)

11.44

(- 20 %)

* p<0.05; *** p<0.001

Conclusions:
Based on the results in this study, the reproduction and developmental NOAEL was defined at 7200 ppm, corresponding to an actual intake of the substance of at least 246 mg/kg bw/day.
Executive summary:

Repeated dose and Reproductive screening tested in the OECD TG 422

Introduction and Method

Potential toxic effects of the test substance after repeated exposure was investigated in an at least 28 -day repeated dose toxicity study with the combined reproduction/developmental toxicity screening test in rats, performed according to OECD guideline and in accordance with GLP principles. Pinyl isobutyraldehyde was administered by daily oral (dietary admixture) to male and female Wistar Han rats at dose levels of 450, 1800 and 7200 ppm (10 rats/sex/dose level, intended doses were 37.5, 120 and 450 mg/kg bw). A control group of 10 rats/sex was given the vehicle (Basic powdered diet). Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated during 2 weeks prior to mating, during mating, during post-coitum, and up to the day before necropsy (i.e. on day 13 of lactation for females delivered, on day 25 of gestation for unmated females and on study Day 55 for failed to mate female).

Results

Dietary exposure analysis: Throughout the pre-mating period, the achieved intake of test item was generally lower, at all dose levels, than the theoretical concentrations for males and females (-18 to -31 %). During the gestation period, the achieved test item concentration was close to the nominal concentration at the low and mid dose level, whereas at the high dose level, the decreased achieved concentration ranged from -18 to -32 %. During the lactation period, due to higher food consumption in treated females, the achieved intake of test item was markedly higher than the theoretical concentrations (+31 to + 121 %). In addition, after 24-hour storage in the food hopper at room temperature, a decrease in test item formulation concentration of approximately -20 % was noted when compared with the nominal concentrations. The intake on a mg/kg bw bases was 21, 83 and 246 mg/kg bw using the lower intake for either males or females.

Clinical signs: There was no death or clinical sign during the study. There were decreased mean body weight gains and food consumption at 1800 and 7200 ppm in both sexes up to the end of the gestation period, leading to a relevant lower body weight at 7200 ppm (246 mg/kg bw).

Functional battery observations: There was no obvious effect of treatment on the auditory, pupillary and righting reflexes or open field tests for the males or females at any dose level. Lower gripping strength in treated rats was considered to be the consequence of slightly lower body weights and not a direct effect of the test item.

Heamatology, Clinical chemistry and T4 levels: There were no test item-related changes.

Organ weights and histopathological effects: For males and females at 7200 ppm (246 mg/kg bw), test item induced changes in organ weights a.o. increase in relative liver weight up to 25% without showing histopathological changes. At this 7200 ppm, relative kidney weights in males only were increased up to 18%. This increase was associated with tubular degeneration and tubular basophilia for males without hyaline droplets and therefore the effects were not considered to be due to alpha-hydrocarbon nephropathy. This means that this effect has to be taken into account for the derivation of the NOAEL.

Fertility and Developmental toxicity:There were no test item-related effects on mating performance for the males and females or on fertility in any group. There was no effect of treatment in length and regularity of the estrous cycle in any group. There were 7, 9, 9 and 8 females that completed delivery in the control, 450, 1800 and 7200 ppm groups 21, 83 and 246 mg/kg bw), respectively. There were no obvious test item-related effects on the number of implantation, pre-birth loss, litter size, pup viability or body weight. There were no test item-related effects on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. Anogenital distance (normalized for body weight) in male and female pups was not affected by test item.

Discussion

In view of the instability of the substance, the intake of the substance was lower than anticipated for all doses. The substance may have volatilised and/or degraded due to oxidation of the aldehyde. At 7200 ppm (246 mg/kg bw) slight (maximum -9%) body weight decrease was considered treatment related. Several organ weights were increased e.g. liver, thyroid and kidney. No histopathological effects were seen in most organs but in kidneys tubular effects were seen in males, which could not be attributed to alpha-hydrocarbon nephropathy in absence of hyaline (eosinophilic) droplets. Based on these results and specifically the histopathological effects in the kidneys, the dose of 1800 ppm, corresponding to at least 83 mg/kg bw/day, respectively, was considered as the parental No Observed Adverse Effect Level (NOAEL). For fertility no adverse effects were seen up to the highest dose level (7200 ppm corresponding to an actual intake of 246 mg/kg bw. For developmental toxicity no adverse effects were seen.

