Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Remarks:
(Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 August 2019 - 24 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Condensation products of phenol and salicylaldehyde
EC Number:
947-320-5
Molecular formula:
Variable as UVCB substance
IUPAC Name:
Condensation products of phenol and salicylaldehyde
Test material form:
solid: flakes
Details on test material:
Description: Red-reddish brown solid flake
Storage conditions: Controlled room temperature (15-25 °C, ≤70 % relative humidity)

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adults, approximately 12 weeks old at the start of treatment and 14 weeks old at mating.
- Weight at study initiation: Males: 424 – 488 g, females: 245 – 314 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
- Housing: Type II and III polycarbonate cage type. Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed (with pups), respectively.
- Diet: ad libitum
- Water: tap water from the municipal supply, as for human consumption from 500 mL bottles, ad libitum.
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY
- Food: the food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Water: water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8200 Veszprém, József Attila u. 36., Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 – 26.3 °C (target range 22 ± 3 °C)
- Humidity (%): 30 – 76 % (target range 30 – 70 %)
- Air changes (per hr): 15 – 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated at appropriate concentrations in the vehicle (as a visibly stable homogenous formulation). Formulations were prepared up to 8 days before use (formulation were kept closed, at room temperature until use).

VEHICLE
- Justification for use and choice of vehicle: Propylene glycol was selected as vehicle for this study based on the formulation and analytical trials. The same vehicle was used in the Dose Range Finding (DRF) study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
> Stability of the test material in the vehicle was assessed in the conditions employed on the study during the analytical method validation. In that study, the formulation samples in the 0.5-25 mg/mL concentration range (using propylene glycol as vehicle) were proven as being stable for at least 8 days when stored at 20 ± 5°C.

> Analysis of test material formulations for concentration and homogeneity was performed using a validated HPLC-UV method:
- Sampling: Duplicate samples of approximately 0.5 mL, accurately weighed were taken from the top, middle and bottom of the test material formulations three times during the study (during the first and last weeks and approximately midway during the treatment). Similarly, two sets of duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
- Analytical method: HPLC-UV
- Analytical conditions:
Instrument: Dionex Ultima 3000 UHPLC with UV detection
Column: Waters Acquity UPLC; BEH C18; 100 x 2.1 mm, 1.7 µm
Column temperature: 50°C
Eluent A: water
Eluent B: Acetonitrile
Flow rate: 0.3 mL/min
Elution: Isocratic, 65:35 (A:B)
Detection: 230 nm
Run time: 10 min
Details on mating procedure:
Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment.
- M/F ratio per cage: 1:1
- Length of cohabitation: 1-14 days
- Proof of pregnancy: presence of vaginal plug or sperm in vaginal smear, referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged: paired during mating and individually housed with pups during gestation/delivery period.
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating). Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.
Frequency of treatment:
Daily
Duration of test:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating). Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control (group 1)
Dose / conc.:
4 mg/kg bw/day (nominal)
Remarks:
Low Dose (group 2)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Mid Dose (group 3)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
High Dose (group 4)
No. of animals per sex per dose:
12 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELETION RATIONALE / PRELIMINARY STUDY
In the preliminary study, dose-dependent test material related adverse effects were observed in all test-material treated groups (75, 150 and 250 mg/kg/day) with a continuous body weight loss with reduced food consumption in the Mid and High dose males. Also, the degree of the increases in the liver weight relative to the body weight in Mid and High dose females, the doses of 150 and 250 mg/kg bw/day were considered unacceptable for a main study. The effects observed in the 75 mg/kg bw/day dose group were considered to be below the MTD (Maximum Tolerable Dose) and not high enough for the High dose level for the Main study. The choice of 100 mg/kg bw/day for the Main study, based on available data from the preliminary study, was considered to be justified. Based on this information and using a factor of 5, the doses of 4, 20 and 100 mg/kg bw/day were deemed suitable for the purpose of the study; to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.

