Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10th July 2019 to 31st January 2020
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Adopted 25th of June 2018
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008
Version / remarks:
L 142, Annex Part B, May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approximately 11-12 weeks old.
- Weight at study initiation:
males: 329 – 379 g (mean: 349.54 g, ± 20 % = 279.63 – 419.45 g)
females: 154 - 245 g (mean: 220.14 g, ± 20 % = 176.11 – 264.16 g)
- Fasting period before study: no
- Housing: IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice, ad libitum.
- Water: tap water, sulphur acidified to a pH of approximately 2.8, ad libitum.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light /12 hours dark

IN-LIFE DATES: not specified

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
dried and de-acidified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in dried and de-acidified corn oil and administered daily.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor on the test item's characteristics.
- Concentration in vehicle: 0; 2.5; 6.25; 12.5 mg/mL
- Amount of vehicle: 0; 2.5; 6.25; 12.5 mg/mL
- Lot/batch no.: MKCH1635
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 187103).
Study pre-start stability analysis was included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 days (2-8 °C) and 10 days (-15 to -35 °C).
Pre-start homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous according to Eurofins Study No. 187103 (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in the first and last week of the study for all doses (8 samples in total).
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 187104) and until then stored under appropriate conditions based on available stability data. The B-samples are retained at below -15 °C at BSL Munich (test facility) and discarded after completion of the final study report.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day.
- M/F ratio per cage: 1:2 (male to female).
- Length of cohabitation: until sperms were observed in the vaginal smear (checked every morning).
- After receiving 92 sperm positive females, the remaining animals were discarded without any observations.
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes, the animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
Daily administration of dose between gestation day 5 to 19.
Frequency of treatment:
A single dose per day, 7 days per week
Duration of test:
from gestation day 5 to 19
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
10 mg/kg bw/day
Remarks:
Low dose (LD
Dose / conc.:
25 mg/kg bw/day
Remarks:
Middle dose (MD)
Dose / conc.:
50 mg/kg bw/day
Remarks:
High dose (HD)
No. of animals per sex per dose:
48 males and 96 females.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a dose range finding study (see Section 7.5.1 for Robust Study Summary). In the 14-day dose range finding study with dichloro(methyl)phenylsilane, male and female Wistar rats were treated at dose levels of 50, 100, 250, 375 and 500 mg/kg bw/day.
Mortality occurred in 2/2 males and 2/2 females at the dose of 500 mg/kg bw/day and 1/2 males and 1/2 females at 375 mg/kg bw/day with inflammatory lesions in stomach, small and large intestine which were considered as the cause of morbidity. Adverse clinical signs were observed at 375 mg/kg bw/day and 500 mg/kg bw/day in both males and females, including increased salivation (slight to moderate), prone position, abnormal breathing, hypothermia, reduced spontaneous activity (slight to moderate), half eyelid closed, slow movements, wasp waist (slight) and dehydration.
No treatment-related mortality or marked clinical signs were observed at 50, 100 and 250 mg/kg bw/day.
Treatment did not affect the body weight gain at 50, 100 and 250 mg/kg bw/day in male throughout the treatment days. The surviving male of group 5 (375 mg/kg bw/day) showed a tendency towards a loss of body weight. Body weight change in females showed no trend towards body weight loss and mean body weight gain was comparable to control.
There were no treatment-related effects on parameters of haematology or clinical biochemistry in both males and females at any dose.
Test item-related gross lesions were noted in the stomach, small and large intestine at ≥ 250 mg/kg bw/day and these were evident during histopathological examination. At the microscopic examination, specific lesions were found, starting at the dose of 50 mg/kg bw/day. These findings included inflammatory and degenerative lesions which consisted of erosions, inflammation, hyperplasia, ulceration, hyperkeratosis, single cell necrosis and epithelial degeneration.
The doses in the main study were based on the results of this dose range finding study and the known corrosive properties of the test item. The highest dose level was selected with the aim of inducing toxic effects, however, not death or severe suffering. Based on the results of the dose range finding study the highest dose was expected to cause some irritation and probably inflammation in the gastrointestinal tract at a level that does not lead to severe distress and suffering of the animals. To avoid suffering due to the known corrosive properties of the test item, no higher doses than 50 mg/kg bw/day were administered. Thereafter, a descending sequence of dose levels were selected to demonstrate any dose-related response and to determine a NOAEL.
- Rationale for animal assignment: randomised

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The health conditions of the animals were recorded at least once a day, preferably at the same time each day. Twice daily, all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded outside the home cage prior to the first administration.