Conclusion

Parental NOAEL is 83 mg/kg bw based on actual substance intake from nominal 1800 ppm feed.

For fertility the NOAEL was > 246 mg/kg bw in absence of adverse effects based on actual substance intake from nominal 7200 ppm feed.

Developmental toxicity the NOAEL was > 246 mg/kg bw in absence of adverse effects based on actual substance intake from nominal 7200 ppm feed.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the present section the repeated dose, fertility and developmental toxicity is addressed to present the fertility and developmental toxicity in the context of the repeated dose effects and not to duplicate the information.

Repeated dose and Reproductive screening tested in the OECD TG 422

Introduction and Method

Potential toxic effects of the test substance after repeated exposure was investigated in an at least 28 -day repeated dose toxicity study with the combined reproduction/developmental toxicity screening test in rats, performed according to OECD guideline and in accordance with GLP principles. Pinyl isobutyraldehyde was administered by daily oral (dietary admixture) to male and female Wistar Han rats at dose levels of 450, 1800 and 7200 ppm (10 rats/sex/dose level, intended doses were 37.5, 120 and 450 mg/kg bw). A control group of 10 rats/sex was given the vehicle (Basic powdered diet). Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated during 2 weeks prior to mating, during mating, during post-coitum, and up to the day before necropsy (i.e. on day 13 of lactation for females delivered, on day 25 of gestation for unmated females and on study Day 55 for failed to mate female).

Results

Dietary exposure analysis: Throughout the pre-mating period, the achieved intake of test item was generally lower, at all dose levels, than the theoretical concentrations for males and females (-18 to -31 %). During the gestation period, the achieved test item concentration was close to the nominal concentration at the low and mid dose level, whereas at the high dose level, the decreased achieved concentration ranged from -18 to -32 %. During the lactation period, due to higher food consumption in treated females, the achieved intake of test item was markedly higher than the theoretical concentrations (+31 to + 121 %). In addition, after 24-hour storage in the food hopper at room temperature, a decrease in test item formulation concentration of approximately -20 % was noted when compared with the nominal concentrations. The intake on a mg/kg bw bases was 21, 83 and 246 mg/kg bw using the lower intake for either males or females.

Clinical signs: There was no death or clinical sign during the study. There were decreased mean body weight gains and food consumption at 1800 and 7200 ppm in both sexes up to the end of the gestation period, leading to a relevant lower body weight at 7200 ppm (246 mg/kg bw).

Functional battery observations: There was no obvious effect of treatment on the auditory, pupillary and righting reflexes or open field tests for the males or females at any dose level. Lower gripping strength in treated rats was considered to be the consequence of slightly lower body weights and not a direct effect of the test item.Heamatology, Clinical chemistry and T4 levels: There were no test item-related changes.

Organ weights and histopathological effects: For males and females at 7200 ppm (246 mg/kg bw), test item induced changes in organ weights a.o. increase in relative liver weight up to 25% without showing histopathological changes. At this 7200 ppm, relative kidney weights in males only were increased up to 18%. This increase was associated with tubular degeneration and tubular basophilia for males without hyaline droplets and therefore the effects were not considered to be due to alpha-hydrocarbon nephropathy. This means that this effect has to be taken into account for the derivation of the NOAEL.

Fertility and Developmental toxicity: There were no test item-related effects on mating performance for the males and females or on fertility in any group. There was no effect of treatment in length and regularity of the estrous cycle in any group. There were 7, 9, 9 and 8 females that completed delivery in the control, 450, 1800 and 7200 ppm groups 21, 83 and 246 mg/kg bw), respectively. There were no obvious test item-related effects on the number of implantation, pre-birth loss, litter size, pup viability or body weight. There were no test item-related effects on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. Anogenital distance (normalized for body weight) in male and female pups was not affected by test item.

Discussion

In view of the instability of the substance, the intake of the substance was lower than anticipated for all doses. The substance may have volatilised and/or degraded due to oxidation of the aldehyde. At 7200 ppm (246 mg/kg bw) slight (maximum -9%) body weight decrease was considered treatment related. Several organ weights were increased e.g. liver, thyroid and kidney. No histopathological effects were seen in most organs but in kidneys tubular effects were seen in males, which could not be attributed to alpha-hydrocarbon nephropathy in absence of hyaline (eosinophilic) droplets. Based on these results and specifically the histopathological effects in the kidneys, the dose of 1800 ppm, corresponding to at least 83 mg/kg bw/day, respectively, was considered as the parental No Observed Adverse Effect Level (NOAEL). For fertility no adverse effects were seen up to the highest dose level (7200 ppm corresponding to an actual intake of 246 mg/kg bw. For developmental toxicity no adverse effects were seen.

