Benzoic acid, C12-15-alkyl esters

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Feb 2021 - 27 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

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:

adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, C12-15-alkyl esters

EC Number:

270-112-4

EC Name:

Benzoic acid, C12-15-alkyl esters

Cas Number:

68411-27-8

Molecular formula:

C19-22 H30-36 O2

IUPAC Name:

Benzoic acid, C12-15-alkyl esters

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females nulliparous and non-pregnant: yes
- Age at receipt: approximately 7 to 8 weeks
- Weight at receipt: 212 to 229 g (males), 178 to 208 g (females)
- Housing: from arrival to pairing animalswere housed up to 5 of one sex to a cage;
during mating animals were housed one male to one female;
after mating the males were re-caged as they were before mating; te females were transferred to individual cages
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 28 days (main groups) and 42 days (recovery groups)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C±2 °C
- Humidity (%): 55%±15%
- Air changes (per hr): approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was weighed and suspended in the vehicle. The preparations were made daily (concentrations of 25, 75 and 250 mg/mL) or at weekly interval, in agreement to the stability data. Dosing preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing. Concentrations were calculated and expressed in terms of test item as supplied.

dose volume: 4 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation:
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in the range from 25 to 250 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation study (r > 0.99; accuracy 80-120%; precision CV < 10%).

Duration of treatment / exposure:

males: for a minimum of 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy, for a total of 42/43 days.

females: for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum for a period comprised from 51 to 65 days

Frequency of treatment:

daily

Details on study schedule:

- F1 animals were not mated (screening study)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10; additional 10 animals in recovery groups (control and high dose)

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and once a week thereafter
- observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). All observations were recorded for individual animals. Animals were examined in an open arena for a minimum of three minutes.

CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during treatment

BODY WEIGHT: Yes
- Time schedule for examinations:
Main groups: Males: weekly from allocation to termination.
Females: weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.
Recovery groups: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
Main groups
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing up to mating. Individual food consumption for mated females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

Recovery groups
The weight of food consumed by each cage of rats was recorded at weekly intervals starting from the first day of dosing


OTHER:
Hematology (for details, see Iuclid section 7.5)
clinical chemictry (for details, see Iuclid section 7.5)
Urinanalysis (for details, see Iuclid section 7.5)
Grip strength and sensory reactivity to stimuli (for details, see Iuclid section 7.5)
Motor activity assessment (for details, see Iuclid section 7.5)

Oestrous cyclicity (parental animals):

main groups:
Vaginal smears were taken in the morning starting from Day 1 of dosing up to positive identification of copulation was made. The vaginal smear data was examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of
mating)

Before despatch to necropsy -Main groups:
Vaginal smears were also taken from all females, before despatch to necropsy and the oestrous cycle phase recorded

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, weight gain, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, Thyroid hormone determination

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, after the mating of all females on Days 43/44
- Maternal animals: All surviving animals, females with live pups were killed on Day 14 post partum
- recovery groups: Animals were killed after 4 weeks of recovery

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

All main group females were examined also for the following:
– number of visible implantation sites
– number of corpora lutea

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the table below were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- Pups that had completed the scheduled test period (Days 4 or 14 post partum)

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
Gonads were inspected from all pups in order to confirm the sex previously determined by external examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered. The thyroid weight was determined after fixation.

Statistics:

Standard deviations were calculated as appropriate.
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups were assessed by the nonparametric version of theWilliams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Reproductive indices:

Copulatory Index, Fertility Index, Pre- implantation loss, Pre-natal loss

Offspring viability indices:

Post natal loss at day 0 post partum, Post natal loss at Day 4 post partum (before culling), Post natal loss at Day 14 post partum (after culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
No treatment-related clinical signs were noted in males and in females of the main groups before the mating period and thereafter during post coitum and post partum periods.
Clinical signs observed at daily intervals were limited to common conditions of the skin and fur (scabs, and/or staining on the body surface, missing of the tail (tip) found in males or females of control and in those receiving 100 or 300mg/kg/day. In any case no more than two animals were affected.

