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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
January 2001
Deviations:
yes
Remarks:
All deviations from the test plan were recorded and evaluated. The deviations were not considered to have impacted the overall integrity of the test results.
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week.
The dose levels in this study were selected to be 0, 10, 30 and 100 mg/kg/day, based on the
results of a 14-day repeated-dose toxicity study. However, based on observed toxicity, dose levels were lowered to 5, 15 and 50 mg/kg/day from Day 5 of treatment onwards.
Details on mating procedure:
Animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling
mating, after at least 10 weeks of treatment. Detection of mating was confirmed by evidence
of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This
day was designated Day 0 post-coitum. Once mating had occurred, the males and females
were separated.
A maximum of 14 days was allowed for mating, after which females who had not shown
evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy and homogeneity were determined for formulations prepared for use up until Day 4
of the Study, from Day 5 of the Study, Week 11, Week 22 and Week 33.
Analytical method: UPLC
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals by once daily oral
gavage 7 days a week.
The first day of dosing was designated as Day 1 (exception: alternate animals used for
replacement after Day 1 was assumed the day of the animal being replaced).
F0-males were treated for a minimum of 12 weeks, including 10 weeks prior to mating and
during the mating and post-mating period, up to and including the day before scheduled
necropsy. F0-females were treated for a minimum of 16 weeks, including 10 weeks prior to
mating, the variable time to conception, the duration of pregnancy and at least 21 days after
delivery, up to and including the day before scheduled necropsy. Females were not dosed
during littering.
Prior to weaning, pups were not treated directly but could potentially be exposed to the test
item in utero, via maternal milk, or from exposure to maternal urine/feces.
From weaning onwards (PND 21 or 22), selected F1-animals for mating were dosed up to and
including the day before scheduled necropsy. The non-selected F1-animals were not dosed.
Frequency of treatment:
daily
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
Clinical observations were performed once daily, beginning during the first administration of
the test item and lasting throughout the dosing periods up to the day prior to necropsy.
Oestrous cyclicity (parental animals):
Estrous stages were determined by examining the cytology of vaginal lavage samples.
Daily vaginal lavage was performed for all F0 females beginning 21 days prior mating, during
mating until evidence of copulation was observed. Vaginal lavage was continued for those
females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.
Sperm parameters (parental animals):
For all surviving males, the following assessments were performed:
Sperm samples were taken from the proximal part of the vas deferens (right) at necropsy.
Sperm motility and progressive motility were assessed from all samples. Sperm smears for
morphological evaluation were fixed from all samples and stained with haematoxylin and
eosin. Abnormal forms of sperm from a differential count of at least 200 spermatozoa (if
possible) per animal was recorded. Evaluation were performed for all samples.
One testis and one epididymis (left) were removed, placed in labeled bags, and kept in the
freezer at =-15°C. After thawing the left testis and epididymis were weighed, homogenized
and evaluated for sperm numbers. Evaluation were performed for all samples.
Litter observations:
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a
litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding
of pups started). The day prior to PND 1 is considered to be the day when the female started
to deliver and is defined as PND 0 and used for recording of delivery. Females that were
littering were left undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and
pregnant females were examined to detect signs of difficult or prolonged parturition or
deficiencies in maternal care.
Postmortem examinations (parental animals):
All animals were subjected to a full post mortem examination, with special attention being
paid to the reproductive organs.
Postmortem examinations (offspring):
Animals surviving until scheduled euthanasia were weighed and deeply anaesthetized using
isoflurane and subsequently exsanguinated and subjected to a full post mortem examination,
with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no
macroscopically visible implantation sites were present, non-gravid uteri were stained using
the Salewski technique in order to detect any former implantation sites and the number of
corpora lutea was recorded in addition.