ANIMAL ASSIGNMENT
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day 0). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

OTHER
- Fasting period before blood sampling for clinical biochemistry: overnight

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for signs of morbidity and mortality. General clinical observations were performed daily (during the pre-treatment and treatment period).
- No general clinical observations were made on those days when detailed clinical observations were made (except on one case on 23 October 2019).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the start of the pre-exposure period and once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment.
Observations included:
- Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern).
- Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
- Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable.
- On gestation day GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly during the pre-exposure period, then on Day 0 for randomisation purposes, afterwards at least weekly and at termination.
- Parental females were weighed on gestation Days GD0, 3, 7, 10, 14, 17 and 20 and on post-partal Days PPD0 (within 24 hours after parturition), PPD4, 7, 10, 13 and 14 (before termination).
- The body weight of the female animals measured on gestational Days GD3, 10 and 17 as well as PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

FOOD CONSUMPTION: Yes
- Animal food consumption was determined weekly by re-weighing the non-consumed diet with a precision of 1 g (on the days of body weight measurements). Food mean consumption values (g/animal/day) were calculated and reported.

POST-MORTEN EXAMINTIONS: Yes
- Sacrifice: High dose females were killed on Day 29 (preterminal euthanasia) and on Days 41-46 (terminal euthanasia) and low and mid dose females were killed on PPD14.
- Organs examined: uterus, cervix, ovary, oviduct and vagina

OTHER: Yes
- Observation of the delivery process, offspring and nursing instinct: Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.
Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach.

- Haematology
- Clinical chemistry
- Thyroid hormone analysis
- Urinalysis
- Neurobehavioural examination (Functional Observational Battery: grip strength / landing foot splay / fore/hind limb grip strength / sensory reactivity (auditory, visual and proprioceptive) / motor activity / general physical condition and behaviour of animals)
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other: Oestrus cycle data
Blood sampling:
- Plasma: Yes
- Serum: Yes
- Volume collected: not specified.
- Other: 3 samples were taken from each animal, one for haematology, one for blood clotting and one to obtain serum.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: thyroid glands
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes
Statistics:
- Data were recorded on the appropriate forms and then tabulated using the Microsoft Office Word and/or Excel, or collected using the software PROVANTIS v.9, as appropriate.
- Group means and standard deviations were calculated from numerical data obtained in the study. The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 (when using Provantis) or with the program package SPSS PC+4.0 (SPSS Hungary Kft., Budapest), as follows:

SPSS PC + 4.0
- Heterogeneity of variance between groups: Bartlett's test.
- Not a significant result: one-way analysis of variance (ANOVA).
- Significant result: Duncan's Multiple Range test is used to assess the significance of inter-group differences.
- Significant heterogeneity: the normal distribution of data is examined by Kolmogorow-Smirnow test.
- Non-normal distribution: non-parametric method of Kruskal-Wallis One-Way analysis of variance
- Normal distribution: inter-group comparisons performed using Mann-Whitney U-test.
- Non-continuous data: Chi-squared test.

SAS 9.2
- Normality and heterogeneity of variance between groups: Shapiro-Wilk and Levene tests.
- No significant heterogeneity: Anova / Ancova (one-way analysis of variance) test.
- Positive result: Dunnett’s (Multiple Range) test.
- Significant heterogeneity: non-parametric analysis is required.
- A Kruskal-Wallis analysis of variance is used after Rank Transformation. If there is a positive result, the inter-group comparisons are performed using Dunn test.
- Non-continuous data: Cochran-Armitage test for trend. Chi-squared test is used for statistical differences relative to control.
- Pathology data (macroscopic and microscopic data): Cochran-Armitage test for trend is applied, then if appropriate, the Chi-squared test homogeneity test.
- If significance is plausible based on a user-defined value (0.05): pairwise test of each treatment group versus the control group.
Indices:
Formulas for calculation of mating and fertility indices: Table 1
Formulas for calculation of pup’s mortality and sex ratio indices: Table 2