Recovery groups
No clinical signs were recorded in treated animals throughout the study, during treatment and recovery periods.
A subcutaneous mass on the mammary area was observed in one female previously dosed at 1000 mg/kg/day (no. X0164097) on Day 29 of the recovery period. This was, however not considered treatment related.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
No significant changes in body weight and body weight gain were observed during the study in the treated males, when compared to controls.
Body weight and body weight gain for female animals were generally comparable between the treated and control groups, before mating and during gestation and post-partum periods.
Before pairing, on Day 8, females receiving 1000mg/kg/day showed a decrease in body weight gain (-45%) compared to the control group. This isolated change was followed by a regular growth of the animals and, hence, it was not considered to be toxicologically relevant.
The negative gain weight noted in all groups on the last measurement was due to the fact that the animals were placed under condition of food deprivation for blood collection.

Recovery groups
During treatment or recovery periods, no relevant differences in body weight and body weight gain were noted between control and treated group, of both sexes.
The differences in body weight gain noted in males receiving 1000 mg/kg/day on Day 8 of treatment was considered unrelated to treatment. Infact, on Day 1 of treatment the mean body weight difference between control and treated groups was already evident (467 g. of Group 6 vs 478 g. of control group).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Main groups
Food consumption was unaffected by treatment in both genders during the study.

Recovery groups
During treatment and recovery periods, no differences were noted in food consumption between control and treated group in both sexes receiving 1000 mg/kg/day.

Haematological findings:

no effects observed

Description (incidence and severity):

for details see Iuclid section 7.5

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

for details see Iuclid section 7.5

Urinalysis findings:

no effects observed

Description (incidence and severity):

for details see Iuclid section 7.5

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

for details see Iuclid section 7.5

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Main groups:
There were no treatment-related microscopic observations at the end of the treatment period.
Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.

Other effects:

no effects observed

Description (incidence and severity):

The determination of T3, T4 and TSH was performed on:

- Samples from all parental males and females from all main groups

- Samples from pups on Day 14 post partum

Parental main groups animals:
No treatment-related changes were recorded. T4 was statistically significantly higher than controls in females dosed at 1000mg/kg/day (19%). Since T4 values were within the range of historical data and no other related changes were recorded (i.e. TSH decrease, thyroid weight or liver/thyroid histopathological changes), this finding was considered to be incidental and judged to be due to biological variation.

Pups:
No treatment-related changes were recorded.
TSH was higher than control in pups of both sexes of Dam no. 67. Due to the minimal incidence, this finding was considered to be incidental.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The total number of oestrous cycles observed in all females before pairing (number of non sequential days in which the females were in oestrous) was similar between control and treated groups and had a mean value of 3/4 times.
Vaginal smears examined on the day of necropsy to determine the stage of the oestrous cycle showed the phase of dioestrous for all females sacrificed on Day 14 post partum.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No treatment related effect was noted on the spermatogenic cycle.
Regular layering in the germinal epithelium was noted, when seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages.

Reproductive performance:

no effects observed

Description (incidence and severity):

Three females, one for each treated group (nos. X1640023, X1640047 and X1640069), although without any positive signs of copulation during the mating phase (mating was not detected) were found pregnant.
The number of females with live pups on Day 14 post partum was 10 in the control group, in the group dosed at 100mg/kg/day, 300mg/kg/day and 1000mg/kg/day.

During the first session of 14 days, the mating was not detected for four females (nos. X1640009, X1640023, X1640047 and X1640069, respectively for Groups 1, 2, 3 and 4). The males were changed within each treatment group and the second pairing combination was monitored until positive signs of copulation was observed. On the basis of the increasing in the body weight the second mating session of the females of the treated groups was stopped after 7 days. These females gave birth after 2-5 days from the end of the second cohabitation suggesting that the mating had been successful with the first assigned male.
At macroscopic observations, no abnormalities were noted in any of the males reported above.
Pre-coital interval values of the treated females were comparable to the control. The majority of females had a spermpositive identification between 1-5 days of cohabitation.
The number of copulation plugs (3 as mean value) was similar between control and treated groups.
Copulatory and fertility indices were considered to be unaffected by treatment with the test item.


Mean gestation period was comparable between control and treated animals, being of 22/23 days.
Corpora lutea, number of implantations sites and live litter size did not show dose-related or treatment-related differences, compared to controls.
All females, both in control and treated groups showed the value of pre-implantation loss equal to zero, with the exception of one female (no. X1640069) receiving 1000 mg/kg/day (25%). This isolate case confirmed that the effect was incidental and not treatment-related.
Pre-natal loss (percentage) was similar between control and treated groups.