Conclusion

Parental NOAEL is 83 mg/kg bw based on actual substance intake from nominal 1800 ppm feed.

For fertility the NOAEL was > 246 mg/kg bw in absence of adverse effects based on actual substance intake from nominal 7200 ppm feed.

Developmental toxicity the NOAEL was > 246 mg/kg bw in absence of adverse effects based on actual substance intake from nominal 7200 ppm feed.

Effects on developmental toxicity

Description of key information

Developmental toxicity (OECD TG 422): NOAEL > 246 mg/kg bw/day (highest dose tested)

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016- 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other:
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated Dose 28-day oral toxicity study in rodents
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
no
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approximately 11 weeks, Females Approximately 13 weeks
- Weight at study initiation: Males: 345 to 401 g; Females: 196 to 247 g
- Fasting period before study: no
- Housing: One air-conditioned room in a barrier protected unit. Animal were housed in plastic cages meeting European directive 2010/63/EU requirements. Cellulose bedding (Serlab, Montataire, France) or dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood. Animals received a small amount (handful) of shredded paper (SDS/Dietex) and the isolated animals had free access to a wooden gnaw block (Aspen Bricks, Dietex France).
- Diet (e.g. ad libitum): Rat powdered complete diet ad libitum (Diet reference A04C10 P2.5) sterilised by irradiation. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
- Water (e.g. ad libitum): Softened and filtered (0.2 µm) mains drinking water was available ad libitum (via an automatic watering system or bottles).
- Acclimation period: 8 days between animal arrival and start of pre-test estrous cycle smears (females) or start of treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3 °C (target range)
- Humidity (%): > 35 % (target)
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 January 2017 To: 27 February 2017
Route of administration:
oral: feed
Vehicle:
other: Basic powdered diet (Diet reference A04C10 P2.5)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
A pre-mix preparation was performed: the test item and the vehicle were weighed. The test item was added slowly to the powder diet and mixed using a spatula. Then the mix preparation was performed: a part of the vehicle was placed into a mixer. The pre-mix preparation was added and the remaining powder diet was added. The formulation was mixed for at least 12 minutes (until a homogeneous mix was obtained).
Test item/diet mixes were formulated at least once weekly and aliquoted into daily aliquots stored frozen (between -15 and -25 °C).Daily aliquots were removed from the freezer just before delivery to the animal unit and were placed at refrigerated temperature (between +2 and +8 °C) and/or placed at
room temperature (between +15 and +25 °C) just before hopper filling. The temperature was outside the targets on 1 occasion (8.8 °C instead of 2-8 °C) during the study. This deviation did not noticeably affect the storage condition of the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity of the test item in the diet mix was confirmed between 250 to 10000 ppm (validation study).
Stability of the test item in the diet mix (250 to 10000 ppm): 48 hours stored refrigerated (2 to 4 °C) and 8 days stored frozen (-15 to -25 °C), in bags. However, the test item formulation was not stable for 24 hours at room temperature (15 to 25°C) in the food hopper (decrease in achieved concentration more than 10% of time 0 concentration) (validation study).
Analysis of preparations:The diets for each dose group including the control prepared to be distributed during the first week of the study were analysed within the stability period for achieved concentrations. Duplicate (2x 30 g) samples were taken from each formulation from the middle of the bulk diets on the day of preparation.The first set of samples was analysed at the Test Facility using a validated method. The transfer and validation of the analytical method was the subject of a separate study plan. Samples were stored frozen (-15 to -25 °C) up to analysis.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as Day 0 of gestation (G0)
- After successful mating each pregnant female was caged (how): individually
Duration of treatment / exposure:
males:14 days before mating, throughout the mating period and up to the day before necropsy (i.e. 31 days for males)
females:14 days before mating, throughout the mating period and up to the day before necropsy.
During gestation (the first day of gestation was designated as G0) and 14 days after parturition up to and including the day before necropsy (the first day of birth is designated as L0). Apparently unmated females for 26 days after the last day of the mating period.
Frequency of treatment:
Continuously
Duration of test:
males:14 days before mating, throughout the mating period and up to the day before necropsy (i.e. 31 days for males)
females:14 days before mating, throughout the mating period and up to the day before necropsy.
During gestation (the first day of gestation was designated as G0) and 14 days after parturition up to and including the day before necropsy (the first day of birth is designated as L0). Apparently unmated females for 26 days after the last day of the mating period.
Dose / conc.:
450 other: ppm; nominal 37.5 mg/kg bw
Dose / conc.:
1 800 other: ppm; nominal 150 mg/kg bw
Dose / conc.:
7 200 other: ppm; 600 mg/kg bw
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: in the preliminary study, test item given in diet at 9000 ppm induced body weight loss during the first days and a decreased body weight gain (associated with the decreased food consumption) over the 14 days of dosing, when compared with the control group. Therefore, the dose of 9000 ppm was considered a maximum tolerated dose for the preliminary 14-day study but was considered too high for a study of longer duration and with pregnant and lactating females. Therefore, for this study, the mid dose of 1800 ppm used in the preliminary study was kept and a factor of 4 was used between each dose (instead of 5 in the preliminary study). Thus, the doses of pinyl isobutyraldehyde in this study were 450, 1800 and 7200 ppm.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights for females were recorded weekly during pre-mating and mating periods (only pre-mating data are reported), on Days 0, 4, 7, 11, 14, 17 and 20 of gestation, and on Days 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 14 of lactation (Day 26 post-coitum for mated females that failed to produce a viable litter; Day 56 for apparently unmated female)
- Organs examined: according to Guideline
Ovaries and uterine content:
Vaginal smears were taken daily and used to determine the stage of estrous for each female from two weeks before treatment, the first 14 days of treatment and during mating until identification of mating or separation from the male.
On the day of necropsy, a vaginal smear was taken to determine the stage of the estrous cycle and allow correlation with histopathology of female reproductive organs.
Weights of ovaries and uterus were determined.
Fetal examinations:
For each F1 litter the following data were recorded:
 number of pups born (live and dead)
 external abnormalities of the pups
 number, weight and sex of pups on Days 1, 4, 7 and 13 post-partum
 Anogenital distance (AGD) was measured on PND1. The AGD was normalized to the cube root of body weight.
 All males in each litter were examined for the number of areola/nipples on PND13.