BODY WEIGHT: Yes
- Time schedule for examinations: once before initiation of pairing. The sperm positive females were weighed during Gestation Days 5, 8, 11, 14, 17 and 20. Males were weighed prior to the initiation of the pairing only.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption was measured in sperm positive females only during Gestation Days 5, 8, 11, 14, 17 and 20.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: all gross lesions, caecum, colon, duodenum, head with paranasal sinuses, ileum, jejunum, larynx, lungs, oesophagus, rectum, stomach and trachea.

OTHER: Thyroid hormones levels from all dams were assessed at the end of the treatment prior to or as part of the sacrifice of the animals.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: the position and number of foetuses in each uterine horn.
Fetal examinations:
- External examinations: Yes: at least 20 litters per group; all fetuses
- Soft tissue examinations: Yes: at least 20 litters per group; one half of each litter
- Skeletal examinations: Yes: at least 20 litters per group; the remaining half of foetuses from each litter
- Head examinations: Yes: at least 20 litters per group; craniofacial examination of the heads of the foetuses used for the soft tissue examination of the first 20 litters per group
Statistics:
A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using an one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
Historical control data:
Yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related clinical signs of toxicological relevance observed in the females of any treatment group. None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Clinical signs observed on a few days during the treatment period of the study included skin and fur, hairless area in one animal (no. 58) of MD group on GDs 9-17 and one animal (no. 84) in HD group on GDs 5-19. These were considered to be an incidental findings and of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality was observed during the treatment period and all animals survived until the end of the study, except one female in control group (animal no. 2) which was found dead on GD16 having previously shown clinical signs of abnormal breathing. The cause of morbidity was not evident at the histopathological investigation and was considered to be most likely accidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight remained unaffected by treatment with the test item and increased with the progress of the study in the control, LD, MD and HD groups throughout the study period.
No statistical significance was achieved in any treatment groups on any day or interval of body weight measurement and all values in the treatment groups were comparable to the controls. However, mean body weight gain was slightly decreased although, without statistical significance, on Gestation Days 0-5 (10.24% below control) in the 25 mg/kg bw/day dose group, on Gestation Days 5-8 in the 10 and 25 mg/kg bw/day dose groups (29.29% and 12.70% below control, respectively) and on Gestation Days 17-20 (10.57% below control) in the 10 mg/kg bw/day group when compared to the control. As this effect on group mean body weight gain was seen in the 10 and 25 mg/kg bw/day dose groups without any dose dependency, it was not considered as related to the treatment with the test item.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption in the LD, MD and HD groups was comparable to the control.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological changes were observed during the macroscopic examination of the any females of the control, LD, MD and HD groups, except for a single incidence of abnormal-colour aorta and lungs and fluid-filled liver in animal no. 2 of control group. These were considered to be incidental findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The test item induced degenerative inflammatory lesions in the forestomach were noted in animals at all dose levels.
At 50 mg/kg bw/day (HD group) the observed degenerative and inflammatory lesions in forestomach consisted of ulcerations and erosions, inflammation (mixed cell infiltrates) and submucosal oedema. Furthermore, reactive changes consisting of hyperkeratosis and squamous hyperplasia were observed in several animals from this group. In addition, submucosal haemorrhages were observed in one animal from this group.
At 25 mg/kg bw/day (MD group) the degenerative and inflammatory forestomach lesions consisted of erosions, inflammation (mixed cell infiltrates) and submucosal oedema. The reactive changes consisted of hyperkeratosis and squamous hyperplasia. Thus, the forestomach changes observed in this group were similar to those observed at 50 mg/kg bw/day but appeared of lower incidence severity.
At 10 mg/kg bw/day (LD group) the findings were similar to those observed at 25 mg/kg bw/day but of lower incidence and consisted of degenerative and inflammatory forestomach lesions consisted of erosions, inflammation (mixed cell infiltrates) and submucosal oedema and submucosal necrosis in one animal. The reactive changes were also represented by increased hyperkeratosis and squamous hyperplasia.
In control animals, minimal hyperkeratosis and squamous hyperplasia mainly located at the limiting ridge and minimal mixed infiltrates (one animal only) were also observed.
The above-mentioned changes in the forestomach observed in all treatment groups were considered as test item related and were dose depended. These changes were most likely induced by the direct irritant effect of the test item on the gastric mucosa.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the 50 mg/kg bw/day dose group, squamous hyperplasia was observed. No other test item related were observed.
The changes were considered to be test item related and dose-dependent. These changes were most likely induced by the direct irritant effect of the test item on the gastric mucosa.
Other effects:
no effects observed
Description (incidence and severity):
In all terminally sacrificed females, no statistically significant or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels and values were comparable with the controls.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The group mean of T3, T4 and TSH hormone levels and values were comparable with the controls.
Details on maternal toxic effects:
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, net weight change from GD 0, AGD, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Successful mating resulted in 22/23 pregnancies in the LD group, 21/23 in the MD group and 21/23 in the HD group compared to 19/23 pregnancies in the control group.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Remarks:
local effects
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: see 'remarks'
Remarks:
Changes induced by the direct irritant effect of the test item on the gastric mucosa.
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
>= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse systemic effects were observed.