The percentage of males in the group receiving 1000mg/kg/day was slightly higher, when compared to the control values at birth, on Days 4 and 14 post partum. In addition the increase became statistically significant on Day 14 post partum for the absolute value. This increase was balanced by a lower number of female pups, so that a comparable total number of pups (for both sexes combined) between control and treated groups, was evident.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

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):

Cold to the touch, apparently no food intake (milk) and small appearance, were in general the mainly clinical signs noted in control and/or treated pups.
These findings are commonly observed in the litters during the lactation period but an increase, in terms of litters affected, was noted in the high dose group. Some pups resulted found dead or missing, possibly for cannibalization after death. One single pup in two different litters (litter nos. 63 and 71) showed some findings (e.g. small in size and/or apparently no food intake and/or cold to touch) during the first days of lactation. In both cases these pups were found dead or missing shortly after birth. In the other cases the findings were occasionally seen with a trend of recovery (litter nos. 67 and 79). Only in one female (no. 65) more than half of its litter was reduced during the first days after birth in the absence of maternal toxicity. It remained unclear, whether this observed effect was treatment-related.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Litter data - Day 0 to Day 4 post partum - before culling:
At birth, the values of post-natal loss (expressed as percentage) of all females were equal to zero in Groups 2 and 4 .
The increase in mean post-natal loss observed in Group 3 females, compared to the control, was attributed to animal no. 49. On Day 1 of lactation, this female had only one live pup (female) compared to the 8 at birth.
On Day 4 post partum, an increase, not significant at statistical analysis, in post natal loss (expressed as mean percentage) was evident in females exposed at the dose level of 1000 mg/kg/day against to control group. A relation to treatment was considered to be unlikely, in view of the comparison with the reference control data.
Moreover looking to the individual data, the litter size of one female (no. 65) was reduced (7 pups on Day 4 vs 16 on the day of birth) by more than half compared to the day of birth.
Infact, during the first days after birth, all the puppies were cold to the touch and with apparently no food intake (milk). Consequently litter weight were slightly below to control of about 14%, but the mean pup weight remaining comparable since the difference was of slight entity (-9%).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

On Day 13 post partum, litter weight and mean pups weight (for both sexes combined) of treated groups were similar with control.
Female no. 65 still showed a reduction in its litter size, litter weight and mean pup weight.
The decrease in live litter size of female pups found at 1000mg/kg/day was associated to the statistical significant decreased in litter and mean pup body weights in the same group.
In particular for two dams (nos. 65 and 69) only one female pup was found in their litter, on Day 13 post partum. On the contrary the litter size for male pups was increased compared to control reaching a significance, at statistical analysis.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No differences in the anogenital distance (value normalized to the cube root of bodyweight), performed on Day 1 post partum, were seen between control and treated groups both for male and female pups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

On Day 13 post partum, the ventral region of male pups was checked for presence of nipples/areolae. At necropsy, on Day 14 post partum, no nipples were found in male pups to be retained.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No differences were observed in the weight of thyroid in treated pups, when compared to controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Autolysed abdominal organs were generally observed in pups found dead at check carried out after birth and in pups which died during the lactation period. Autolysed process, normally occurs when pups are found dead some time after death, therefore it is not considered to be treatment-related. No significant findings were seen in pups killed on Days 4 and 14 post partum.

Description (incidence and severity):

The percentage of males in the group receiving 1000mg/kg/day was slightly higher, when compared to the control values at birth, on Days 4 and 14 post partum. In addition the increase became statistically significant on Day 14 post partum for the absolute value.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

SUMMARY OF REPRODUCTION DATA – GROUP DATA

	Dosage Levels mg/kg/day
Observations	0	100	300	1000
No. Dams Inseminated	10	10	10	10
Oestrus cycle (mean number)	4	3	4	3
No. Dams That Conceived	10	10	10	10
Percent Dams Conceived	100	100	100	100
Conceiving (1-5 days)	9	9	8	8
Conceiving (6-15 days)	1	0	1	1
Mating not detected	0	1	1	1
No. Dams Died During Study	0	0	0	0
No. Dams with Resorptions only	0	0	0	0
No. of Litters	10	10	10	10
Total No. Corpora Lutea	170	176	164	164
Mean No. Corpora Lutea/Pregnancy	17.0	17.6	16.4	16.4
Total No. Implantation	170	176	164	161
Mean No. Implants/Pregnancy	17.0	17.6	16.4	16.1
Mean No. Resorptions/Pregnancy	0	0	0	0
Pre implantation Loss (%)	0	0	0	2.5
Pre natal Loss (%)	15.2	5.5	18.3	10.2

 

SUMMARY OF LITTER DATA – GROUP DATA

	Dosage Levels mg/kg/day
Observations	0	100	300	1000
Paired (no.)	10	10	10	10
Dams with live pup at birth (no.)	10	10	10	10
Dams with live pups at Day 4 pp (no.)	10	10	10	10
Live litter size at birth (mean)	14.4	16.6	13.0	14.6
Live litter size at Day 4 (mean)	14.0	16.5	12.5	13.1
Sex ratio (%) at birth (mean)	46.7	54.3	50.3	53.8
Sex ratio (%) at Day 4 (mean)	46.3	54.6	47.7	55.4
Litter weight at Day 1 (mean)	111.0	127.3	108.7	99.6
Litter weight at Day 4 (mean)	157.7	177.7	154.4	135.2
Pup weight at Day 1 (mean)	7.9	7.7	8.0	7.3
Pup weight at Day 4 (mean)	11.5	10.8	11.6	10.4
Pup weight at the Day of AGD (Day 1pp)
mean males	8.14	7.86	8.25	7.50
mean females	7.68	7.54	7.89	6.95
Pup AGD normalized (Day 1pp)
mean males	2..35	2.48	1.30	2.45
mean females	1.34	1.41	1.30	1.36
Male pup nipple present at Day 13 (no.)	0	0	0	0
Pup weight at Day 13 (mean) – Combined sex	36.4	35.5	35.7	33.2
PUPS WITH MAJOR ABNORMALITIES AT NECROPSY
Dams with 0	0	0	0	0
Dams with 1	0	0	0	0
Dams with >/= 2	0	0	0	0
Pre-natal loss (implantations minus live births)(no.)
Females with 0	1	2	4	3
Females with 1	2	7	1	2
Females with 2	2	0	2	2
Females with >/=3	5	1	3	3
Post-natal loss (live births minus live at Day 1) (no.)
Females with 0	9	10	7	7
Females with 1	1	0	3	1
Females with 2	0	0	0	0
Females with >/=3	0	0	0	2
Post-natal loss (live births at Day 4 after culling minus live Day 14) (no.)
Females with 0	9	10	10	9
Females with 1	1	0	0	1
Females with 2	0	0	0	0
Females with >/=3	0	0	0	0

 

Applicant's summary and conclusion

Conclusions:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.

Executive summary:

In a a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD Guideline 422 (adopted on 29 July 2016) Sprague Dawley rats of both sexes were treated with repeated doses of Benzoic acid, C12-15-alkyl esters to assess any general systemic toxic effects of the test item on the animals, as well as possible particular effects on male and female reproductive performance, such as gonadal function, mating behaviour, conception, parturition and early lactation of the offspring related were investigated.

Upon conclusion of the treatment, a 4 week treatment-free period was allowed for groups 5 and 6 in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

The vehicle was corn oil. All doses of 100, 300 and 1000 mg/kg/day were administered at a constant volume of 4 mL/kg body weight.

 

Main groups

Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 42/43 days. Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, for a period comprising from 51 to 65 days.
The following investigations were performed in all groups: mortality check, determination of body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology comprising coagulation, and clinical chemistry in five animals/sex/group randomly selected, urinalysis in males only), macroscopic observations
determination of organweights. In addition, for main phase groups only (parental animals), oestrous cycle evaluation for parental females (2 weeks before dosing, during pre-mating and mating phases, prior to necropsy), assessment of mating performance, thyroid hormone
determination (males and females) and the assessment of litter data were performed. The assessment of clinical signs and measurement of the anogenital distance were performed in all pups. All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum and those sacrificed on Day 14 post partum were subjected to an external examination and the sex was determined by internal gonads inspection.
Thyroid weight and thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.
Routine histopathological examination was performed only in control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in five males.

 

Recovery groups

Recovery males were treated for up to 4 consecutive weeks and killed after 4 further weeks of recovery (treatment free) period.
Recovery females were treated for up to 9 weeks. The females were sacrificed after 4 further weeks of recovery (treatment free) period.
The following parameters were evaluated in the recovery group animals: mortality check, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to stimuli), body weight and food consumption. Clinical pathology investigations (haematology comprising coagulation, clinical chemistry and urinalysis for females only) at the end of treatment, macroscopic observations and organ weights were also determined.