Blood samples were collected from two surplus pups where possible, pooled and used for determination of serum T4 levels. No pups were eliminated when litter size dropped below the culling target (8 pups/litter). When there is only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible serum T4 assessments.
Statistics:
The best transformation for the data (none, log or rank) was determined depending upon  the kurtosis of the data  the probability of the Bartlett's test for homogeneity of the variances and  an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
The locomotor activity in an open field and the estrous cycle, pre-coital interval and anogenital distance data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test. Microsoft Excel® 2010 was employed to present certain results and perform associated calculations.
Indices:
Pre-coital interval (in days): sum of days until successful insemination/ number of inseminated females
Irregularity index: Mean standard deviation of length of the oestrous cycle/ √ (length of the oestrous cycle)
Days in estrus: (number of estrus days/ number of smears) x100
Copulation index (%): (number of inseminated females/ number of paired animals) x100
Fertility index (%): (number of pregnant females/ number of inseminated females) x100
Pre-birth loss (%): (number of implantations - number of offspring born/ number of implantations) x100
Live birth index (in %): (number of pups born alive/ number of pups born) x 100
Viability index (in %): (number of pups alive on PND 4 (before culling)/ number of pups alive at birth) x 100
Lactation index (%): (number of pups alive on PND 13/ number of pups alive on PND 4 (after culling)) x 100
Sex ratio (proportion of male) (in %): (number of males/ number of pups) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
Torn claw, localized hairloss, sores, scabs or nodule on the tail were considered incidental.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths among F0 rats.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During the pre-mating period, there was a lower mean body weight gain for all treated female groups during the first 8 days of dosing when compared with the control group. Changes were not dose-related.
During the gestation period (G0 to G20), there was a dose-related lower mean body weight gain at 1800 ppm (mainly between G0 and G14) and at 7200 ppm, when compared with the control group.
During the lactation period, the mean body weight change between L1 and L13 was similar for all groups, although a mean body weight gain at 1800 and 7200 ppm slightly lower than controls between L1 and L4.
These decreased in body weight gains induced minor effects on body weight at 1800 ppm (-4 % and -2% at the end of the gestation (G20) and lactation (L13) periods, respectively, for females).
(see tabel mean body weight changes of females in section any other results including tabels)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a lower mean food consumption for females treated at 7200 ppm between Days 1 and 4 during the pre-mating period (up to -44 %) and a dose-related lower mean food consumption for females treated at 1800 and 7200 ppm between Gestation Days 1 and 20 (-11 and -21 %, respectively), compared with the control group.
During the lactation period, there was no test item effect in food consumption in any group. At 450 and 1800 ppm, the mean food consumption was even higher than in controls (+22% and +14 %, respectively) and at 7200 ppm, it was comparable with controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects in haematology and coagulation parameters in any group. The slightly lower mean fibrinogen concentration for females treated at 1800 and 7200 ppm was considered of no toxicological relevance in view of the low magnitude of the change and since mean values were within the background control range.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no relevant test item-related effects in serum clinical chemistry parameters in any group. The following statistically significant differences compared with controls, were considered of no toxicological significance in view of their low magnitude or since mean values remained close or within the normal historical control range:
- increased individual phosphorus levels in 2 females at 1800 and 3 females at 7200 ppm.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There was a dose-related lower mean grip strength in all treated groups for both sexes compared with the control groups (-7%, -30% and -27% in males dosed 450, 1800 and 7200 ppm, respectively, and
-16%, -27% and -38% in females dosed 450, 1800 and 7200 ppm, respectively). Lower body weights in treated groups might be the cause of lower strength in treated groups. This effect was therefore
considered most probably incidental.
There were no test item-related changes in auditory, pupillary or righting reflexes.
There were no test item-related changes in open field data for males and females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher mean absolute and relative liver weights were noted in males (+24%) and females (+26%) at 7200 ppm, as were thyroid with parathyroid glands (+25%) and kidney (+18%) weights in males at the same dose. Lower mean absolute and relative spleen (-18%), ovaries (-18%) and uterus (-24%) weights were seen in females at 7200 ppm. Of these changes, only the kidney weight variation could be accounted for histologically by tubular degeneration and basophilia, noted in all males at 7200 ppm only. Although the liver and thyroid weight variations had no histological correlate, they are likely to be attributable to an adaptive hepatocellular hypertrophic change with secondary thyroid follicular cell hypertrophy.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic observations that were considered to be associated with administration of the test item for treated males and females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
observed in male rats only
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Observations in males only: Slightly higher total T4 levels were noted for F0-males given 450 and 1800 ppm and for male PND 13 pups in the 450 ppm dose group, compared with concurrent control animals.

There was no effect of treatment in length and regularity of the estrous cycle in any group. There was an acyclic female (no. 74) in the 7200 ppm group, before mating during the treatment period. This isolated finding was considered incidental.

There was no test item-related effect on mating performance in any group. All animals mated with the exception of one pair in the 450 ppm group. All mated females showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal estrous cycle). The mean pre-coital interval was therefore normal (less than 4 days) in all groups.
There was no effect of treatment on fertility in any group. There were 3, 1 and 2 mated females in the control, 1800 ppm and 7200 ppm groups respectively that did not become pregnant. These isolated cases, including controls, were considered incidental.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The percentage of pre-birth loss was slightly higher in the 7200 ppm group (11.86 %) compared with the concurrent control (7.16 %) but remained comparable with the historical control data range (2.5 to 13.4 %, calculated with the pre-birth loss formula). Moreover, individual values did not indicate any test item-related trend. This change was therefore considered as unrelated to test item.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
There were 2 litters in the 1800 ppm group with 1 and 4 stillborn pups, respectively, compared with none in the concurrent control group. With no similar finding in the high dose group, this was considered incidental. The mean live litter size and live birth index were comparable in all groups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 800 ppm
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean pup body weight was lower for pups at 7200 ppm than the mean pup control body weight. The difference increased during the lactation period. The mean body weight was approximately 14 % lower than controls for both sexes on PND 13. This difference may be related to the lower mean body weight of dams treated at 7200 ppm, during the gestation and lactation periods.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The mean live litter size and live birth index were comparable in all groups.
The viability and lactation indices were comparable in all groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no test item-related effect on the sex ratio.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
The mean pup body weight was lower for pups at 7200 ppm than the mean pup control body weight. The difference increased during the lactation period. The mean body weight was approximately 14 % lower than controls for both sexes on PND 13. This difference may be related to the lower mean body weight of dams treated at 7200 ppm, during the gestation and lactation periods.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The mean live litter size and live birth index were comparable in all groups.
The viability and lactation indices were comparable in all groups.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related effects on areola/nipple retention. No nipples were observed in the examined male pups at PND 13.
The mean anogenital distance (normalized for body weight) in male and female pups was comparable in all groups.
Slightly higher total T4 levels were noted for male PND 13 pups in the 450 ppm dose group, compared with concurrent control animals. These differences reached statistical significance, but were not considered test item-related in the absence of a dose-related distribution, because no increase in total T4 levels were observed in the animals in the 7200 ppm groups.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 7 200 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of mean achieved concentrations throughout the study period (deviations from theoretical concentrations in brackets)

