Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: approx 10 weeks at start of treatment
- Weight at study initiation: males 413-495 g, females 224-287 g
- Fasting period before study: no
- Housing: up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or indivdually housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
animals received ssniff SM R/M complete diet for rats and mice, Soest, Germany
animals received tap water from municipal supply.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.9
- Humidity (%): 31 - 64
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: propylene glycol with 1% polysorbate 80
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:


The test item was formulated in the vehicle propylene glycol containing 1% polysorbat 80 (as a visibly stable homogenous suspension). Formulations were prepared daily at appropriate concentrations in the vehicle.

The calculated volume of Propylene glycol / 1% Tween 80 vehicle was added into a beaker containing the calculated amount of test item, it was mixed vigorously for at least an hour by a magnetic stirrer. Then formulation was incubated overnight at room temperature and mixed again by a magnetic stirrer for approximately 2 hours. Formulations were mixed continually with a magnetic stirrer until the end of the treatment procedures

- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
Details on mating procedure:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed photometrically using a validated NIR method. Recovery of nominal concentrations ranged from 99% to 107%.
Duration of treatment / exposure:
males were dosed for 28 days starting 14 days prior to mating. Females were dosed for 14 days prior to mating, during mating and gestation until day 13 post partum.
Frequency of treatment:
once daily
Details on study schedule:
- Age at mating of the mated animals in the study: approx. 12 weeks
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a Dose Range Finding study no effects were observed at 1000 mg/kg bw/day.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: food consumption was determined

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

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

Oestrous cyclicity (parental animals):
Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments started. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: weights and histological examination of testis, epididymis, prostate, seminal vesicles with coagulating glands
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
- If yes, maximum of [...] pups/litter ([...]/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, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 28 days of dosing
- Maternal animals: All surviving animals on post natal day 14.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively: see "pathology and histopathology investigations" attached as background material
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: macroscopic

GROSS NECROPSY
- Gross necropsy consisted of external examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
The statistical evaluation of data (labelled as † in the lists below) was performed with
the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2
(when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between
groups was checked by Bartlett's test. Where no significant heterogeneity was
detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained
result was significant, then Duncan's Multiple Range test was used to assess the
significance of inter-group differences. Where significant heterogeneity was found,
the normal distribution of data was examined by Kolmogorow-Smirnow test. In the
case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-
Way analysis of variance was applied. If a positive result was detected, the inter-group
comparisons were performed using Mann-Whitney U-test. The Chi-squared test was
used for non-continuous data.
In case of the SAS v9.2 software package (within the validated Provantis system) the
following decision tree was applied automatically for statistical evaluation of
continuous numeric data. The normality and heterogeneity of variance between groups
was checked by Shapiro-Wilk and Levene tests using the most appropriate data format
(log-transformed when justified). Where both tests showed no significant
heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out.
If the obtained result was positive, Dunnett (Multiple Range) test was used to assess
the significance of inter-group differences; identifying differences of <0.05 or <0.01
as appropriate.
Reproductive indices:
mating, fertility, gestation indices were determined for parental animals
Offspring viability indices:
survival, pre-implantation mortality, intrauterine mortality, total mortality, sex ratio indices were determined for the F1 generation
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
one female of the low dose group was found dead on Day 27 due to a gavage accident.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see Tables and Figures on body weight attached as background material
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see Tables and figures on food consumption attached as background material
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, non-treatment-related
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
see Table on oestrous cycles attached in background material
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
based on organ weights and histological examinations

see Table on organ weights, males attached in background material
Reproductive performance:
no effects observed
Description (incidence and severity):
see Table on reproductive parameters of males and females attached in background material
Key result
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: NOAEL = highest dose tested
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of viable pups on PND0, 4 and 13 as well as pups’ survival indices on PND0, 4 and 13 were comparable to control values in each dose group. Overall, there were no treatment-related effects on pup mortality and on the viability of pups on PND0, 4 and 13.

There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.