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related clinical signs were observed during the study. There were no abnormal clinical signs in any animal during the study, excluding one female in the Mid dose group where a whole body tonic convulsion was observed on sporadic occasions between Day 26 to Day 43. In the absence of any other similar findings at this dose level or higher, this finding was considered to be incidental, and not related to the test material.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was observed during the study.
Two High dose females were euthanized (on Day 29), because they had no evidence of positive mating. Tissues were examined to try to identify any reason for lack of mating/pregnancy. No cause could be identified after microscopic examination and the uterus of one of these animals had signs of oestrus present.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the first 7 days of treatment (pre-mating period) the Low dose had a lower mean weight gain than controls (5.7 g and 16.3 g for the low dose and controls, respectively) but it was not statistically significant and during the second week, the weight gain was very similar to controls (approx. 3 g for low dose and controls).
The Mid and High dose groups lost body weight during the first week of the treatment period (-4.8 g and -13.2 g in the Mid and High dose, respectively), but both groups gained weight in the second week. By the end of the pre-mating period, the females of the control group had gained an average of 19.1 g bodyweight, low-dose females had gained 8.7 g, mid-dose females had lost an average of 1.2 g and high-dose females had lost an average of 0.3 g, compared to body weights at the start of dosing.
During the mating period, Control and Low-dose females generally recorded gains in body weight; Mid and High dose females showed static bodyweights or body weight losses. However, negative values are very common in the historical control database (~20 % of rats) so this group difference was not an adverse effect.
As the High dose females were not pregnant and therefore had no offspring, body weight data for pregnancy and lactation periods were not comparable with controls. Only the Low and Mid dose body weight and body weight gain values of the treated females were evaluated statistically during the gestation and lactation periods.
Group mean body weights of the Mid dose females were statistically significantly lower than controls on day 0, 14 and 20 of gestation. During the gestation period the Mid dose had significantly lower weight gain (by 25%, p<0.01) than Controls, such that group mean body weight of Mid dose females (387.3 g) was 14.8% lower than Control (454.6 g) on day-20 of gestation. The low dose was not affected.
During the lactation period the Control, Low and Mid groups gained a similar weight. However, group mean body weight for Mid-dose females remained significantly lower than controls for the whole period (days 0 to 13, 13.7% to 11.2% lower than controls, respectively).
The observed body weight or body weight gain differences between the female groups showed no adverse effects during gestation or lactation in the Low dose, though markedly less weight was gained during gestation (-25 %) in the Mid dose. - This effect was considered as adverse. The High dose female data was not comparable with controls due to a lack of pregnancy in this group, but during pre-mating period there was a significant but transient body weight loss which though not as severe as the effect in males at the same dose, was still considered to be an adverse effect of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The Low dose had slight but statistically significant lower food intake as a total in the first 14 days (-16.2 %) and during gestation (-10.3 %), however the percentage differences were relatively small and were not considered to represent an adverse effect of treatment. The Mid dose group had food intake values of about 20-40 % lower than controls during each phase of the study, this was considered to be an adverse effect of treatment. High dose females had an intake of over 60 % lower than control in the first week and almost 50 % lower for the total of the first 2 weeks which was considered as an adverse effect (no further comparisons were made since this group were not pregnant and not comparable with control).
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):
No test material-related adverse changes were observed in the haematology parameters.
High dose females were not pregnant during this study, therefore the Study Director decided parameters were not comparable with pregnant controls, so the Low and Mid dose group were sampled only.
Slightly decreased MCHC and increased numbers of Large Unstained Cells (LUC) achieved statistical significance in Low and Mid dose females respectively, but also within historical control range. The significant differences were considered to be incidental as there was no relationship with dose, and therefore these differences were considered to not reflect an effect of the test material.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
For all statistically significant differences, either the histopathology results (with non-adverse adaptive responses) confirm a lack of treatment related adverse effects, or they were considered to be incidental, with no relationship with dose and/or all recorded values were near or within the historical control ranges. Hence, it is considered that the test material had no adverse effects on Clinical Chemistry parameters, statistical differences were considered to incidental and unrelated to treatment.
High dose females were not pregnant during this study, therefore the Study Director decided parameters were not comparable with pregnant controls, so the Low and Mid dose group were sampled only.
Endocrine findings:
no effects observed
Description (incidence and severity):
Thryoid hormone analysis: The test material had no effect on measured thyroid hormone levels.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No clear test material-related changes were observed in the urinalysis parameters. The significant differences were considered to be incidental, not reflecting an effect of the test material.
High dose females were not pregnant during this study, therefore the Study Director decided parameters were not comparable with pregnant controls, so the Low and Mid dose group were sampled only.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted during the assessment of grip strength, foot splay or locomotor activity.
The occasional statistically significant differences in grip strength compared to control in the Low and Mid dose females were not considered as test material related effects as the observed values were in the middle of the historical control range, with control values close to the maximum value of the historical control data.
All females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. High dose females appeared to be slightly more active; but all mean results were within the historic control range. There was no statistical significance between the test material treated animals and the Control when evaluating the overall distance travelled. The test material was not considered to have had a significant effect on locomotor activity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Terminal body weights of Mid and High Dose animals were statistically significantly lower (by 14.9% or 27.2 %, respectively, though high dose females were nulliparous and not comparable to the lactating controls).
As High dose females were nulliparous, organ weights were not statistically analysed for this group being non-comparable to controls, but the mean and percentage difference to control were tabulated and evaluated by the Study Director.