Period

Target dose level in mg/kg BW/day (dose in ppm)

37.5 (450)

150 (1800)

600 (7200)

Pre-mating

D1-D8

Males

 

31

(- 18 %)

 113

(- 25 %)

445

(- 26 %)

Females

30

(- 19 %)

165

(+ 10 %)

413

(- 31 %)

Pre-mating

D8-D15

Males

 

27

(- 28 %)

 104

(- 25 %)

412

(- 31 %)

Females

28

(- 27 %)

115

(- 23 %)

465

(- 22 %)

Gestation

G0-G20

(Females)

Range

32 to 36

(- 3 to – 15 %)

114 to 136

(- 10 to – 24 %)

410 to 492

(- 18 to - 32 %)

Lactation

L1-L4

(Females)

Range

56 to 83

(+ 51 to + 121 %)

214 to 309

(+ 43 to + 106 %)

784 to 1167

 (+ 31 to + 95 %)

 Actual lowest dose   

 Female

 21

 83

 323

 Male

 21

 94

 246

Formulation analysis:All analyzed samples of fomulations prepared at nominal concentrations of 450, 1800 and 7200 ppm of Pinyl iso Butyraldehyde alpha in basic powder diet (A04C10 P2.5) taken from each preparation, including the control, one day during the first week of treatment were in agreement with acceptance criteria (± 20 %), except group 2 formulation at 450 ppm showing a deviation from nominal concentration of -20.2 % slightly above acceptance criteria of +/-20 %. The deviations from the nominal concentrations of formulation at 1800 and 7200 ppm were -18.0 % to -18.4 %. Investigation of the result outside acceptance criteria did not allowed identifying a root cause which may explain the low concentration observed. Duplicate sample from group 2 were not reanalyzed as stability of test item was limited to 5 days at room temperature (+15 to +25 °C) in open feeder and showed degradation for further duration storage.

No test item was present in the vehicle sample.

Achieved dose:Throughout the pre-mating period, the achieved intake of test item, calculated from the body weight and food consumption data, was lower, at all dose levels, than the theoretical concentrations, for males and females (-18 to -32 %), except at the mid-dose where there was a +10 % higher achieved dose during the first week for females only.

During the gestation period, the decreased achieved intake of test item was more slight at the low and mid dose level (-3 to – 24 %), than during the pre-mating period. At the high dose level, the decreased achieved concentration ranged from -18 to -32 %.

During the lactation period, due to higher food consumption for treated females (see section 11.7), the achieved intake of test item was markedly higher than the theoretical concentrations (+31 to + 121 %).

In addition, after 24-hour storage in the food hopper at room temperature, a decrease in test item formulation concentration of approximately -20 % was noted when compared with the nominal concentrations.

  Summary of salient mean body weight changes of females (in g - percent difference from concurrent controls in brackets)

Period

Dose level (ppm)

0

450

1800

7200

Pre-mating

D1-D8

7.26

2.62 *

(- 64 %)

4.77 *

(- 34 %)

1.04 ***

(- 86 %)

Gestation

G0-G20

110.34

-

99.83

(- 10 %)

78.88 ***

(- 29 %)

Lactation

L1-L4

14.33

-

10.83

(- 24 %)

11.44

(- 20 %)

* p<0.05; *** p<0.001

Conclusions:
Based on the results in this study, the reproduction and developmental NOAEL was defined at 7200 ppm, corresponding to at least 246 mg/kg bw/day.
Executive summary:

In the study record on fertility also the developmental toxicity is presented and will not be repeated here.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The information is presented in the fertility section.

Mode of Action Analysis / Human Relevance Framework

In absence of fertility and developmental toxicity in the OECD TG 422 the substance does not have a specific mode of action for reproductive toxicity.

Justification for classification or non-classification

Based on the available data and in absence of adverse fertility and developmental toxicity effects Pinyl isobutyraldehyde does not need to be classified for these reproductive toxic effects in accordance with EU CLP (EC no 1272/2008 and its amendments).