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: forestomach
Description (incidence and severity):
At histopathological evaluation, ulceration, hyperkeratosis, squamous hyperplasia, submucosal edema, hemorrhages and mixed cell infiltrates were observed in HD group. Further, in LD and MD groups, hyperkeratosis, squamous hyperplasia and minimal mixed inflammatory infiltrates (one in MD group only) were also noticed, statistically relevant only in the MD and HD group.

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
In male foetuses statistically significantly lower foetuses body weight and cube root of foetuses body weight was observed in LD group when compared with the controls. As there was no dose dependency and consistency in decrease in foetuses body weight, this finding is not considered to be treatment related.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Following 10 mg/kg bw/day, male foetuses had a statistically significant lower pup weight and cube root of pup weight when compared with the controls. However, since no dose-dependent relationship was observed, the findings were regarded as not test substance related.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In all dose groups including the control group, the head, subcutaneous haematoma, the cerebellum and increased perimeningeal space were observed. Although, these findings were considered to be spontaneous in nature and not treatment-related. Statistical analysis of the data revealed no statistical significance for any of these findings.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Following skeletal examination, incidences occurred which were either slightly lower or higher compared to the control group. A statistically significantly lower incidence of incomplete ossification of vertebra sacral arches occurred in the 10, 25 and 50 mg/kg bw/day dose groups (10%, 5% and 0% respectively compared to 47 % in control). Similarly, statistically significantly higher incidence of unossified forelimb phalanx occurred in the 10, 25 and 50 mg/kg bw/day dose groups (85%, 100% and 100% respectively compared to 11 % in control).
A statistically significantly lower litter incidence for unossified forelimb proximal phalanx in the 10, 25 and 50 mg/kg bw/day dose groups (5%, 25% and 20% respectively, compared to 90 % in control) and statistically significantly higher litter incidence for unossified skull mandible in the 10, 25 and 50 mg/kg bw/day dose groups (35% (not significant), 65% and 90% respectively, compared to 5% in control) were observed. Moreover, a statistically significantly higher litter incidence for incomplete ossification of skull frontal in HD groups (15%) was observed when compared to 0 % in control. Furtermore, a statistically significantly higher litter incidence for incomplete ossification of skull interparietal in LD groups (70%) was observed when compared to 26% in control. Finally, statistically significantly lower litter incidence for incomplete ossification of sternebra (5th) in LD group (15%) was observed when compared to 58% in control. All the above findings are considered to be spontaneous in nature as well as being within the historical control data.