 

Mortality and fate of females

No mortality occurred throughout the study. The number of females with live pups on Day 14 post partum was 10 of 10 in the control group and in the treated groups at all dose levels.

 

Clinical signs

Main groups and Recovery groups

No treatment-related clinical signs occurred during the study.

 

Neurotoxicity assessment (removal of animals from the home cage and open arena)

Main and recovery groups

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal any changes attributable to the test item.

 

Motor activity, Grip strength and sensory reactivity to stimuli

Main and recovery groups

No treatment-related alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment or recovery period.

 

Body weight and body weight gain

Main groups

No relevant differences were found in body weight and body weight gain between control and treated males and females throughout the study.

 

Recovery groups

During treatment or recovery periods, no differences in body weight and body weight gain were noted between control and treated group of both sexes.

 

Food consumption

Main groups

No effects on food consumption were observed in either males or females during the treatment period.

 

Recovery groups

In recovery animals no relevant changes were noted in food consumption during the treatment or recovery period.

 

Haematology

Main groups

No changes of toxicological significance were recorded in haematological parameters and coagulation.

 

Clinical chemistry

Main groups

No treatment-related changes at clinical chemistry examinations were recorded between control and treated groups.

 

Urinalysis

Main groups males

No treatment-related changes were recorded.

 

Recovery groups females

No treatment-related changes were recorded.

 

Thyroid hormone determination

Adult males and females

No treatment-related changes were recorded in parental males and females.

 

Pups

No relevant changes were recorded in pups of both sexes at Day 14 post partum.

 

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital intervals and copulatory index did not show any differences between groups. Fertility and copulatory indexes of males and females were similar in all groups.

 

Implantation, pre implantation loss, pre-natal loss data and gestation length of females

Main groups

Gestation periods were similar in treated groups and controls. All pregnant dams gave birth between Days 22 and 23 post coitum. Corpora lutea, number of implantations sites, live litter size, pre-implantation and pre-natal losses of treated groups appeared comparable to control values.

 

Litter data at birth, on Day 1, Day 4 (before culling) and Day 13 post partum of females and sex ratio of pups

Litter data

No relevant differences were observed in litter data between control and treated groups from the day of birth and Day 4 post partum.
After culling litterweight and mean pupsweight (for both sexes combined) of treated groups remained similar to control.
On Day 13 post partum, the statistically significant increase of the presence of male pups in the litters of the group dosed with 1000mg/kg/day matched the decrease of the presence of female pups. A decrease in litter weight and mean body weight of females pups was observed. Only one female pup was present in the litters nos. 65 and 69.

 

Sex ratio

The percentage of males in the group receiving 1000mg/kg/day was slightly higher, when compared to the control values at birth, on Days 4 and 14 post partum. A statistically significant increase in the absolute value of male pups was noted on Day 14 post partum. This increase was balanced by a lower number of female pups, so that a comparable total number of pups (for both sexes combined) between control and treated
groups, was evident.

 

Clinical signs of pups

The pre-weaning clinical signs found in control and treated groups are commonly observed and were comparable between treated and control groups, although an increase in terms of litter affected was noted in Group 4 compared to control. One single pup in two different litters (litter nos. 63 and 71) showed some findings (e.g. small in size and/or apparently no food intake and/or cold to touch) during the first days of lactation. In both cases these pups were found dead or missing shortly after birth. In the other cases the findings were occasionally seen with a trend of recovery (litter nos. 67 and 79). Only in one female (no. 65) more than half of its litter was reduced during the first days after birth in absence of maternal toxicity. It remained unclear whether this observed effect was treatment-related.

 

Anogenital distance and nipple count

No differences in the anogenital distance (normalised value), that can be related to treatment, were noted in pups on Day 1 post partum.

 

Pups thyroid weights

No significant differences were observed at statistical analysis in the weight of thyroid in treated pups, when compared to controls.

 

Necropsy findings in pups and nipple check

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 14 post partum did not reveal any treatment-related effect. No nipples were observed in male pups.

 

Terminal body weight and organ weights

Main and recovery groups

No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls.

 

Macroscopic observations

Main and recovery groups

No remarkable differences were noted at post mortem examination in treated animals, when compared to the controls.

 

Microscopic observations

Main groups

No treatment-related changes were observed in treated animals, when compared to the controls.

 

Conclusion

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000 mg/kg/day for males and females.