No treatment-related adverse effects on organ weight were observed in Mid and Low dose females when compared with controls. The body weights of the Mid dose females were about 15 % lower than control, so similarly to the cases with smaller males, a range of organs showed statistical differences related to body weight differences. However, these sporadic, statistically significant differences (absolute and/or relative to body and brain, of the adrenals, kidneys, uterus and ovaries) lacked any histopathology changes.
The High dose females were about 27 % lower bodyweight, taking this into account, there were no organs showing evident changes in weight due to a direct test material effect. The lack of any histopathological changes in organs at the High dose confirms this conclusion.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
PRETERMINAL EUTHANASIA
Two High dose females were pre-terminally euthanized on Day 29 because they did not mate during the mating period. No test material-related macroscopic findings were seen at necropsy. The uterine body and horns of one animal was dilated, filled with clear fluid and was considered as background change.
- NON PREGNANT FEMALES
Necropsy did not show any test material related change in the non-pregnant animals.

TERMINAL / PARENTAL GENERATION
No maternal changes, changes were considered incidental or a common background finding.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
PRETERMINAL EUTHANASIA
No test material-related microscopic findings were observed. In the uterus of the animal with the dilated uterine body and horns, signs of oestrus was seen and considered to be normal background for non-pregnant animals and consistent with other sex organ histology for this animal.
- NON PREGNANT FEMALES
Organs from the reproductive system (ovary, oviduct, uterus, cervix) were microscopically examined from these animals and did not reveal any test material-related changes.

TERMINAL
Females:
The ovaries and uterus of High dose animals were not different from tissues of normal non-pregnant rats. The ovaries and uterus of Mid and Low dose animals were not different from tissues of normal pregnant rats. The ovary, uterus and vagina histology were checked against the vaginal smear taken at necropsy to check for any signs of desynchrony. Generally, there were no unusual findings although a non-specific change of an increased incidence of mucification in the vagina was identified; in these cases the degree of mucification was slightly above the normal control range. A Mild increase in mucification was observed in 9/12 Mid dose females, a similar change in the vagina was seen in 2/12 High dose females. None were seen in the Low dose. This change could indicate abnormal cycles, but it is not a clear adverse effect and was not associated with any other detectable changes in sex organs. This mucification did not reflect the proestrus phase.
Mid and Low dose males and Low dose females had normal reproductive tissues with no differences in incidences or findings compared with controls.
All other changes were seen in control and/or treated animals, or without meaningful differences in severity and incidence, therefore were regarded as incidental or a common background.