For animals that died on study a necropsy was conducted within 24 hours, and specified
tissues were saved but not weighed.
Reproductive indices:
Mating index (%) : (Females mated / Females paired) * 100
Fertility index (%) : (Pregnant females / Females mated) * 100
Gestation index (%) : (Females with living pups on Day 1 / Pregnant females) * 100
Offspring viability indices:
Post-implantation survival index (%) : (Total number of offspring born/Total number of uterine implantation sites) * 100
Live birth index (%) : (Number of live offspring on Day 1 after littering/Total number of offspring born) * 100
Viability index (%) : (Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100
Lactation index (%) : (Number of live offspring on Day 21 after littering/Number of live offspring on Day 4 (after culling) * 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the first 4 days there were 3 males found dead in the dose group of 100 mg/kg bw/day. On day 5 the doses were lowered to 0-5 and 50 mg/kg bw/day and no dose related adverse effects were observed.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the first 4 days there were 3 males found dead in the dose group of 100 mg/kg bw/day. On day 5 the doses were lowered to 0-5 and 50 mg/kg bw/day and no dose related adverse effects were observed.
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
not examined
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):
effects observed, treatment-related
Description (incidence and severity):
Clinical signs observed among surviving animals (and animals found dead or those that were
sacrificed) at 100 mg/kg/day primarily consisted of lethargy and piloerection and/or ptosis in
most animals, and at lower incidence posture changes (cramped/flat/hunched or ventro-lateral
recumbency), decreased locomotor activity, tremors and/or uncoordinated movements,
essentially between Days 1 and 3 of treatment for males and Day 1 for females.
On day 5 the dose was lowered to 50 mg/kg bw/day. Clinical signs occurred on Day 5 of treatment in a few males only (lethargy, hunched posture, uncoordinated movements, decreased
locomotor activity, and/or ptosis). These clinical signs may have occurred secondary to the
dosing at 100 mg/kg/day on previous days and had resolved from Day 6 of treatment
onwards.
There were no adverse effects observed in the lower dose groups.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 100-50 mg/kg/day, a lower number of implantation sites was recorded (11.3 vs 13.7 in the
control group). This was in line with the lower litter size observed at this high dose level (see
F1-Developmental results). The mean remained well within the historical control data range
and the control mean was considered to be slightly high based on this range. However, this
change was considered to be adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 50 mg/kg/day, treatment-related but non-adverse changes in body weights and food intake
were recorded. Given the generally slight degree of the observed variations in body
weight and food intake and the absence of any clinical signs indicative of ill-health and delivery
of normal litters at this dose level, the variations in body weight and food intake were
considered not to be adverse.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day, an adverse delay in time to vaginal opening was recorded (36.1 days vs
32.5 days in the control group; mean exceeded the historical control data range). For a total of
12/24 animals (vs 3/23 animals in the control group) the time to vaginal opening exceeded
historical control data range. Based on this recorded change in F1 animals, anogenital distance
was measured for F2 animals but this parameter was considered not affected by treatment with
the test item. However, this delayed time to vaginal opening was considered to represent an
adverse effect of the test item, given the type of developmental change observed.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day, a lower number of implantation sites was recorded (10.1 vs 13.7 in the
control group). This was in line with the lower litter size observed at this high dose level (see
F1/F2-Developmental results). The mean remained well within the historical control data
range, and quantitative evaluation of primordial and small growing follicles and corpora lutea
from selected F1-females of Groups 1 and 4 did not reveal any treatment-related changes.
However, as described under F1/F2-Developmental results this was considered to represent an
adverse effect.
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 100-50 mg/kg/day, a non-adverse but treatment-related lower mean pup body weight was
recorded for male and female pups on lactation Days 13 and 21 (0.93x and 0.94x of control
mean for male and female pups, respectively, on lactation Day 21). There was no apparent
relationship between body weight gain of dams and offspring (i.e. dams with the lowest
weight gain during lactation did not necessarily have litters with the lowest PND 21 body
weight). It was therefore not considered likely that these lower pup weights occurred
secondary to the lower body weights of the dams. However, except for slightly lower postweaning body weights and food intake, their development did not reveal any adverse changes.
Also, given that the degree of change was relatively minor, these lower pup body weights
were considered not to represent an adverse effect.
Body weights of pups at 30-15 and 10-5 mg/kg/day were considered not affected by treatment
with the test item.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 50 mg/kg/day, lower mean pup body weights were recorded on lactation Days 7, 13 and 21
(0.87x and 0.88x of control mean for male and female pups, respectively on lactation Day
21). There was no apparent relationship between body weight gain of dams and offspring (i.e.
dams with the lowest weight gain during lactation did not necessarily have litters with the
lowest PND 21 body weight). It was therefore not considered likely that these lower pup
weights occurred secondary to the lower body weights of the dams. Overall, given the
magnitude of change, these lower pup body weights were considered to represent an adverse
effect of the test item.
At 15 mg/kg/day, non-adverse lower mean pup body weights were recorded on lactation
Day 21 (0.97x and 0.96x of control mean for males and females, respectively). Given the
minor degree of this change (reaching no statistical significance), it was considered not
adverse at this dose level.
Body weights of pups at 5 mg/kg/day were considered not affected by treatment with the test
item.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Behaviour (functional findings):
no effects observed
Developmental immunotoxicity:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
reliable guideline study

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
15 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
fully reliable guideline study

Justification for classification or non-classification

In an animal study the substance affected fertility and is therefore classified as a class 2 reproductive chemical (H361). 

Additional information