One female in the High dose group had 14 pre-natal losses out of the 16 implantation sites and had only two liveborns, which died before PND4. This resulted in an 88% pre-natal mortality and a 100% PND4 post-natal mortality for this animal. The High dose group means without this animal are shown in brackets in the Table. The rest of the animals in the dose group were comparable to the control group, but this animal caused a major change in the mean of the High dose group in several parameters, but without reaching statistical significance. As the higher pre-natal mortality seen in the High dose group is not statistically significant and was related to a single litter, it is within the normal range and is considered as not test item related.

see Table on offspring viability attached in background material
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see Table on litter body weight development attached in background material
Gross pathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in the adult male dose groups. Regarding the PND13 pups, significantly higher (p<0.01) T4 concentration was recorded in the High dose. As the recorded value is within the historical control range and due to the lack of any supporting evidence of thyroid gland weight or histopathology changes in these animals, this difference was considered to not reflect any adverse effects of the test item.

There were no statistically significant differences in the anogenital distance between the test item treated groups (males/females) and the control on PND0. There was no nipples/areolae presence seen in any of the male pups on PND13.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
dams were dosed
Sex:
male/female
Basis for effect level:
other: NOAEL = highest dose tested
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD 422 reproduction / developmental toxicity screening study
Version / remarks:
2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx 10 weeks at start of treatment
- Weight at study initiation: males 413-495 g, females 224-287 g
- Fasting period before study: no
- Housing: up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or indivdually housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
animals received ssniff SM R/M complete diet for rats and mice, Soest, Germany
animals received tap water from municipal supply.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.9
- Humidity (%): 31 - 64
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: propylene glycol containing 1% polysorbat 80
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:

The test item was formulated in the vehicle propylene glycol containing 1% polysorbat 80 (as a visibly stable homogenous suspension). Formulations were prepared daily at appropriate concentrations in the vehicle.

The calculated volume of Propylene glycol / 1% Tween 80 vehicle was added into a beaker containing the calculated amount of test item, it was mixed vigorously for at least an hour by a magnetic stirrer. Then formulation was incubated overnight at room temperature and mixed again by a magnetic stirrer for approximately 2 hours. Formulations were mixed continually with a magnetic stirrer until the end of the treatment procedures

- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed photometrically using a validated NIR method. Recovery of nominal concentrations ranged from 99% to 107%.
Details on mating procedure:
- If cohoused:
- M/F ratio per cage: 1 : 1
- Length of cohabitation: up to 5 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug / sperm in vaginal smea] referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
males were dosed for 28 days starting 14 days prior to mating. Females were dosed for 14 days prior to mating, during mating and gestation until day 13 post partum.
Frequency of treatment:
once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a Dose Range Finding study no effects were observed at 1000 mg/kg bw/day.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations:on gestation days 0, 3, 7, 10, 14, 17, and 20 and on post-partal days 0, 4, 7, 10, 13

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post partum day # 14
- Organs examined: see Table on pathology and histopathology investigations attached as background material
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other: number of pre-implantation mortality
Fetal examinations:
Females were allowed to litter and rear their offspring until post natal day 13. The delivery process was observed as carefully as possible. All observations were recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0) and on PND4 and PND13, with accuracy of 0.01g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). Presence of nipples/areolae in male pups were recorded on PND13.

All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on PND4.
Statistics:
The statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One- Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
one animal of the low dose group died due to a gavage accident
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see Tables and Figures on body weight attached as background material
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see Tables and figures on food consumption attached as background material
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:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
One female in the High dose group had 14 pre-natal losses out of the 16 implantation sites and had only two liveborns, which died before PND4. This resulted in an 88% pre-natal mortality and a 100% PND4 post-natal mortality for this animal. The High dose group means without this animal are shown in brackets in the Table. The rest of the animals in the dose group were comparable to the control group, as were all animals of the mid and low dose groups. As the higher pre-natal mortality seen in the High dose group is not statistically significant and was related to a single litter, it is within the normal range and is considered as not test item related.