Other findings include slightly higher litter incidences, but without achieving statistical significance were observed in the 50 mg/kg bw/day for incomplete ossification of skull parietal (50 % compared to 26 % in control), skull frontal (15% compared to 5% in control), 6th sternebra (65% compared to 53% in control), skull supraoccipital (25% compared to 11% in control), un-ossified vertebra caudal centrum (70% compared to 42% in controls), un-ossified forelimb metacarpals (50% compared to 42% in controls), pelvic girdle caudal shift (B) (50% compared to 37% in controls) and rib-rudimentary (14th, R) (45% compared to 37% in controls).

The observed incomplete ossification without achieving statistical significance of a few bones and a few other skeletal findings in the 50 mg/kg bw/day group were either marginally lower or higher or within historical control data range. Generally delayed ossification is not regarded to persist postnatally and is not associated with long-term consequences on survival, general growth and development and therefore, is not considered to be adverse.
There was no statistical significance and no indication of a test item-related trend in the type and/or incidences of other skeletal findings and therefore, considered to be spontaneous in nature.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As the observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.
All litter incidences were statistically insignificant when compared with the control except lower litter incidence for long thymus in the 10, 25 and 50 mg/kg bw/day groups (5%, 5% and 10%, respectively, compared to 21% in control). Lower litter incidences were observed for a few visceral findings like umbilical artery malpositioned (30% in the 50 mg/kg bw/day compared to 37% in control), higher incidences of testes malpositioned (45% in 50 mg/kg bw/day compared to 11% in control), lower incidences of abdomen internal haemorrhage (65% in 50 mg/kg bw/day, 91% in 25 mg/kg bw/day and 70% in 10 mg/kg bw/day when compared to 74 % in control), neck subcutaneous haemorrhages (0% in 50 mg/kg bw/day compared 5% in control), lower incidences of liver, supernumerary lobe (35 % in 50 mg/kg bw/day compared to 53 % in control), lower incidences of azygos vein (bilateral) lobe (45 % in 50 mg/kg bw/day compared to 53 % in control) and renal pelvis dilated (35 % in 50 mg/kg bw/day compared to 11% in control) in 50 mg/kg bw/day group without achieving statistical significance. However, findings were either minor variations or values were well within the historical control data range (47.37 % -renal pelvis dilated) and therefore not considered to be adverse. Dilated renal pelvis or ureter is a common finding in rodent studies and is classified as a variation as it is transient and likely to be postnatally reversible. Furthermore, values were within the historical control data and thereby, not considered as toxicologically relevant.
Other effects:
no effects observed
Description (incidence and severity):
In males, the absolute and relative anogenital distance (AGD) in treatment groups remained unaffected and no statistical significance was observed in any of the treatment groups. However, statistically significant higher (10%) absolute and relative anogenital distance in the 50 mg/kg bw/day dose group and higher relative anogenital distance in the 25 mg/kg bw/day group (8.3%) compared to the controls. In females, the absolute and relative AGD was comparable to the control group. The respective means were within the range of historical control data (absolute anogenital distance: between 1.77 mm and 3.71 mm; relative anogenital distance: between 1.15 and 2.40), hence this is not considered to be treatment related effects. In females, the absolute and relative anogenital distance (AGD) was comparable to the control group. Considering the mean values were within the historical control data, the changes in the anogenital distance were not considered to be toxicologically relevant.

All male foetuses were checked for indication of incomplete testicular descent/cryptorchidism and evaluation revealed completion of testicular descent in all male foetuses from all the test groups.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In a prenatal developmental toxicity study, conducted according to OECD TG 414 and in compliance with GLP, the LOAEL for maternal local effects was concluded to be 10 mg/kg bw/day based on changes in the forestomach. No systemic effects were observed and no adverse effects were detected in foetuses therefore the developmental and maternal systemic NOAELs were concluded to be greater than or equal to 50 mg/kg bw/day.