There were no indications from the female reproductive tissues of the High dose group as to why there were longer time to mating and no implantations at this dose level.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Oestrus cycle:

PRE-EXPOSURE PERIOD
- Each female selected for the study showed largely acceptable cycles before starting the treatment period.

EXPOSURE PERIOD (PRE-MATING AND MATING PERIODS)
- In High dose females there was an indication of a test material related effect on oestrous cyclicity data during the pre-mating period which persisted into the pairing phase, with fewer days in cycle, more irregular cycles, and more pseudo pregnancy than the concurrent controls; it was outside the expected historic range.
- After pairing for mating, the mean time taken to mate exceeded the historical control mean and was longer than controls at the High dose, although this was not statistically different it was considered to be an effect of treatment.
- The High dose females had a high incidence of persistent dioestrus/pseudo pregnancy in the treatment period before and during mating (11/12 females, with the remaining High dose female cycling irregularly) which would likely increase the numbers of animals with no oocytes released to achieve a positive fertilisation after mating, despite evidence of a positive copulation which would typically indicate oestrus.
- The Mid and High dose females had a clearly lower body weight than controls, a maximum of 10.2 % below controls up to the day of mating; it is plausible this could have affected the oestrus cycles, although a key publication on the topic (Terry et al, 2005) states that “In general, body weight differences of 10-15 % by themselves were not adverse to normal reproduction”. This would suggest that although chronic low body weights are known to be adverse for oestrus cycling, a short term secondary, adverse effect caused by reduced body weights of a maximum of ~10 % is not highly likely, but the sudden weight loss in the days after initiation of treatment may have had unexpected consequences on oestrus.
- Taking into account the High dose effects in both sexes, with significant effects in the secondary sex organs of High dose males and weight loss of females, the cause for the lack of pregnancy at the high dose could not be clearly ascertained.
- The parameters were normal for Mid and Low dose females, when taking account of the historical control data.
Details on results:
In High dose females there were significant body weight losses during the first week followed by an increased weight gain for the following 14 days, but from this point the body weights were practically static. Although there are no comparable concurrent non-pregnant female body weights, the lack of weight gain from about the 3rd week of treatment is considered to be a treatment effect. Changes in oestrous cycling (persistent dioestrus/pseudo pregnancy) and increased time to mating, with no implantations in any of the High dose female animals may have been influenced by body weight, although the degree of weight change does not conclusively imply a causal effect.
Histology of all primary sex organs of both sexes were normal. Histologically a mild increase in vaginal mucification was found in High dose females without dose response, which was of unclear significance; considering the successful mating at the mid dose (which also had mucification), and a lack of any other microscopic abnormalities in the reproductive tract of High dose females. Whether effects on oestrous cyclicity at the high dose reflected a direct effect of test material or secondary effect of the body weight loss is unclear. Furthermore, as females were acyclic before and during mating, indicating a lack of ovulation for fertilisation, this may have also contributed to the infertility at the high dose.
In the Mid dose group, all measured parameters were within a range for control animals but some statistical differences were seen between the concurrent controls and the Mid dose for body weight, prostate and seminal vesicle weights (but no histological evidence of any change) and a slightly lower number of pups born. Mid dose as well as High dose had an increased incidence of vaginal mucification, but the expert opinion of the histopathologist and peer reviewer was that this was not a clearly adverse effect. Overall, at the Mid dose, there were a number of apparent trends in endpoints related to reproduction, but careful analysis of the data in relation to current and historic controls, did not identify any change which could be considered clearly as an adverse effect of the test material. The statistically lower number of pups born is considered of equivocal relationship with treatment; taking a conservative approach, this finding should be considered as a potential effect when assessing the NOAEL

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of treatment noted during the gestation period in the Low or Mid dose groups. In the High dose there were no implantations observed from the 10 mated females. Whilst there were no visible corpora lutea, without fertilisation of the oocyte, it would be unlikely that corpora lutea would be visible at macroscopic examination. Following microscopic examination at histopathology the ovaries had the normal corpora lutea of non-pregnant females, and the reproductive tract had no structural abnormalities.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
not specified
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no differences between the control and Low or Mid dose groups with regard to duration of pregnancy.
Changes in number of pregnant:
not specified
Description (incidence and severity):
High dose females were not pregnant during this study therefore the evaluation of the reproductive ability was limited in this dose group. From the 12 High dose pairs, 10 had sperm detected in the smear (although mating was generally later than in controls); none of these females had implantations or litters.
There were no differences between the control and test material treated Low and Mid dose groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test material.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
- Mating:
The mating indices were 100% in Control, Low and Mid groups, in the High group, 2/12 pairs failed to mate (no sperm positive vaginal smears). The male and female fertility indices were normal (83-100%) in the Control, Low and Mid dose groups. In the High dose group the fertility index was zero (no pregnant animals). It is concluded that the test material did not have an effect on the reproductive parameters for the Low or Mid dose animals but there was an adverse outcome for High dose pairs.
Test material administration was considered to have no impact on the duration of the mating period for the Low and Mid dose groups; in the High dose group there was a delay in the time taken to mate (not statistically significant but outside the expected control range). Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 1-14 days of pairing (cohabitation) for all Control, Low and Mid females (data was all close to historical values and no adverse histology was found, hence these groups were considered as normal). Only 4/12 High dose females had sperm positive smears in the first 4 days, 6 females were positive on days 7 or 8, 2 pairs failed to mate. There was considered to be an adverse effect at the High dose.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Whilst pups were all born at a similar weight, there were statistically significant differences in the offspring body weight gain in the treated groups (Low and Mid dose) when compared to the controls, but with no clear dose response. All values were within the range commonly recorded for this strain and age, with control litter weights at the upper end of the historical control data range, therefore it is considered that these statistical differences are not test material related.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
The number of viable pups on PND0, 4 and 13 as well as pup survival indices on PND0, 4 and 13 were comparable to control values in the Low dose group.
Statistically significant lower numbers of born/live born pups were observed in the Mid dose group compared to the concurrent control group (p<0.05) although all individual values were within the normal historical control range. To evaluate further if this may be a random finding, the difference was also statistically compared to 4 contemporaneous study control groups. The Mid dose group was statistically lower than 3 of the 4 contemporaneous control groups. The number of liveborn is related to the number of implantations (maximum feasible litter size if 100% survive) and the prenatal mortality; both these numbers were also lower than the concurrent control but not statistically different and also remained within the normal range. The decreased number of implantations, their survival and causal effect of a smaller litter size (total number of pups born alive or dead) therefore does not show a clearly adverse effect, with all parameters remaining within the historical control range. However, considering the changes seen at the High dose, a test material effect on the reproductive performance cannot be excluded which may be secondary consequence of the systemic toxicity also apparent in these females.
The number of cannibalised pups between PND5-13 (10 pups) and on PND0-13 (19 pups) is significantly higher in the Mid dose relative to the Control or Low dose group consequently the survival of pups up to PND 13 was lower. The main reason for this difference was that one dam cannibalised all remaining pups in the litter on Day 7 and in one further litter, pups had all died by PND4 but as only one pup was born, this has biased the mean. When taking account of the number of affected litters per group, it is considered that there was no effect of treatment and the viability of the other litters in the Mid dose group was comparable with controls.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There was no effect on sex ratio at any dose level.
Changes in litter size and weights:
not examined
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the anogenital distance between the test material treated groups (males/females) and the control on PND0.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
It is considered that the test material did not affect the Mid dose pup post-natal survival.
External malformations:
no effects observed
Description (incidence and severity):
No test item related macroscopic changes were seen in the F1 offspring generation euthanized and examined externally at scheduled termination on PND13.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
4 mg/kg bw/day (nominal)
Treatment related:
not specified
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Summary of the pre-implantation and gestation periods

Parameters

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Mean number of implantations

14.33

14.08

12.50

0**

Number of Corpora lutea

15.83

14.83

14.75

0**

Duration of Pregnancy, mean (days)

22.60

22.60

22.58

-

Pre-natal mortality, mean

2.60

1.00+

2.25

-

Pre-natal mortality (%), mean

15.80

6.21

20.80

-

Number of pups born, mean

14.80

15.90

10.50*

-

Number of live born pups, mean

14.60

15.90

10.25*

-

Note: *= p<0.05; **= p<0.01; Duncan’s multiple range test;+=p<0.05; U= Mann- Whitney U-test. The Pre-natal mortality (%) of Mid dose was above historic control range but was not statistically different to control. Number of corpora lutea = number visible macroscopically at necropsy.

 

Summary of the pre-natal and post-natal periods with survival indices

Parameters

Group /Concentration (mg/kg bw/day)

Control (0)

Low (4)

Mid (20)

High (100)

Mean number of implantations

14.33

14.08

12.50

0**

Number of pups born, mean

14.80

15.90

10.50 *

-

Number of live born pups, mean

14.60

15.90

10.25 *

-

Pre-natal mortality, mean

2.60

1.00+

2.25

-

Pre-natal mortality (%), mean

15.80

6.21

20.80

-

Post-natal mortality on PND0-4, mean

0.40

0.60

0.50

-

Post-natal mortality on PND0-4 (%), mean

2.39

3.51

11.01

-

Total mortality on PND4, mean

3.00

1.60

2.75

-

Total mortality on PND4 (%), mean

17.89

9.63

25.39

-

Post-natal mortality on PND0-13, mean

0.60

0.80

1.33

-

Post-natal mortality on PND0-13 (%), mean

4.12

4.70

18.35

-

Total mortality on PND13, mean

3.20

1.80

3.58

-

Total mortality on PND13 (%), mean

19.26

10.79

32.13

-

Survival index on PND0(%)

98.99

100.00

93.75

-

Sex ratio (% of females) on PND0

51.88

55.20

44.59

-

Survival index on PND4(%)

97.61

96.49

88.99

-

Sex ratio (% of females) on PND4

51.41

54.89

48.73

-

Survival index on PND13(%)

(from PND4 after culling)

97.78

98.66

90.15

-

Sex ratio (% of females) (PND13)

51.58

55.73

50.36

-

Note: *= p<0.05; Duncan's multiple range test; p<0.01

 += p<0.05; Mann-Whitney U-test

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the NOAEL for developmental toxicity of the test material was considered to be 4 mg/kg bw/day.
Effects on fertility and reproduction were only seen at dose levels with overt and adverse parental toxicity in the OECD 422 study. As such, in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to reproductive or developmental toxicity.
Executive summary:

The developmental toxicity of the test material was investigated in accordance with the standardised guideline OECD 422 under GLP conditions.The study was conducted with repeated (daily) administration of the test material (0, 4, 20 and 100 mg/kg bw/day) by oral gavage to Wistar rats. The study included a reproductive / developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum. Dose levels were based on the results of a dose range finding study. During the study, groups of 12 male and 12 female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

Parameters measured during the study included signs of morbidity and mortality twice daily, daily general and/or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessments, including a functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal day (PND13).

At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals and/or offspring. The thyroxine (T4) levels in the Day 13 pups and adult males were also assessed. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

Under the conditions of the study, daily administration of the test material did not result in test material-related mortality and no treatment related clinical signs were noted.

The body weight, body weight gain, and food consumption of the High dose animals were significantly and adversely affected. Males lost body weight in the first 14 days of treatment, with some recovery, but the mean weight remained below the starting weight at termination at the High dose. Male High dose body weight on Day 28 was 12.4 % below controls. Females of the High dose also lost weight in the first 7 days, but regained their original mean weight at about day 14. However, weight gain was lower than control; from about the third week of treatment, High dose females failed to continue gaining bodyweight. Since High dose females were not pregnant, subsequent statistical comparison of their body weight with the pregnant control females was not made. Markedly lower food intake of High dose animals of both sexes reflected the body weight effects.

Mid dose males and females also lost body weight in the first 7 days. Whilst some weight was gained thereafter, in males overall weight gain was still 50 % lower than controls, and in females, lower gain was evident in the mating, gestation and lactation periods resulting in a mean body weight approximately 12 % lower than controls by the time of termination. The body weight effect was considered to be transient but adverse in Mid-dose group males and females. Food intake values of the Mid dose reflected the body weight in both sexes. There was no significant effect on the Low dose for body weight or food intake.

At the functional observation battery (FOB) there were no adverse changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different type of stimuli in the control or test groups. No clearly adverse test material-related findings were seen in the clinical pathology parameters of animals that were assessed. There were no effects on thyroid hormone levels of adult males.

There was a significant effect of treatment on reproductive parameters in High dose animals. Oestrous cycling data for High dose females showed 11/12 females were in a state of persistant dioestrous/pesudopregancy before and during pairing, and mating was delayed beyond historical control maximum periods. Although, 10/12 females recorded sperm positive smears during the mating period (lower than controls), no High dose females were pregnant and at necropsy there was no evidence of uterine implantations, indicating that the 10 mated females probably did not have fertilised ova. Considering the adverse effects on body weight in the first 7 days, and lower body weights that were evident in the pre-mating and mating period for females of this group, it is unclear whether effects on oestrous cyclicity could be a direct effect of treatment or a secondary effect of body weight.

There were no differences between the control and Low or Mid dose groups with regard to mating, reproductive ability, gestation indices or duration of pregnancy.

There were no effects on the number of Low dose pups born, or live born, or on F1 offspring viability, clinical signs or at observations following euthanasia. At the Mid dose, the number of pups born and born alive was statistically less than the controls, but individual values remained within the normal historical control range. There were statistical differences in pup growth (body weight gain) at the low and mid-dose levels; however, body weight and body weight gain values were within the normal range, and there was not a dose response; therefore, this was not considered to be a treatment effect. Considering the changes in reproductive parameters at the high dose, a test material effect on the number of pups born and live born pups in the Mid dose group could not be excluded; however, the relationship with the test material was considered to be equivocal since this dose level was also responsible for an adverse effect on body weight in adult females. It was considered that the test material did not affect the Mid dose pup post-natal development/survival.

There were no treatment related changes in endocrine sensitive endpoints in the pups at any of the dose levels (anogenital distance, nipple retention, thyroid gland weights, thyroid hormone level).

There were no adverse effects of treatment on organ weights of either sex at any dose level other than for lighter male secondary sex glands (prostate and seminal vesicles) at the high dose; higher liver weights in High dose males were considered to be adaptive though there was no histopathological change and statistical differences in kidney and other organ weights were considered as secondary to body weight differences.

At histopathology, High dose prostate and seminal vesicles showed atrophy, reflecting the organs weight effects; this was considered secondary to the body weight loss with low food intake throughout the study in males. There was no evidence of a direct effect of the test material on the other reproductive organs in males, with the testes and epididymis normal. There were no histological changes seen in the Mid or Low dose secondary sex organs.

Careful microscopic examination of all the female reproductive organs/tissues found no adverse effect of treatment in the High dose group. Histologically, a mild increase in vaginal mucification was found in Mid and High dose groups which was of unclear significance and lacked clear dose response.

In High dose males, the main observed effect was on the secondary sex glands with prostate and seminal vesicle atrophy evident. Whilst this may have contributed to the reproductive failure as it could be expected to reduce semen fluid quantity/quality, semen was visible in the vaginal smears. Furthermore, as females were acyclic before and during mating, indicating a lack of ovulation for fertilisation, this may have also contributed to the infertility at the high dose.

Other than the histological effects seen in High dose male secondary sex organs, there were no histological adverse changes attributed to treatment with the test material in any group of either sex (the sex organs of all groups were examined).

Based on an equivocal relationship with reduced litter size at the Mid dose the NOAEL for developmental toxicity of the test material was considered to be 4 mg/kg bw/day.

Categories Display