see table on pre-natal and post-natal periods attached in background material
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
see Table on offspring viability attached as background material
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOAEL = highest dose tested
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
see Table on litter body weight development attached as background material
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
see Table on pre-natal and post-natal survival indices attached as background material
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
dams were dosed
Sex:
male/female
Basis for effect level:
other: NOAEL = highest dose tested
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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:
2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: approx 10 weeks at start of treatment
- Weight at study initiation: males 413-495 g, females 224-287 g
- Fasting period before study: no
- Housing: up to 3 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or indivdually housed
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
animals received ssniff SM R/M complete diet for rats and mice, Soest, Germany
animals received tap water from municipal supply.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 24.9
- Humidity (%): 31 - 64
- Air changes (per hr): 15 -20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: propylene glycol containing 1% polysorbat 80
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:


The test item was formulated in the vehicle propylene glycol containing 1% polysorbat 80 (as a visibly stable homogenous suspension). Formulations were prepared daily at appropriate concentrations in the vehicle.

The calculated volume of Propylene glycol / 1% Tween 80 vehicle was added into a beaker containing the calculated amount of test item, it was mixed vigorously for at least an hour by a magnetic stirrer. Then formulation was incubated overnight at room temperature and mixed again by a magnetic stirrer for approximately 2 hours. Formulations were mixed continually with a magnetic stirrer until the end of the treatment procedures

- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg body weight
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and homogeneity was performed photometrically using a validated NIR method. Recovery of nominal concentrations ranged from 99% to 107%.
Duration of treatment / exposure:
males were dosed for 28 days starting 14 days prior to mating. Females were dosed for 14 days prior to mating, during mating, gestation until day 13 post partum.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
according to guideline OECD 422 (2016)

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: food consumption was determined

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

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

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes pentobarbital
- Animals fasted: Yes
- How many animals: 5 per sex and dose group
- Parameters checked in table [No.?] were examined: see Table on haematology attached as background material

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: just before necropsy
- Animals fasted: Yes
- How many animals: 5 per sex and dose group
- Parameters checked in table [No.?] were examined.: see Table on clinical chemistry and urinalysis attached as background material

URINALYSIS: Yes
- Time schedule for collection of urine: just before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined: see Table on clinical chemistry and urinalysis attached as background material

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity.

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

see "pathology and histopathology investigations" attached as background material
Statistics:
The statistical evaluation of data (labelled as † in the lists below) was performed with the program package SPSS PC+4.0 (SPSS Hungary Kft, Budapest) or SAS v9.2 (when using Provantis).
In case of the SPSS PC+4.0 program package, the heterogeneity of variance between groups was checked by Bartlett's test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One- Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test. The Chi-squared test was used for non-continuous data.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
one animal of the low dose group died due to a gavage accident.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see Tables and Figures on body weight attached as background material
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see Tables and figures on food consumption attached as background material
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):
Significantly lower (p<0.05) prothrombin time (PTT) was recorded in the Low dose males. As the recorded value is within the historical control range and there was no dose response, this difference was considered to not reflect any adverse effects of the test item.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no statistically different results in any of the dose groups compared to the control.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly higher urine volume was collected from the Low dose males (p<0.05) and from the High dose females (p<0.01). The collected urine volume is usually highly variable. All study data was considered to be normal, therefore this numerical difference was considered to not reflect any adverse effects of the test item. This fact is confirmed by the lack of any supporting evidence of any changes in these animals (clinical chemistry, other urinalysis parameters, etc.)
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly higher (p<0.01) absolute brain weights in the male Mid dose group and significantly lower (p<0.05) relative kidney weights in the female Low and High dose groups were recorded. However, as the observed values were near the middle of the historical control range and there were no clear dose responses observed in these parameters, these statistical differences were considered to have no toxicological significance.

see Tables on weights of organs attached as background material


Besides this, there were no statistically different results in any other of the dose groups compared to the control.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item-related findings were seen at dose levels up to 1000 mg/kg bw/day.

Based on the low incidence and/or severity and/or distribution cross control and dosed animals the following observations were considered incidental or a common background: minimal or slight tubular basophilia in the kidney (#1001, 4506), minimal multifocal casts in the kidney (#1511), minimal tubular multifocal mineralization in the kidney cortex (#1504, 4503), minimal focal congestion or haemorrhage in the stomach (#1506), minimal multifocal congestion or haemorrhage in the thymus (#1001, 1504).
Other effects:
no effects observed

Effect levels

Key result
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: NOAEL = highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion