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Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

No effects on fertility were observed in the two studies available (OECD 422 and OECD 443) in rats. Any treatment related effects as found in the screening study were seen at maternally toxic doses and related to generic inflammation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 July 2001 - 28 January 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to internationally accepted guidelines and under GLP. No CoA attached to study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Alpk:APfSD (Wistar-derived)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rodent Breeding Unit, Alderley Park, Macclesfield, Cheshire, UK.
- Age at study initiation: 9- 10 weeks
- Weight at study initiation: females 200-240g, males 300-350g
- Fasting period before study: Not applicable
- Housing: The rats were housed, sexes separately, in multiple rat racks. They were housed up to 5 per cage initially, and two males or two females per cage after they had been assigned to experimental groups and during the pre-mating period: females were housed individually during pregnancy and lactation and provided with bedding material
- Diet (e.g. ad libitum): ad libitum, Diet (CTI)
- Water (e.g. ad libitum): ad libitum, mains water
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: 24 July 2001 - 21 September 2001
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was formulated in Mazola corn oil. Dosing preparations were shaken prior to dosing, and stirred, using a magnetic stirrer, during
dosing as required.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 10, 35 or 100 mg/ml (nominal), actual concentration ± 5%
- Amount of vehicle (if gavage): 1.0ml/100g bodyweight
- Lot/batch no. (if required): Y00790/007
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: The pre-coital interval was less than 4 days for all animals i.e. all animals mated within the first oestrus cycle
- Proof of pregnancy: sperm in vaginal smear referred to as [day 0 / day 1] of pregnancy
- Further matings after two unsuccessful attempts: Not applicable, did not occur
- After successful mating each pregnant female was caged (how): females were housed individually during pregnancy and lactation and provided with bedding material
- Any other deviations from standard protocol: none
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from all dose levels (including controls) were taken at intervals throughout the study and analysed quantitatively for Aquapel 364. The homogeneity of Aquapel 364 in the dosing formulations was determined by analysing samples from the low and high dose levels. The chemical stability of Aquapel 364 in corn oil was determined at concentrations of 1 and 100mg/ml in a preliminary study (RRO905). The method of analysis used for the quantitative determination of Aquapel 364 in Mazola Corn Oil is HPLC.
Duration of treatment / exposure:
Exposure period: At least 28 days
Premating exposure period (males): Males were dosed two weeks prior to mating continuing throughout day 29 or 30.
Premating exposure period (females): Females were dosed two weeks prior to mating, during mating, throughout pregnancy and until 4 days post partum (day 4 of lactation).
Frequency of treatment:
Once per day
Remarks:
Doses / Concentrations:
0 (control), 100, 350 or 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose levels selected for this study were based on the results of a dose range finding study (RR0905) in the same strain of rat carried out in this Laboratory and included an anticipated no-observed effect level and a level where Aquapel 364 was expected to produce toxicity in the parents.
- Rationale for animal assignment (if not random): random
- Other: None
Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of the study, all rats were examined to ensure that they were normal. During the study all rats were observed daily and changes in clinical condition were recorded. Rats requiring euthanasia were killed and subjected to an examination post mortem.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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

OTHER:
Functional observational battery
Detailed clinical assessments (during which each rat was removed from its cage and physically examined for changes in general health status) and quantitative assessments of muscle weakness (fore- and hindlimb grip strength) and sensory perception (tail-flick test) were made in week 4 of the study for five animals per sex per group. The observations were made by one observer who was 'blind' with respect to the animal's treatment, and recorded on a computer system by personnel not directly involved in the clinical observations. The presence and/or absence of all listed observations was recorded and the degree of condition noted (slight, moderate or extreme) where appropriate.
The clinical observations included, but were not restricted to, the following list of measures:
a) Assessment of signs of autonomic functions, e.g. lachrymation, salivation, piloerection, exophthalmus, urinary incontinence, diarrhoea, pupillary response to light and ptosis.
b) Description, incidence and severity of any convulsions, tremors or abnormal motor movements, both in the home cage and standard (open) arena.
c) Ranking by severity, the subject's reactivity to general stimuli such as removal from the cage or handling.
d) Ranking by severity, the subject's arousal level or state of alertness during observations of the unperturbed subject in the standard (open) arena.
e) Descriptions and incidence of posture and gait abnormalities observed in the home cage and in the standard (open) arena.
f) Assessment of audition by response to a sudden sound (e.g. clicking fingers).
g) Description and incidence of any unusual or abnormal behaviours, excessive or repetitive actions (stereotypes), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose or mouth, and any other observations that may facilitate interpretation of the data.

Motor activity
Locomotor activity was monitored by an automated activity recording apparatus. Five animals per sex per group were tested in week 4 of the study. Each observation period was divided into ten scans of five minutes duration. Treatment groups were counter balanced across test times and across devices. Motor activity was assessed in a separate room to minimise disturbances.

Urine clinical chemistry
The following parameters were measured, or determined semi-quantitatively on urine samples collected from all animals (10/sex/group) during week 4.
colour, volume, specific gravity, glucose, bilirubin, appearance, pH, protein, ketones, blood

Haematology
The following measurements were made or determined on samples collected from all animals (10/sex/group) using EDTA as an anticoagulant:
red blood cell count, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, total white blood cell count, differential count, platelet count.
Measurements of potential clotting (prothrombin time and activated partial thromboplastin time with kaolin) were made on samples of blood collected into tubes containing trisodium citrate as an anticoagulant. Blood cell morphology, including a differential white blood cell count was assessed by automated methods for all animals. A blood film was prepared from all animals and examined where the results from the automated analysis suggested this was necessary.

Blood clinical chemistry
The following measurements were made on the plasma from blood samples collected from all animals (10/sex/group) into tubes containing lithium heparin as an anticoagulant:
urea, creatinine, glucose, albumin, total protein, albumin/globulin ratio, cholesterol, triglycerides, sodium, potassium, chloride, calcium, total bilirubin, phosphorus (as phosphate), gamma-glutamyl transferase activity, creatine kinase activity, alkaline phosphatase activity, aspartate arninotransferase activity, alanine arninotransferase activity
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
% of pups live born
% of pups surviving to day 5
Bodyweights - Individual pup bodyweights were recorded within 24 hours of birth (day 1) and on day 5 post partum. Since pups were not individually identified, data were recorded by sex and litter.

GROSS EXAMINATION OF DEAD PUPS:
no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on day 29/30 of the study.
- Maternal animals: All surviving animals day 4 of lactation.

GROSS NECROPSY
- All animals were subjected to a full exarnination post mortem. A full exarnination post mortem is defined in this Laboratory as one which involves an external observation and a detailed examination of all cranial, thoracic and abdominal organs and structures. For all females (10/sex/group) which had been mated the number of implantation sites was recorded and the number of corpora lutea counted.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

From all animals (10/sex/group) surviving to scheduled termination, the following organs were removed, trimmed free of extraneous tissue and weighed (Paired organs were weighed together):
adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, thymus, testes, ovaries

At necropsy the full range of tissues, see below was taken on all animals (i.e. 10/sex/group) but the tissues from odd numbered animals were stored for possible future examination. Removal and storage of tissues.
abnormal tissue, adrenal gland, bone marrow (femoral bone), brain (cerebrum, cerebellum, pons), caecum, cervix, colon, duodenum, epididymis, heart
ileum, jejunum, kidney, liver, lung, lymph nodes (cervical and mesenteric), mammary gland (females only), ovary, Peyer's patches, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicle including coagulating gland, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina

Tissue processing and examination
All submitted tissues from all animals found dead or killed intercurrently and for 5 males and 5 females (even numbered animals) per group at scheduled termination were trimmed, embedded in paraffin wax and 5pm sections cut, stained with haematoxylin and eosin and examined by light microscopy. Compound-related changes were observed in some tissues, listed below, in the high dose group and therefore relevant tissues from the mid and low dose groups (even numbered animals) were trimmed, embedded in paraffin wax and 5 p sections cut, stained with haematoxylin and eosin and examined by light microscopy. Tissues processed from the mid and low dose groups:
Males: Kidney, liver, mesenteric lymph node, spleen and heart.
Females: Adrenals, cervix, heart, jejunum, kidney, liver, cervical and mesenteric lymph nodes, Peyer's patch, spleen, thymus and uterus.
Postmortem examinations (offspring):
None
Statistics:
All analyses were carried out in SAS (1999). For Fisher's Exact Tests the proportion in each treated group was compared to the control group proportion. Analyses of variance and covariance allowed for the replicate structure of the study design.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One control female was killed for humane reasons following paturition, no further mortalities. There were no compound related clinical chenges.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Males days 11-29
Bodyweights in males given 1000mg/kg/day were slightly lower than controls during the last week of the study. There were no other compound-related effects on bodyweights in males.
- Females pre-mating
There were no compound-related effects on bodyweights in females.
- Females during gestation
Group mean female bodyweights during gestation were similar to control values.
- Females during lactation
During the lactation period there were no effects that were attributable to treatment with Aquapel 364. Daily bodyweights were recorded prior to littering for animals number 59 71 and 79 and since littering was not completed during the working day, bodyweightspost partum were not recorded on day 1 for these animals. The pre-littering bodyweights were excluded from the data analysis.

- Males pre-mating
Food consumption in all treated male groups was similar to controls during the pre-mating period.
- Females pre-mating
There were no adverse effects on food consumption during the pre-mating period.
- Females during gestation
Group mean food consumption during gestation was similar to control values.
- Females during lactation
Food consumption in females during lactation showed some variation between the groups but there was no clear indication of a compound-related effect.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating
The pre-coital interval was less than 4 days for all animals i.e. all animals mated within the first oestrus cycle.
- Fertility
There were no compound-related effects.
- Gestation
There was no effect on the length of gestation.
- Reproductive performance
In females, however, there was an inflammation of the ovaries, uterus and cervix. Along with the inflammations, the ratio of corpora lutea to implantation sites was reduced. This pre-implantation loss, was observed in all treated groups, but the effect was not dose-related. . The number of corpora lutea was slightly higher in females given 1000 mg/kg b.w./day than in the control, 100 and 350 mg/kg b.w./day dose groups. However, the ratio of implantation sites to corpora lutea was similarly reduced in all treated groups when compared to controls. There were no effects on post-implantation loss. The litter size was smaller in all treated groups with the effect being most marked at the low- and mid-dose where the number of corpora lutea and hence the number of implantation sites, was lower than at the high-dose (see table 3) Individual animal data (see Table 4) seems to confirm a correlation between inflammation of the uterus and cervix and the observed pre-implantation losses. Five animals per group were subjected to micropathology. To analyse the data, a threshold for significant pre-implantation losses was set to five pre-implantation losses, since it is the mean + S.D. for pre-implantation losses in the control group.
- Whole litter losses
There was one whole litter loss, in the 100mg/kg/day group. There were no whole litter losses in the higher dose groups and therefore this is not a compound-related effect.
- Pups live born
There were no compound-related effects.
- Litter size
There were no compound-related effects.

ORGAN WEIGHTS (PARENTAL ANIMALS) (table 1)
There was a dose-related elevation in spleen weights at all dose levels in males and females. In females there was an elevation in kidney and liver weights at all dose levels and an elevation in ovary weights in animals given 1000 or 350 mg Aquapel 364kg/day

GROSS PATHOLOGY (PARENTAL ANIMALS)
All animals survived to the scheduled termination except for one control female which was killed on humane grounds. Implantation sites were absent from one(350 mg Aquapel 364kg) or two (1000 mg Aquapel 364kg) females in the two higher dose groups whereas all controls and females receiving 100 mg Aquapel 364kg had implantation sites visible at necropsy. Enlargement of the spleen and a number of lymph nodes were observed in females in all treatment groups. The affected lymph nodes were as follows: hepatic (100, 350 and 1000mg Aquapel 364/kg), mesenteric (350, 1000mgkg), pancreatic (1000 mgkg), renal (100, 350 and 1000 mg/kg), thymic (1000 mg/kg). In most cases the incidence of the observation was related to dose level. No change which could be related to treatment was observed in males.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment related inflammatory changes including histiocytosis were observed in a variety of tissues of animals at all dose levels. Overall the incidence, severity and distribution of the changes were greater in females. In females the liver and mesenteric lymph node were most consistently affected i.e. in 100% of females at all dose levels. The other tissues affected (listed in order of decreasing frequency with which they were involved) were cervix, kidney, Peyer's patch, spleen, uterus, hepatic lymph node, cervical lymph node and jejunum. Isolated incidences of changes similar to those seen in other tissues were also seen in the adrenal gland, ileum, pancreatic, renal and thymic lymph nodes and thymus. In males the range of tissues affected was smaller. Changes similar to those observed in females were present in the mesenteric lymph node and liver at all dose levels. Other tissues affected were kidney in animals receiving 350 and 1000 mg Aquapel 364/kg and spleen in animals receiving 1000 mg Aquapel 364kg only. No treatment related changes were observed in the male reproductive organs. Increased extramedullary haemopoiesis in the spleen was also observed in all treated females at all dose levels and in the majority of males receiving 350 and 1000 mg Aquapel 364/kg. In addition there was a small increase in incidence of minor changes in the heart (degenerative cardiomyopathy and mononuclear cell infiltration) in treated animals of both sex compared with controls although this was not closely related to dose level. In all tissues the inflammatory changes consisted predominantly of mononuclear cell infiltration often with focal accumulation of macrophages. The latter feature was particularly prominent in the liver of females where large multinucleate giant cells were also frequently observed. Similar accumulation of macrophages was observed as histiocytosis in the spleen, lymph nodes and Peyer's patches of a number of animals at the affected dose levels. In affected lymph nodes histiocyte accumulation was most prominent in the paracortex and medullary cords. In the heart inflammatory cell infiltration was sometimes associated with degenerative changes; in these cases the term degenerative cardiomyopathy was used. In male livers the change consisted of a few very small foci of mononuclear cells without macrophage accumulation. The term "mononuclear cell infiltration" was used to describe this to differentiate it from the considerably more diffuse change (diffuse mononuclear cell hepatitis) seen in females. There was no consistent dose response; especially in females where the incidence and severity of most changes was not dose related. In males a dose response was a little more evident with changes at the lowest dose level (100mg Aquapel 364/kg) restricted to the liver and mesenteric lymph node.

OTHER FINDINGS (PARENTAL ANIMALS)
- Urine clinical chemistry
There was a slight reduction in urine pH in males given 1000mg Aquapel 364 kg/day. This change is slight and is considered to be unrelated to administration of Aquapel 364. All other urine parameters in males and females were similar to controls.
- Haematology (table 2)
There were no effects on red blood cell parameters in either sex. The decreased platelet count seen in females receiving 350 mg/kg/day was not sustained at 1000 mgkglday so was considered not to be treatment-related. A dose-responsive increase was seen for white blood cell, lymphocyte, large unstained cells and monocyte counts in both sexes at all doses, although in males, the lymphocyte and monocyte counts were only statistically significantly raised at 1000mg/kg/day. In females there was a dose related increase in basophil numbers, but whilst all dose groups of males had increased basophil numbers compared with controls this increase was only statistically significant at 100 mg/kg/day. The automated haematology analyser indicated alerts for atypical white blood cells in individual animals, which triggered the manual examination of blood smears. The manual verification of blood films demonstrated the presence of immature myeloid cells and atypical monocytes. Only a single male at each of 100 and 350 mg/kg/day required evaluation, but all dosed females except for one female at each of 100 and 350 mg/kg/day required evaluation. In females there was a dose-related increase in immature myeloid cells, but the incidence of atypical monocytes did not vary across the groups. Prothrombin times were slightly lower than control values in both sexes given 1000 mg Aquapel 364kg/day and in males given 350 mg/kg/day. These changes were considered not to be adverse and are of no toxicological significance.
- Blood clinical chemistry
In females, increases were seen for albumin and total protein in animals receiving 350 and 1000 mg/kg/day, leading to a decreased albumin globulin ratio in these groups. Increases were also seen for plasma calcium values at these doses in this sex.
Following exclusion of high values for females 60 and 70 in the intermediate dose groups, total bilirubin was increased in both sexes receiving 1000 mg/kg/day. Increases were also seen for cholesterol in both sexes receiving 1000 mgkglday and in females receiving 350 mg/kg/day. Enzyme activity was increased for alkaline phosphatase in females receiving 350 and 1000 mg/kg/day and males receiving 1000 mg/kg/day, for alanine aminotransferase in males receiving 350 and 1000 mg/kg/day and females receiving 1000 mg/kg/day and for aspartate aminotransferase in both sexes receiving 350 and 1000 mg/kg/day. Generally these increases showed a dose response. The increased gamma glutamyl transferase activity in females receiving 1000 mgkglday was small in magnitude and considered not to be of any biological or toxicological significance.
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
< 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: LOAEL based on inflammatory changes in a variety of tissues in both genders.
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
< 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
reproductive performance
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
LOAEL
Remarks:
reproductive performance
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: LOAEL based on reduced proportion of implantation sites compared to the numbers of corpora lutea.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen
Remarks on result:
other: No effects at highest dose 1000 mg/kg bw/day
Reproductive effects observed:
not specified

Table 1: Body weights and Organ weights (only organs with significant changes are listed) 

 

Dose [mg/kg b.w./day]

 

0

100

350

1000

TERMINAL B.W.

 

 

 

 

MALES

 

 

 

 

Mean

467.9

 463.5

 459.2

 446.3

S.D.

30.7

31.4

33.1

33.1

FEMALES

 

 

 

 

Mean

331.2

338.9

330.0

319.8

S.D.

11.3

13.1

25.1

26.2

LIVER

 

 

 

 

MALES

 

 

 

 

MEAN

18.4

18.2

18.5

18.5

S.D.

1.5

1.6

1.8

1.5

Organ Weight Adjusted

18.1

18.0

18.5

19.0*

For Bodyweight

 

 

 

 

FEMALES

 

 

 

 

MEAN

13.4 15.7*

17.0**

16.8**

 

S.D.

 1.0

1.1

2.8

2.4

Organ Weight Adjusted

13.3

14.9**

17.0**

17.6**

For Bodyweight

 

 

 

 

KIDNEYS

 

 

 

 

MALES

 

 

 

 

Mean

3.21

3.08

3.20

3.20

S.D.

0.23

0.27

0.23

0.38

Organ Weight Adjusted

3.16

3.05

3.20

3.28

For Bodyweight

 

 

 

 

FEMALES

 

 

 

 

Mean

1.94

2.18**

2.10*

2.09*

S.D.

0.11

0.17

0.20

0.13

Organ Weight Adjusted

1.93

2.13**

2.10**

2.15**

for Bodyweight

 

 

 

 

SPLEEN

 

 

 

 

MALES

 

 

 

 

Mean

0.966

1.096*

1.184**

1.200**

S.D.

0.100

0.132

0.156

0.116

Organ Weight Adjusted

0.943

1.084**

1.184**

1.235**

for Bodyweight

 

 

 

 

FEMALES

 

 

 

 

Mean

0.708

1.110**

 1.403**

1.405**

S.D.

0.085

0.203

0.227

 0.253

Organ Weight Adjusted

1.93

2.13**

2.10**

2.15**

for Bodyweight

 

 

 

 

OVARIES

 

 

 

 

Mean

0.107

0.118

0.130**

0.127**

S.D.

0.015

0.014

 0.015

 0.012

Organ Weight Adjusted

0.107

0.116

0.130**

0.129**

for Bodyweight

 

 

 

 

** Statistically significant difference from the control group mean at the 1% level (Student's t-test, two sided)

 

Table 2: Haematology(in cell counts x10-9/L) (only parameter with significant changes are listed)

 

 

 

Dose in mg/kg b.m./day

Gender

Parameter

control

100

350

1000

day 30

White blood cells

7.56

9.02*

9.50**

10.29**

 

Neutrophil

1.46

2.34**

2.36**

2.10**

 

Lymphocyte

5.71

6.02

6.49

7.41**

 

Monocyt

0.123

0.174

0.192

0.290**

 

Large unstained cells

0.092

0.238**

0.246**

0.275**

day 5
post partum

White blood cells

5.41

8.32**

9.96**

11.40**

 

Lymphocyte

53.23

5.29**

6.73**

7.55**

 

Monocyt

0.066

0.332**

0.395**

0.536**

 

Basophil

0.012

0.133**

0.152**

0.160**

 

Large unstained cells

0.063

0.609**

0.697**

0.922**

** Statistically significant difference from the control group mean at the 1% level (Student's t-test, two sided)

Table 3: Corpora lutea and implantation sites and incidences of pre-impantation losses

 

Dose level of Aquapel 364 [mg/kg b.w./day]

 

0 (control)

100

350

1000

No. of corpora lutea

 

 

 

 

 MEAN

13.9

 14.1

12.2

16.0

 S.D.

2.4

 2.9

4.5

3.1

 N

9

10

9

8

No. of implantations

 

 

 

 

 MEAN

11.2

 9.3

9.1

11.3

 S.D.

3.6

4.6

 3.1

 3.4

 N

9

9

8

8

Pre-implantation loss

 

 

 

 

 Prop. of implants affected

24/125

54/141**

36/110*

47/128**

 Percentage

 

 

 

 

   MEAN

19.8

39.4

28.1

35.6

   S.D.

19.8

27.8

27.1

29.9

   N

9

10

9

8

No. of live + dead pups

 

 

 

 

 MEAN

 11.2

 7.0*

7.1

9.1

 S.D.

3.7

4.5

 4.7

 4.9

 N

10

 10

 9

 8

Table 4: Pre-implantation losses and inflammations of uterus and/or cervix in animals with micropathological examination (five animals per group were examined)

Dose

Animal no

Cervix

Uterus

Corpora lutea

Implantations

Pre-implantation. lossesa

 

 

 

 

Left

Right

Left

Right

 

0

42

0

0

4

10

4

8

2

44

0

0

9

7

3

6

7

46

0

0

3

8

3

5

3

48

other

 

6

10

6

9

1

50

0

0

6

6

5

0

7

100

52

0

0

6

5

0

5

6

54

1

1

6

7

2

3

8

56

2

1

10

8

2

2

14

58

0

0

3

9

3

9

0

60

other

other

5

4

0

3

6

350

62

1

1

4

9

2

4

7

64

2

1

6

8

2

4

8

66

2

2

7

5

3

4

5

68

1

1

small

small

1

0

0

70

0

0

small

small

0

0

0

1000

72

1

1

8

8

8

5

3

74

1

1

5

8

1

1

11

76

1

1

7

7

4

7

3

78

1

1

small

small

0

0

0

80

1

0

5

10

5

10

0

 a bold letters indicate losses above threshold of 5 (mean+S.D. of control), Italic, red fonts indicate significant losses with inflammation of uterus and/or cervix

Cervix/Uterus

 “severity of the lesions”

1 minimal

2 slight

3 moderate

4 marked

Conclusions:
Oral administration of 1000 mg/kg/day Aquapel 364 for at least 28 days in males and throughout pregnancy to day 5 lactation in females produced inflammatory changes in a number of tissues together with increases in specified organ weights, increases in white blood cell counts and splenic extramedullary haemopoiesis. In addition there was a mild perturbation of some clinical chemistry parameters and a decreased proportion of implantation sites compared to the observed number of corpora lutea. There were no adverse effects on pup parameters.
The majority of changes were seen at decreased severity and/or incidence in animals given 350 or 100 mg Aquapel 364/kg/day. However some of the histopathological changes and the reduced ratio of implantation sites to corpora lutea showed no evidence of a dose-response relationship.
A NOAEL was not achieved in this study.
Executive summary:

A 28 day repeated dose combined with repoductive/developmental screening study was performed according to OECD 422 and under GLP.

Study design

Groups of 10 male and 10 female (parents) Alpk:APfSD (Wistar-derived) rats were dosed with 0 (control), 100, 350 or 1000mg/kg/day Aquapel 364 in Mazola Corn Oil. After 2 weeks, the animals were mated and allowed to rear the ensuing litters to 5 dayspost parturn. Males were dosed during mating and to termination on day 29/30 of the study. Females were dosed during mating, gestation and to day 4 of lactation. Litters were weighed and examined on days 1 and 5 postpartum.

In addition the following were carried out on all animals. Clinical observations, bodyweights and food consumption were measured throughout the study. Urine samples were collected during week 4 for the assessment of urine clinical pathology. Detailed clinical observations, including quantitative assessments of sensory perception and muscle weakness, and assessment of motor activity were performed on day 28 on 5 animals /sex/group. At the end of the scheduled period, the animals were killed and subjected to an examination post mortern. Cardiac blood samples were taken for clinical pathology, selected organs were weighed, corpora lutea were counted in pregnant females and specified tissues were taken for

subsequent histopathology examination.

Results

The analyses of stability, achieved concentration and homogeneity of the test substance in the dosing preparations were satisfactory. At a dose level of 1000 mg Aquapel 364/kg/day in adults there were no mortalities, no clinical changes and no adverse effects on bodyweight or food consumption and no neurotoxic effects. There were no effects on breeding parameters with the exception of a reduced proportion of implantation sites compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there was an increase in basophils. There were increases in total bilirubin and cholesterol in both sexes and in albumin, total protein and plasma calcium in females. In addition there were increases in some enzymes in both sexes. In both sexes there was an increase in spleen weight and in liver, kidney and ovary weights in females. Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the

overall incidence, severity and distribution was greater in females. There were no effects on pups. At a dose level of 350mg Aquapel 364/kg/day in adults there was a reduced proportion of implantation sites compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there was an increase in basophils. There was an increase in cholesterol, albumin, total protein and plasma calcium in females. In both sexes there was an increase in spleen weight and in liver, kidney and ovary weights in females.

Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the overall incidence, severity and distribution was greater in females. At a dose level of 100mg Aquapel 364kg/day in adults there was a reduced proportion of implantation sites compared to the numbers of corpora lutea. In both sexes there was an increase in total white blood cell count due to increases in large unstained cells, lymphocytes and monocytes and in females there was an increase in basophils. There was an increase in spleen weight and in liver and kidney weights in females. Macroscopic enlargement of the spleen and lymph nodes was seen in females. Microscopically, inflammatory changes were seen in a variety of tissues in both sexes but the overall incidence, severity and distribution was greater in females.

Conclusion

Oral administration of 1000 mg/kg/day Aquapel 364 for at least 28 days in males and throughout pregnancy to day 5 lactation in females produced inflammatory changes in a number of tissues together with increases in specified organ weights, increases in white blood cell counts and splenic extramedullary haemopoiesis. In addition there was a mild perturbation of some clinical chemistry parameters and a decreased proportion of implantation sites compared to the observed number of corpora lutea. There were no adverse effects on pup parameters. The majority of changes were seen at decreased severity and/or incidence in animals given 350 or 100mg Aquapel 364/kg/day. However some of the histopathological changes and the reduced ratio of implantation sites to corpora lutea showed no evidence of a dose-response relationship.

A NOAEL was not achieved in this study. A LOAEL for reproductive performance was defined at 100 mg/kg bw based on reduced proportion of implantation sites compared to the numbers of corpora lutea.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October 2019 to
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
request by ECHA (decision CCH-D-2114440086-55-01/F )
Qualifier:
equivalent or similar to guideline
Guideline:
other: • OECD guidance document supporting OECD test guideline 443 on the extended one-generation reproductive toxicity test, No. 151, July 2013.
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
Based on ECHA decision number CCH-D-2114440086-55-01/F (14 September 2018)
Specific details on test material used for the study:
At the time of the study the substance was still defined as 2-Oxetanone, 3-C12-16-alkyl-4-C1317-alkylidene derivs (CAS number: 84989-41-3)
Species:
rat
Strain:
Wistar
Details on species / strain selection:
This rat is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive and toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain: Wistar Crl: WI(Han)
- Age at study initiation: (P) 6 wks; (F1) dosing after weaning on day 21
- Weight at study initiation: (P) Males: 159-192 g; Females: 110-141 g; (F1) Males: 44-66 g; Females: 40-65 g
- Fasting period before study: NA
- Housing:
F0 pre-mating (males post-mating) and F1 5/sex/group in polycarbonate cages (Macrolon type IV; height 18 cm or 2000P; 61x43.5x21.5 cm).
Mating: 1 male/1 female in Macrolon plastic cages (type III; height 18 cm).
Females during gestation and lactation individually in Macrolon plastic cages (type III, height 18 cm). Pups were housed with the dam until termination or weaning (on PND 21).
The cages contained appropriate bedding and were equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: Municipal tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean 22 °C
- Humidity (%): 45-54%
- Air changes (per hr): at least 10 with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% Aqueous carboxymethyl cellulose with 0.1% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized by mixing with an ultra-turrax to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 24 hours after adding the vehicle to the test item. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on pre-test trials
- Concentration in vehicle: 0,2,10 and 50 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1 male +1 female
- Length of cohabitation: maximum 14 days; actual 1-4 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post-coitum
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
UPLC/UV

Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Acquity UPLC PDA detector (Waters)
Column Acquity UPLC BEH C18, 50 mm x 2.1 mm i.d., dp =1.7 µm (Waters)
Column temperature 40°C ± 1°C
Injection volume 1 and 2 µL
Mobile phase Methanol
Flow 0.6 mL/min
UV detection 220 nm

Method development
Calibration: at 80-600 mg/mL, r> 0.99 (y = 0.108 x - 0.48)
QC samples: at 1 mg/ml 98-104% of nominal; at 200 mg/mL 99-102% of nominal
Accuracy: day of preparation: at 1 mg/mL 99% of nominal; at 200 mg/mL 93% of nominal
Homogeneity: confirmed CV at 1 mg/mL 5.4%; CV at 200 mg/mL 0.9 %
Stability in refrigerator: 3 days at 1 mg/mL 83% of nominal (CV 10%); 3 days at 200 mg/mL 97% of nominal (CV 0.94) --> not stable -->daily formulation

Sample analyses (week 1, 11 and 22):
Calibration: at 80-600 mg/mL r> 0.99 (no details)
QC: at 2 mg/mL 87-106% of nominal; at 50 mg/mL 103-121% of nominal
Sample analyses (week 13):
Calibration: at 80-600 mg/mL r> 0.99 (no details)
QC: at 1 mg/mL 100% of nominal; at 3 mg/mL 104% of nominal

Accuracy: at 50 and 250 mg/kg bw: 88-93% and 87-112% of nominal, respectively
Homogeneity: at 250 mg/kg bw: CV 2.6-5.7%, respectively
Accuracy: at 10 mg/kg bw: 55-102% of nominal in week 1, 11 and 22; 72% of nominal in week 13
Homogeneity: at 10 mg/kg bw: CV 14-23% in week 1, 11 and 22; 10%, respectively of nominal in week 13

The accuracy at 10 mg/kg bw was below the 85% specification. Therefore the concentration tested at 10 mg/kg bw could have been lower than indicated. This does not affect the study outcome, as a NOAEL of 250 mg/kg bw was derived. For this NOAEL all analytical specifications were met.

Needxs to be extended
Duration of treatment / exposure:
F0: males and females 10 weeks before mating and during mating; females during gestation and lactation (day 21 post-coitum)
F1:
Cohort 1A: males and females from weaning day 21 post-natal until day 89-95
Cohort 1B: males and females from weaning day 21 post-natal until day 97
Cohort 1C: males and females from weaning day 21 post-natal until day 41-42 (males) and 31-32 (females)
Frequency of treatment:
once daily (pups during lactation were not dosed directly)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
measured 87-112% of nominal
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
measured 88-93% of nominal
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
measured <10 mg/kg bw
No. of animals per sex per dose:
F0: 25 males + 25 females per group
F1: 20 males + 20 females per group
(surplus: 10 males +10 females per group for thyroid hormone assessment on post natal day 22)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels in this study were selected based on the results of two toxicity studies with oral exposure of the substance in rats and in an attempt to produce graded responses to the test item.
In a OECD 422 study (28-day study combined with the reproduction and developmental screening test), Alpk:APfSD (Wistar-derived) rats were dosed by gavage at 0 (corn oil only), 100, 350 or 1000 mg/kg/day. There were no mortalities or clinical symptoms, no adverse effects on body weight or food consumption and no adverse effects on reproductive or developmental parameters up to 1000 mg/kg/day. However, in both sexes at 1000 mg/kg/day, inflammatory changes were observed microscopically in a variety of tissues (overall incidence, severity and distribution was greater in females) in combination with an increase in total white blood cell counts (due to increases in large unstained cells, lymphocytes and monocytes), increased liver enzyme activity, increases in spleen, liver, kidney and ovary weight and splenic extramedullary haemopoiesis. In addition, the spleen and lymph nodes of females were enlarged at necropsy. The majority of these effects were also seen although at decreased severity and/or incidence in animals dosed at 350 or 100 mg/kg/day, but some of the microscopic changes were not observed in a dose-related relationship. Nevertheless, no NOAEL was established in this study.
In an OECD 408 study (90-day study), Alpk:APfSD (Wistar-derived) rats received 0, 65, 650 or 6500 ppm by dietary administration, resulting in dose levels of 0, 6-7, 63-70 or 645 691 mg/kg/day. There were no mortalities or clinical symptoms up to 6500 ppm. A decrease in body weight and food consumption was noted in animals at 6500 ppm. Similar effects were described at 6500 ppm as observed during the OECD 422 study, increased white blood cell counts, increased liver enzyme activity, increased spleen and liver weights and treatment-related inflammatory changes in a variety of tissues in both sexes. The inflammatory changes included foci of inflammation and histiocytosis. In addition, a decrease of red blood cells in males and females and a higher kidney weight in females only was reported at this dose level.
At 650 ppm, there were similar findings but of lesser severity and/or incidence when compared with the 6500 ppm group and at 65 ppm there were no changes of biological or toxicological relevance when compared with controls.
In the current study, the total duration of exposure was longer than in the OECD 408 study and a 10-week premating period was included as opposed to the 2-week premating period in the OECD 422 study. In addition, the F0-females would become pregnant and lactating during the current study which would also burden their livers in addition to the test item related effects on the liver as observed in previous studies (i.e. increased liver weight, increased liver enzyme activity and inflammatory changes) and the F1-generation will be dosed from a younger age (i.e. 3 weeks) onwards when compared with the age at start treatment in the OECD 408 and 422 studies. Considering this information and the results of the previously performed toxicity studies, 10 mg/kg/day was selected as low dose as this dose level is anticipated to be a ‘No Adverse Effect Level’ (NOAEL). A dose level of 250 mg/kg/day was selected as high dose, as this was considered likely to result in some toxicity similar to the effects observed in previous studies. A mid dose level of 50 mg/kg/day was selected as this was at an appropriate interval between the low and high dose.
The high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
Positive control:
NA
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality/moribundity, twice daily for clinical signs (pre- and post dosing)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly in a standard arena

BODY WEIGHT: Yes
- Time schedule for examinations: weekly including mating period; Mated females on day 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post-natal day 1, 4, 7, 14 and 21.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly including mating period; Mated females on day 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post-natal day 1, 4, 7, 14 and 21.

WATER CONSUMPTION Yes
- Time schedule for examinations: qualitative examination

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes up to 24 hours (water supplied)
- How many animals: 10 males + 10 females per group
- Parameters: White blood cells (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Red blood cells, Reticulocytes (absolute), Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets, PT, APTT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes up to 24 hours (water supplied)
- How many animals: 10 males + 10 females per group
- Parameters checked : phosphatase (ALP), Total protein, Albumin, Total bilirubin, Bile acids (in serum), Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic phosphate (Inorg. Phos)

THYROID HORMONES: Yes
- Time schedule for collection of blood: day of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes up to 24 hours (water supplied)
- How many animals: serum of 10 males + 10 females per group
- Parameters checked : TSH, T4

URINALYSIS: Yes
- Time schedule for collection of urine: on day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes up to 15-20 hours (water supplied)
- How many animals: serum of 10 males + 10 females per group
- Parameters: Volume, Specific gravity, Clarity, Colour, pH, Blood, White blood cells (WBC), Bilirubin, Urobilinogen, Protein, Ketones, Glucose, Nitrite
Sediment: White blood cells (WBC-sed.), Red blood cells (RBC-sed.), Casts, Epithelial cells, Crystals, Bacteria, Other

Oestrous cyclicity (parental animals):
Daily vaginal lavage from 14 days prior to mating and during mating until evidence of copulation was observed. On day of termination vaginal lavage of all surviving females.
Sperm parameters (parental animals):
testis weight, epididymis weight, sperm count in epididymides, sperm (progressive) motility, sperm morphology
Postmortem examinations (parental animals):
F0 and F1 see attached table
Organ weights: see table for all F0 and unscheduled deaths
Macroscopic investigations: all F0 and unscheduled deaths,
Histopathology details: see attached table

Based on possible treatment-related microscopic findings, additional tissues were prepared
and examined of animals of the 10 and 50 mg/kg/day groups:
F0-Generation: Kidneys of both sexes, eyes of males and adrenal glands of females.
Postmortem examinations (offspring):
F0 and F1 see attached table
Organ weights: see table for all F1 Cohort 1A animals and unscheduled deaths
Macroscopic investigations: F1 Cohort 1A animals and unscheduled deaths; reproductive organs and sternum of Cohort 1B and 1C
Histopathology details: see attached table

Based on possible treatment-related microscopic findings, additional tissues were prepared
and examined of animals of the 10 and 50 mg/kg/day groups:
F1-Generation-Cohort 1A: Eyes of females. The mesenteric lymph nodes of selected rats of
both sexes were already defined by protocol.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
- Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
- Incidence: An overall Fisher’s exact test was used to compare all groups. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
Mating index males (%); Mating index females (%); Fertility index males (%); Fertility index females (%); Gestation index (%)
Offspring viability indices:
Live birth index (%); Viability index (%); Weaning index (%):
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control male was sacrificed in extremis on day 30 (wound in neck correlating with ulcer)
One male at 50 mg/kg/day was sacrificed in extremis on day 72, due to a gavage error
One female at 250 mg/kg/day was sacrificed due to total litter loss/difficult parturition on post-coitum Day 23. Necropsy of this animal showed one late resorption in the left uterine horn and one late resorption and one live male fetus (without abnormalities) in the right uterine horn. Since none of the other females at this dose level displayed a similar condition, this was considered to be an incidental occurrence.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males at 250 mg/kg bw:
end of mating period BW 6% below controls (statistically significant) and BWG over the whole period 11% below controls (statistically significant)

These changes were within normal variations
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:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
females at 250 mg/kg bw: increase of white blood cells (21% ns), increase of number of lymphocytes (25% ns) and decrease of reticulocytes (15% ns)
females at 50 mg/kg bw: increase of white blood cells (17% ns), increase of number of lymphocytes (18% ns) and decrease of reticulocytes (16% ns)
females at 10 mg/kg bw: increase of white blood cells (21% ns) and increase of number of lymphocytes (21% ns)

All values were within historical control values
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Incidental significantly higher sodium and potassium levels were considered not treatment related effects;
for details on the results on thyroid hormones see attached table
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males and females at 250 mg/kg bw: inflammatory cell infiltrate in kidneys (most obvious at the corticomedullary junction)
Females at 250 mg/kg bw: inflammatory cell infiltrate in adrenals
Males at 250 mg/kg bw: inflammatory cell infiltrate in uvea of the eyes

Infiltrates were mainly lymphocytes (details see attached table)
It cannot be excluded that these findings were treatment related, however considered non-adverse
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
(benign) myelolipoma in the cortex of the adrenal gland of one male at 250 mg/kg/day, that was considered spontaneously
Other effects:
no effects observed
Description (incidence and severity):
No treatment related effects on TSH and T4 levels (see attached table)
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Mean oestrus cycle was regular at ca 4 days in all treatment groups and controls
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects on testis weight, epididymis weight, sperm count in epididymides, sperm (progressive) motility, sperm morphology.
Normal progression of the spermatogenic cycle in males of all treatment groups and controls
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects on pre-coital interval, Mating index (% mated), no of pregnancies, Gestation length, Gestation index (%), no of live litters born, no of implantations, litter size (before cull) and offspring bw (M/F before cull)
For details see attached table
Possible test item-related microscopic findings after treatment with the substancel gland of females and eyes of males at 50 and/or 250 mg/kg/day. In previous studies with this test item up to 1000 mg/kg/day, inflammatory cell infiltrates were present in many organs and were related with changes in white blood cell count, liver enzymes, several organ weights and splenic extramedullary hematopoiesis. In this study, the increased incidences and/or severity of mainly lymphocytic inflammatory cell infiltrates were noted in a few organs and were recorded at relatively low degrees (up to slight). In contrast to the previous studies, there were no test-item related changes in white blood cell count.
Furthermore, the presence of inflammatory cell infiltrates did occur at similar degrees but at lower incidences in the concurrent control rats (adrenal gland and kidneys) or are described in the literature as background finding. Taking the previous studies and the arguments stated above into consideration, it remains inconclusive whether the (increased incidences) of these infiltrates were related to the test item. Therefore, they were considered as possible test item-related. However, based on their low severities, occurrence in the concurrent control group and absence of any additional related degenerative or proliferative findings, these findings were considered non-adverse.
No test item-related changes were noted in any of the other F0-Generation parameters investigated in this study (i.e. mortality, clinical appearance, haematology, coagulation parameters, total TSH and T4 levels, urinalysis, macroscopic examination, and organ weights).
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No treatment related adverse effects were seen
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
250 mg/kg bw: 2 males (Cohort 1B and 1C)
50 mg/kg bw :1 male (Cohort 1A) and 1 female (Cohort 1C)
controls: 1 male (Cohort 1A)
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
250 mg/kg bw: males significantly decreased during week 3-6 (<5%); females significantly decreased during week 2-7 (<7%)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A

Females at 250 mg/kg bw: increase of white blood cells (22% ns) and, increase of number of lymphocytes (23% ns) --> outlier
Males at 250 mg/kg bw: non significant decrease of white blood cells and lymphocytes
Females at 50 mg/kg bw: increase of white blood cells (5% ns) and increase of number of lymphocytes (8% ns)
Females at 10 mg/kg bw: increase of white blood cells (16% ns) and increase of number of lymphocytes (15% ns) --> outlier

All values were within historical control values
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A

250 mg/kg bw: Females urea 13% increase (ns); Males: ASAT increase 9% (ns); ALP decrease 12% (ns); urea significantly increase 17% (mean remained within the historical control range)
50 mg/kg bw: Males: ASAT increase 9% (ns)
Urinalysis findings:
no effects observed
Description (incidence and severity):
Cohort 1A
Sexual maturation:
no effects observed
Description (incidence and severity):
Balanopreputial separation on day 41.7, 41.9, 41.0 and 42.3 at controls, 10, 50 and 250 mg/kg bw
Vaginal patency on day 31.5, 31.0, 30.9 and 31.6 at controls, 10, 50 and 250 mg/kg bw
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Measured on post-natal day 1
AGD (corrected for cube root of BW) (M/F): 1.54/0.68 mm,1.50/0.64 mm, 1.45/0.63 mm, 1.50/0.68 mm at 0, 10, 50 and 250 mg/kg bw
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Measured on post-natal day 13
no nipples seen in males of any of the dose groups or controls
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A

250 mg/kg bw: males rel weight of mesenteric lymphnodes significantly increased (61%); females abs weight of mesenteric lymphnodes increased 19% (ns)
10 mg/kg bw: females abs weight of mesenteric lymphnodes increased 39% (ns)

Effect in males could be possibly related to treatment (inconclusive)

Cohort 1B
250 mg/kg bw: males abs weight of pituitary increased 12%(ns)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A and 1B
250 mg/kg bw: 1 male (Cohort 1A) thickened mesenteric lymph node (increased macrophage aggregates);
250 mg/kg bw (not considered treatment related) females: reduced size of the thyroid gland (4/20 Cohort 1B); 1 female (Cohort 1A) with lesions in the lungs related to the gavage procedure
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A

Mesenteric lymphnodes increased macrophage aggregates in 4/10 females at 50 mg/kg bw, 8/10 males and 9/10 females at 250 mg/kg bw
Eyes: Inflammatory cell infiltrate (minimal) in the uvea in 1 female at 250 mg/kg bw

The effect in the lymphnodes could be treatment related.

see attached table
Other effects:
no effects observed
Description (incidence and severity):
No treatment related effects on TSH and T4 levels (Cohort 1A)
No treatment related effects on spermatogenesis in males and follicle/corpora lutea numbers in females
No treatment related effects on onset and duration of oestrus cycle in females
Developmental immunotoxicity:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1 A
Splenic Lymphocyte Subpopulation Analysis: no treatment related effects on percentage of T-cells, T-helper cells, T-cytotoxic cells and B-cells
At 250 mg/kg bw significant increase of NK-cells in males (51%)

The higher relative NK-cell counts occurred in absence of substance-related changes in differential white blood cell counts in blood and in the absence of apparent changes in lymphoid cellularity of the spleen as revealed by histopathology evaluation. Moreover, any inflammatory reactions in the F1 generation were absent. Additionally, this change in NK-cell count which occurred in males only could not be linked to the increased macrophage aggregates in mesenteric lymph nodes recorded in both F1 sexes at similar severities.
The results mentioned here are for the F1 pups during lactation until day 21 (weaning);
Live litters born: 25, 24, 21 and 22 at 0, 10, 50 and 250 mg/kg bw
Litter size ( day 1 before cull): 12.2, 10.7, 11.1 and 11.6 at 0, 10, 50 and 250 mg/kg bw
Offspring bw (M/F before cull): 6.6/6.3, 6.9/6.6, 6.7/6.4 and 6.7/6.3 g at 0, 10, 50 and 250 mg/kg bw
Sex ratio (% M before cull): 47, 47, 47 and 56% at 0, 10, 50 and 250 mg/kg bw
Post implantation index (%): 96, 93, 90 and 93% at 0, 10, 50 and 250 mg/kg bw
Postnatal loss (%): 0.3, 0.8, 0.4 and 1.2% at 0, 10, 50 and 250 mg/kg bw
Viability index (day 1-4 before cull): 100, 99, 100 and 99% at 0, 10, 50 and 250 mg/kg bw
Litter size (day 4 after cull): 8.0, 7.8, 7.9 and 7.7 at 0, 10, 50 and 250 mg/kg bw
Litter size (day 21): 8.0, 7.8, 7.9 and 7.7 at 0, 10, 50 and 250 mg/kg bw
Sex ratio (day 21 % males): 50, 47, 46 and 52% at 0, 10, 50 and 250 mg/kg bw
Weaning index (%): 100, 100,100 and 100% at 0, 10, 50 and 250 mg/kg bw
Body weight day 21: 50.8, 53.5, 52.7 and 51.2 g at 0, 10, 50 and 250 mg/kg bw
Body weight gain: no treatment related effects
Clinical signs: no treatment related effects
AGD (corrected for cube root of BW) (M/F): 1.54/0.68 mm,1.50/0.64 mm, 1.45/0.63 mm, 1.50/0.68 mm at 0, 10, 50 and 250 mg/kg bw
Nipple number: none at any of the treatment groups and controls
Thyroid hormones (PND 4) -T4 : no treatment related effects (see table)
Thyroid hormones (PND 22) -T4 (cohort surplus): no treatment related effects (see table)
Organ weights (cohort surplus): Brain, thymus and spleen weight no treatment related effects
Macroscopy: no treatment related effects
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
NOAEL for effects on pups during gestation and lactation
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No effects that could be clearly attributed to treatment
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Results F0 (males until termination after mating, females until weaning F1)

Dose (mg/kg bw)

0

10

50

250

Treatment related

 

Endpoint

M

F

M

F

M

F

M

F

 

 

Mortality

1/25

0/25

0/25

0/25

1/25

0/25

0/25

1/25*

No

 

Clinical signs

NTRE

 

Body weight

 

 

 

 

 

 

↓6 (end of mating)

 

No

 

Body weight gain

 

 

 

 

 

 

↓≤ 11%

 

No

 

Food consumption

NTRE

No

Haematology

 

 

 

WBC ↑21% ns

Lymph ↑21% ns

 

 

WBC ↑17% ns

Lymph ↑18% ns

Retic ↓ 16% ns

 

WBC ↑21% ns

Lymph ↑25% ns

Retic ↓ 15% ns

No

(within HC)

 

Clinical Biochemistry

NTRE

No

 

Thyroid hormones

-TSH

 

NTRE

No

 

 

-T4

NTRE

No

 

Urinalysis

NTRE

No

 

Organ weights

 

 

 

 

 

Thymus ↑18% rel.

 

Thymus ↑18% rel.

No

 

Sperm parameters

NTRE

No

 

Estrous circle

NTRE (4 days)

No

 

Macroscopy

NTRE

No

 

Histopathology

-  Eye uvea infiltrate

-  Adrenals lymphocyte infiltrate

- Kidneys inflammatory cell infiltrate

 

1/25

 

7/25

 

 

6/25

7/25

 

 

 

11/25

 

 

1/25

9/24

 

1/25

 

10/25

 

 

5/25

6/25

 

3/25

2/25

17/25

 

 

12/25

18/25

 

 

No

 

 

 

 

 

 

 

F

F

F

F

 

 

Pre-coital interval

NTRE (1-4 days 96-100%)

No

Mating index (% mated)

100%

100%

100%

100%

No

 

Pregnancies

25

24

22

24

No

 

Gestation length

NTRE (21-23 days) 100%

No

Gestation index (%)

100%

100%

95%

92%

No

 

Live litters born

25

24

21

22

No

 

Live birth index

100%

100%

100%

100%

No

 

Implantations

12.8

11.5

11.8

11.5

No

 

Litter size (before cull)

12.2

10.7↓

11.1↓

11.6

No

 

Offspring bw (M/F before cull)

6.6/6.3

6.9/6.6

6.7/6.4

6.7/6.3

No

 

Sex ratio (% M before cull)

47

47

47

56

No

 

                       

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

HC = historical conrols

% compared to controls

*total litter loss/ difficult parturition on day 23

 

Lactation F1

Dose (mg/kg bw)

0

10

50

250

Treatment related

 

Endpoint

M

F

M

F

M

F

M

F

 

 

Litter size (day 1)

12.2

10.7↓

11.1↓

11.6

No

 

Post implantation index (%)

96%

93%

90%

93%

No

 

Postnatal loss (%)

0.3

0.8

0.4

1.2

No

 

Viability index (day 1-4 before cull)

100

99

100

99

No

 

Litter size (day 4 after cull)

8.0

7.8

7.9

7.7

No

 

Litter size (day 21)

8.0

7.8

7.9

7.7

No

 

Sex ratio (day 21 % males)

50%

47%

46%

52%

No

 

Weaning index (%)

100%

100%

100%

100%

No

 

Body weight day 21

50.8

53.5 ↑*

 52.7

51.2

No

 

Body weight gain

NTRE

No

 

Clinical signs

NTRE

 

 

AGD (corrected for cube root of BW)

1.54 mm

0.68 mm

1.50 mm

0.64 mm

1.45 mm

0.63 mm

1.50 mm

0.68 mm

No

 

Nipple number

NTRE (0 nipples in males)

No

Thyroid hormones (PND 4)  -T4

NTRE

No

Thyroid hormones (PND 22)  -T4 (cohort surplus)

NTRE

No

Brain, thymus and spleen weight (cohort surplus)

NTRE

No

Macroscopy

NTRE

No

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

*expected to be related to the lower litter size

 

Results F1 until termination COHORT 1A/1B/1C

Dose (mg/kg bw)

0

 

10

 

50

 

250

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Mortality

1/60 (1A)

0/60

0/60

0/60

1/60(1A)

1/60 (1C)

2/60(1B/1C)

0/60

No

Clinical signs

NTRE

No

Body weight

 

 

 

 

 

 

↓<5% (WK3-6)

↓<7% (wk 2-7)

No

Food consumption

NTRE

No

Balanopreputial separation (day)

41.7

 

41.9

 

41.0

 

42.3

 

No

Vaginal patency (day)

 

31.5

 

31.0

 

30.9

 

31.6

No

Macroscopy

NTRE

No

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

 

Results F1 until termination COHORT 1A

Dose (mg/kg bw)

0

 

10

 

50

 

250

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Haematology

 

 

 

WBC↑ (16%) ns *

Lymph ↑ (15%) ns *

 

WBC↑ (6%) ns

Lymph ↑ (8%) ns

WBC↓(11%) ns

Lymph↓(14%) ns

WBC↑ (22%) ns *

Lymph

↑ (23%) ns *

No

Clinical Biochemistry

 

 

 

 

ASAT↑(9%) ns

 

ASAT↑(9%) ns

ALP ↓(12%) ns

Urea ↑ (17%)

Urea ↑ (13%) ns

No

Urinalysis

NTRE

No

Splenic lymphocytes

 

 

 

 

 

 

NK cells↑ (51%)

 

No (not adverse)

Thyroid hormones

-TSH

-T4

NTRE

No

Organ weights

 

 

M-lymph ↑ 39% ns

 

 

 

M-lymph ↑ 60% ns (Rel  61%)

M-lymph ↑ 19% ns

Possible (males)

Sperm analysis

NTRE

No

Follicle/corpora lutea

NTRE

No

First estrous cycle (day)

 

33.5

 

35.1

 

33.8

 

35.8

No

Length estrous cycle  between day 75 and 88.

NTRE (4-5 days)

 

Macroscopy

NTRE

No

Histopathology

-M-lymph Increased macrophage aggregates

 

 

 

 

 

 

4/10

 

8/10

 

 

9/10

 

Possible

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

*outlier 553 (10 mg/kg bw) and 692 (250mg/kg bw)


Results F1 until termination COHORT 1B

Dose (mg/kg bw)

0

 

10

 

50

 

250

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Organ weights

 

 

 

 

 

 

Pituitary ↑(12%) ns

 

No

Macroscopy

NTRE

No

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

 

Results F1 until termination COHORT surplus

Dose (mg/kg bw)

0

 

10

 

50

 

250

 

Treatment related

Endpoint

M

F

M

F

M

F

M

F

 

Thyroid hormones

-TSH

-T4

 

NTRE

 

No

Organ weights

NTRE

No

NTRE= no treatment related effects

↑/↓= significantly increased/decreased

% compared to controls

 

 

Conclusions:
NOAEL General toxicity (F0): 250 mg/kg/day.
NOAEL Reproductive toxicity (F0): 250 mg/kg/day.
NOAEL General toxicity (F1): 250 mg/kg/day.
NOAEL Developmental toxicity (F1): 250 mg/kg/day.
Executive summary:

The objective of this study was to provide an evaluation of the pre- and postnatal effects of the substance on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring of Wistar Han rats.  Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. 

In addition, the study provided and/or confirmed information about the effects of the substance on the integrity and performance of the adult male and female reproductive systems.  Specifically, but not exclusively, the following parameters were considered: gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, and lactation. 

The study follows the protocol as described in OECD 443.

The dose levels in this study were selected to be 0, 10, 50 and 250 mg/kg/day.

For the F0-generation, the following parameters and end points were evaluated in this study:  mortality/moribundity, clinical signs, body weight, food consumption, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis, organ weights and histopathologic examinations. 

For the F1-generation, the following parameters and end points were evaluated in this study:  mortality/moribundity, clinical signs, body weight, food consumption, vaginal patency and balanopreputial separation, day of first estrus, estrous cycle determination, clinical pathology including measurement of thyroid hormones and urinalysis, gross necropsy findings, sperm analysis and splenic lymphocyte subpopulation analysis, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy and measurement of thyroid hormones).

Test formulations for the 50 and 250 mg/kg/day groups were prepared accurately, and homogeneity of the 250 mg/kg/day formulations was confirmed. Homogeneity, as well as accuracy, for the 10 mg/kg/day test formulations were outside the acceptance criteria (accuracy and homogeneity values were 102±14, 55±23, 72±10 and 56±19% for Weeks 1, 11, 13 and 22, respectively). Based on the NOAEL values derived from the study results, obtained analytical results were considered adequate for the purpose of this study.

F0-Generation:

No adverse parental effects were observed up to the highest dose level tested (250 mg/kg/day).

No mortality occurred that was considered to be related to treatment with the test item. A total of three F0 animals were sacrificed before scheduled necropsy. One control male was sacrificed in extremis on treatment Day 30 due to a large wound in the neck region. One male at 50 mg/kg/day was sacrificed in extremis on treatment Day 72, due to severe breathing difficulties. Based on necropsy and histopathological findings, this death was attributed to the gavage procedure. One female at 250 mg/kg/day was sacrificed due to total litter loss/difficult parturition on post-coitum Day 23. Since none of the other females at this dose level displayed a similar condition, this was considered to be an incidental occurrence.

Males at 10, 50 and 250 mg/kg/day showed a minor lower weight gain, the duration of which appeared dose-dependent. At 250 mg/kg/day, weight gain remained reduced throughout treatment, along with an occasional lower food consumption during the premating period. At 10 and 50 mg/kg/day, weight gain essentially recovered to control values from Week 4 and Week 9 of treatment, respectively, and food intake appeared unaffected by treatment. At 50 and 250 mg/kg/day, mean body weight gain of males at the end of the treatment duration was 0.94x and 0.89x of the control mean, respectively, and at 10 mg/kg/day, weight gain at the end of treatment was similar to control values. Females at 250 mg/kg/day displayed an occasional higher mean body weight gain during the premating period only. Since these changes in body weight and food consumption were of a minor degree, they were considered not adverse.

At 50 and 250 mg/kg/day, higher potassium was recorded in males, but a clear dose-related trend or histopathological correlates were absent. As such, this change was considered not adverse.

Possible test item-related microscopic findings after treatment with Alkyl Ketene Dimer consisted of increased incidences of inflammatory cell infiltrates in the kidneys of both sexes, adrenal gland of females and eyes of males at 50 and/or 250 mg/kg/day.  In previous studies with this test item up to 1000 mg/kg/day (information provided by the Sponsor), inflammatory cell infiltrates were present in many organs and were related with changes in white blood cell count, liver enzymes, several organ weights and splenic extramedullary hematopoiesis. In this study, the increased incidences and/or severity of mainly lymphocytic inflammatory cell infiltrates were noted in a few organs and were recorded at relatively low degrees, in few cases slight). In contrast to the previous study, there were no test-item related changes in white blood cell count.

Furthermore, the presence of inflammatory cell infiltrates did occur at similar degrees but at lower incidences in the concurrent control rats (adrenal gland and kidneys) or are described in the literature as background finding (eyes: Ref. 2). Taking the previous studies and the arguments stated above into consideration, it remains uncertain whether the (increased incidences) of these infiltrates were related to the test item. Therefore, they were considered as possible test item-related. However, based on their low severities, occurrence in the concurrent control group and absence of any additional related degenerative or proliferative findings, these findings were considered non-adverse.

No test item-related changes were noted in any of the other F0-Generation parameters investigated in this study (i.e. mortality, clinical appearance, haematology, coagulation parameters, total TSH and T4 levels, urinalysis, macroscopic examination, and organ weights).

Reproduction results – F0-generation

No test item-related changes were noted in any of the reproductive parameters investigated up to the highest dose level tested (250 mg/kg/day), i.e. sperm motility, sperm concentration and morphology, mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs including stage-dependent qualitative evaluation of spermatogenesis in the testis.

Developmental results – F0 generation / F1 generation (pre-weaning)

No test item-related changes were noted in any of the developmental parameters investigated up to the highest dose level tested (250 mg/kg/day) during the gestation and lactation phases.

A few pups were found dead or missing between PND 1 and 4. One pup was found dead at first litter check. As this occurred in the control group, this was unrelated to treatment with the test item. One pup of the control group, two pups at 10 mg/kg/day, one pup at 50 mg/kg/day and three pups at 250 mg/kg/day were found dead or missing between PND 2 and 4. Pups missing were most likely cannibalized. These incidences of dead/missing pups were considered not related to treatment since a dose-related trend was absent and incidences remained within the range considered normal for pups of this age.

No test item-related changes were noted in any of the following developmental parameters investigated in this study: gestation index and duration, post-implantation survival index, live birth, viability and weaning indices, parturition, sex ratio, litter size, maternal care and early postnatal pup development consisting of mortality, pup clinical signs, pup body weight, anogenital distance, areola/nipple retention, thyroid hormone levels of pups (T4 of PND 4 pups,  T4 and TSH of PND 22 pups), macroscopic examination and brain, thymus and spleen weight of pups sacrificed at the end of the lactation period.

Developmental results – F1 generation (post-weaning)

No developmental toxicity was observed up to the highest dose level tested (250 mg/kg/day).

No mortality occurred that was considered to be related to treatment with the test item. A total of five animals died prior to scheduled necropsy. One control male was sacrificed in extremis on Day 12 due to severe eye lesions. One female at 50 mg/kg/day died spontaneously with unknown cause on Day 18 of treatment; a relationship with the test item of this single occurrence in F1-females was regarded unlikely. One male at 50 mg/kg/day was sacrificed in extremis on Day 30 of treatment due to a large wound. Two males at 250 mg/kg/day died spontaneously on Day 14 of treatment and were related to the gavage procedure.

At 250 mg/kg/day, mean body weights of males and females were slightly lower between Weeks 3 and 6 or Weeks 2 and 7 for males and females, respectively. These changes were minor and body weights became similar to control values as treatment progressed. Also, food consumption was considered not affected by treatment with the test item. These temporary changes in body weight were therefore considered not adverse.

At 250 mg/kg/day, higher urea concentration was recorded for males (1.17x of control). In absence of a histopathological correlate and given the minor degree of change, this was considered not adverse.

Higher relative NK-cell counts in spleens were recorded for Cohort 1A males at 250 mg/kg/day. Overall, since the higher NK-cell counts occurred in absence of corroborative findings indicative of an affected immune system, this shift was considered not to represent an adverse immunological effect.

A test item-related but non-adverse increase in macrophage aggregates of the mesenteric lymph node were recorded in the F1-Generation at 50 mg/kg/day in females and at 250 mg/kg/day in rats of both sexes (up to marked degree). These macrophage aggregates correlated at 250 mg/kg/day in males to higher mesenteric lymph node weights and to the macroscopic finding of a thickened mesenteric lymph node in a single male. For females at 250 mg/kg/day an increase in mean mesenteric lymph node weight was recorded which was comparable to or even lower than that seen in males at 10 and 50 mg/kg/day, but a relationship to treatment with the test item could not be excluded since this organ was identified as target organ during histopathological examinations.

The mesenteric lymph node is the first draining lymph node after exposure of test item by oral gavage. It is known that macrophages can accumulate and form aggregates when they cannot completely degrade ingested macromolecules (Ref. 3). These macrophage aggregates were only observed in test item dosed animals and were without other test item-related associated degenerative changes or inflammations of the mesenteric lymph node or its drained areas. Therefore, this finding was regarded as non-adverse.

One female at 250 mg/kg/day showed an increased incidence of inflammatory cell infiltrates in the uvea of the eyes. Since this lesion occurred unilaterally and at minimal degree in a single animal, this finding was considered non-adverse.

No treatment-related effects on F1-animals were recorded for other developmental parameters including mortality, clinical signs, body weight and food consumption, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrus, time between vaginal opening and first estrus, length and regularity of the estrous cycle, sperm motility, sperm concentration and morphology, haematology, coagulation parameters, total T4 and TSH levels, urinalysis, splenic lymphocyte subpopulation data (other than NK-cell counts), follicular and corpora lutea count, macroscopy, organ weights and stage-dependent qualitative evaluation of spermatogenesis in the testis).

In conclusion, based on the results of this extended one generation reproductive toxicity study (including Cohort 1), the following no-observed-adverse-effect levels (NOAEL) of the substance were established:

General toxicity (F0):                    250 mg/kg/day.

Reproductive toxicity (F0):           250 mg/kg/day.

General toxicity (F1):                    250 mg/kg/day.   

Developmental toxicity (F1):        250 mg/kg/day.

The highest dose level of 250 mg/kg/day was based on available data from previous toxicity studies in rats. Based on these data, and considering the treatment duration of the current study and possible increased sensitivity of pregnant females, it was judged that this high dose level would result in some toxic effects, but not death nor obvious suffering. Indeed, some toxic effects were recorded at the highest dose level in this study (i.e. lymphocytic inflammatory cell infiltrates recorded in eg. adrenal glands and kidneys in F0 animals). These  were in line with reported effects in previous toxicity studies in rats where (adverse) inflammatory changes were observed microscopically in a variety of tissues. However, the observed inflammatory changes in the current study occurred at a higher dose level than in previous toxicity studies, which may be related to differences in study design.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The NOAEL derived is a worst case estimation based on the effects seen in the OECD 422 study. These effects were not confirmed in the OECD 443 study, but were nevertheless taken into account in the derivation of the NOAEL in a weight of evidence approach
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Available studies


Effects on reproductive performance were evaluated in a combined repeated dose toxicity study with reproductive and developmental toxicity screening in Wistar-derived rats (10/sex/dose) according to OECD Test Guideline 422. The test was performed with Aquapel 364 at doses of 100, 350 or 1000 mg/kg bw/day, which was administered by oral gavage two weeks prior to mating until day four of lactation. There was no effect on male and female reproductive performance and no effect on male reproductive organs. Based on data from five females from each dose group, AKD caused inflammation of the ovaries, uterus and cervix, which was associated with a statistically significant, but not dose related, increase in pre-implantation losses (the ratio of corpora lutea to implantation sites was reduced in all treated groups compared to control group). Few animals, however, showed pre-implantation losses without inflammation. This was most often observed in the control group.
The authors of the study conclude that a NOAEL for reproductive performance could not be achieved in this study due to pre-implantation losses at the lowest dose tested. However, the increased pre-implantation losses (the number of implantation sites compared to corpora lutea was reduced), that are observed at all doses, is correlated with of cervix/uterus inflammations, is most likely a secondary to the general organ inflammation and therefore not a specific reproductive effect. No direct effects on fertility were observed. No effects on male reproductive organs and pup development were observed.


In the EOGRTS according to OECD 443 Wistar rats received 0, 10, 50 and 250 mg/kg bw in 1% CMC with 0.1% Tween 80 during 10 weeks pre-mating. Males were dosed during mating and females were dosed during mating, gestation and lactation. The F1 animals were dosed from weaning day 21 post-natal until day 89-97 (3 cohorts).
No treatment related adverse effects were observed up to the highest dose level tested in the F0 generation (250 mg/kg/day). No test item-related changes were noted in any of the developmental parameters investigated up to the highest dose level tested (250 mg/kg/day) during the gestation and lactation phases. In the F0 animals there were no treatment related effects on estrous cycle, sperm analysis, gestation index and duration, post-implantation survival index, parturition, live births and maternal care. 
For pups there were no treatment related effects on pup mortality, viability and weaning indices, sex ratio, litter size, early postnatal pup development consisting of mortality, pup clinical signs, pup body weight, anogenital distance, areola/nipple retention, thyroid hormone levels of pups (T4 of PND 4 pups,  T4 and TSH of PND 22 pups), macroscopic examination and brain, thymus and spleen weight of pups sacrificed at the end of the lactation period. 
For the F1 as dosed after weaning no developmental toxicity was observed up to the highest dose level tested (250 mg/kg/day). No treatment-related effects on F1-animals were recorded for mortality, clinical signs, body weight and food consumption, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous, length and regularity of the estrous cycle, sperm motility, sperm concentration and morphology, haematology, coagulation parameters, clinical pathology, total T4 and TSH levels, urinalysis, splenic lymphocyte subpopulation data (other than NK-cell counts), follicular and corpora lutea count, macroscopy, organ weights and stage-dependent qualitative evaluation of spermatogenesis in the testis). 
Based on the findings the NOAEL for reproduction toxicity and developmental toxicity is 250 mg/kg bw.


In conclusion, there is no indication of effects on fertility, on male reproductive organs or on pup development. The pre-implantation loss as seen in the OECD 422 study could not be confirmed in the EOGRTS. In this last study no effects at all were seen on reproduction in the F0 and no abnormalities on sexual development were seen in the F1. Therefore it can be concluded that the effects seen in the OECD 422 were indeed secondary to the inflammatory changes in the female reproductive organs. In a weight of evidence and in a worst case approach the NOAEL for reproductive performance is set at 50 mg/kg bw.


 


 


 


 

Effects on developmental toxicity

Description of key information

There were no effects on development in studies according to OECD 422 (rat), 414 (rats and rabbits) and 443 (rat) at the highest dose tested.


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 June 2020 to 26 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
request by ECHA (decision CCH-D-2114440086-55-01/F )
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Strain: New Zealand White
- Age at study initiation: 18-20 weeks
- Weight at study initiation: 2981-4112 g at dosing start
- Fasting period before study: NA
- Housing: individually in cages with perforated floors equipped with water bottles, enriched with shelters and wooden sticks
- Diet: Pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Swizerland) ad libitum
- Water: tap water ad libitum
- Acclimation period: NA

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 19°C
- Humidity (%): 51-95%
- Air changes (per hr): at least 10/h (fresh air)
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% Aqueous carboxymethyl cellulose with 0.1% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: daily as a suspension
Substance dosing formulations (w/w) were homogenized using an ultra-turrax to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 24 hours after adding the vehicle to the test item.
Substance dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

VEHICLE
- Justification for use and choice of vehicle:based on trial preparations
- Concentration in vehicle: 20, 60 and 200 mg/mL at 100, 300 and 1000 mg/kg bw
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
UPLC/UV

Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Acquity UPLC PDA detector (Waters)
Column Acquity UPLC BEH C18, 50 mm x 2.1 mm i.d., dp =1.7 µm (Waters)
Column temperature 40°C ± 1°C
Injection volume 1 and 2 µL
Mobile phase Methanol
Flow 0.6 mL/min
UV detection 220 nm

Method development
Calibration: at 80-600 mg/mL, r> 0.99 (see attached document)
QC samples: at 1 mg/ml 98-104% of nominal; at 200 mg/mL 99-102% of nominal
Accuracy: day of preparation: at 1 mg/mL 99% of nominal; at 200 mg/mL 93% of nominal
Homogeneity: confirmed CV at 1 mg/mL 5.4%; CV at 200 mg/mL 0.9 %
Stability in refrigerator: 3 days at 1 mg/mL 83% of nominal (CV 10%); 3 days at 200 mg/mL 97% of nominal (CV 0.94) --> not stable -->daily formulation

Sample analyses:
Calibration: at 80-600 mg/mL r> 0.99 (see attachment)
QC: at 20 mg/mL 105% of nominal; at 200 mg/mL 99% of nominal
Accuracy: at 100, 300 and 1000 mg/kg bw: 96, 91 and 94% of nominal, respectively
Homogeneity: at 100 and 1000 mg/kg bw: CV 4.3% and 3.9%, respectively
Details on mating procedure:
NA, time-mated females
Duration of treatment / exposure:
Day 7 to Day 28 post-coitum, inclusive
Frequency of treatment:
daily
Duration of test:
21 days
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:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: based on a dose range finding studies where 6 time-mated female rabbits/group were exposed to the substance at 0, 100, 300 and 1000 mg/kg bw by gavage from day 7 to 28 post-coitum. There were no effects on body weight (gain), food consumption and macroscopy. Reduced faeces production was seen among all dose groups. At 1000 mg/kg bw an increase of implantation loss and resorptions with concomitant lower litter size was reported. Fetal weights were above historical control values at 1000 mg/kg bw. No external abnormalities were observed at any dose level.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily day 7 to 28

BODY WEIGHT: Yes
- Time schedule for examinations: day 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: daily

WATER CONSUMPTION : Yes
- Time schedule for examinations: daily visual observation of water bottles

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- Organs examined: macroscopic external, thoracic and abdominal examination, with special attention being paid to the reproductive organs
Ovaries and uterine content:
• The number of corpora lutea.
• The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of early and late resorptions.
Fetal examinations:
gross external examination of late resorptions
external, visceral and skeletal examinations of litters of surviving females
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Tests conducted, where applicable: Dunnett-test (many-to-one-t-test), Steel-test (many-to-one rank test), Mann Whitney test, Kruskal-Wallis nonparametric ANOVA test and Fisher’s exact test.
Indices:
Pre-implantation loss (%)
Postimplantation loss (%)
Historical control data:
included in the report: fetal examinations
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced faeces production in all groups and controls during the observation period.
Piloerection was seen occasionally in all groups and controls.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female was sacrificed after early delivery on day 23
One female at 300 mg/kg bw was killed in extremis on day 28 (gavage error)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight of treated animals was similar to the control level over the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
measured qualitatively
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No effect on uterus weight.
No other organs examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Watery cyst(s) on the oviducts or ectopic splenic tissue were considered to be unrelated to treatment in absence of a dose response relationship (see table)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Two females did not survive until scheduled necropsy (one in the control group, one at 300 mg/kg/day). As these premature deaths were considered to be incidental or have occurred as the result of the gavage procedure, they were unrelated to treatment with the test item.
No maternal toxicity was observed in the 100, 300 and 1000 mg/kg/day groups.
Number of abortions:
no effects observed
Description (incidence and severity):
One early delivery in the control group
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The dosing has started on day 7 post-coitum, when implantation is expected to be completed, therefore any effects on this parameter cannot be attributed to exposure to the substance.

Pre-implementation loss (%): 9.9, 15.8, 13.0 and 12.6 % in controls and at 100, 300 and 1000 mg/kg bw/day (mean no. per litter 1.3, 1.5, 1.7 and 1.7 in controls and at 100, 300 and 1000 mg/kg bw/day)
Post-implementation loss (%): 9.3, 4.1, 3.5 and 4.0% in controls and at 100, 300 and 1000 mg/kg bw/day
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
None
Early or late resorptions:
no effects observed
Description (incidence and severity):
Early resorptions (mean/dam): 0.6, 0.4, 0.3 and 0.4 in controls and at 100, 300 and 1000 mg/kg bw/day
Late resorptions (mean/dam): 0.5, 0.1, 0.1 and 0.1 in controls and at 100, 300 and 1000 mg/kg bw/day
Dead fetuses:
no effects observed
Description (incidence and severity):
none
Changes in pregnancy duration:
not examined
Description (incidence and severity):
not applicable
Changes in number of pregnant:
not examined
Description (incidence and severity):
not applicable, as time-mated females were used.
no of pregnant females: 20, 18, 21 and 19 in controls and at 100, 300 and 1000 mg/kg bw/day
Details on maternal toxic effects:
No effects on maternal developmental toxicity. The slightly, but non-significantly increased pre-implantation loss depicted as percentage could be attributed to individual females and as, dosing started on day 7 post-coitum, when implantation is expected to be completed, any effects on this parameter cannot be attributed to exposure to the substance.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: No adverse effects observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No treatment related effects up to 1000 mg/kg bw in both sexes
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio young (% males): 54.5, 50.0, 43.6 and 52.5% in controls and at 100, 300 and 1000 mg/kg bw/day
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean viable fetuses/dam: 9.4, 9.1,9.4 and 9.6 in controls and at 100, 300 and 1000 mg/kg bw/day
Mean fetal weight: 40.3, 40.2, 40.5 and 40.6 g in controls and at 100, 300 and 1000 mg/kg bw/day
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Incidental findings (no dose response and/or within historical controls):

At 1000 mg/kg/day: spina bifida and flexure of both tarsals in one fetus, short tail in one fetus and malrotated hindlimbs in one fetus.
At 300 mg/kg/day: distended abdomen (related to various cardiovascular defects) in one fetus
At 100 mg/kg/day: multiple malformations, namely meningocele, brachydactyly, malpositioned digits and carpal flexure in one fetus.
Controls: anencephaly (with matching skeletal findings) and carpal flexures in one fetus

Late resorptions in control: carpal flexure, besides mass in neck in one and umbilical hernia in another resorption
Skeletal malformations:
no effects observed
Description (incidence and severity):
Incidental findings (No dose response and/or within historical controls):

At 1000 mg/kg bw/day: fused skull bones in one fetuse, vertebral anomaly in one fetuse, vertebral centrum anomaly in the fetus with spina bifida.
Controls: vertebral anomaly or costal cartilage anomaly in two littermates
Visceral malformations:
no effects observed
Description (incidence and severity):
Incidental findings (no dose response and/or within historical controls):

At 1000 mg/kg/day: malpositioned and fused kidneys in the fetus with the short tail
At 300 mg/kg/day: malpositioned kidney in one fetus, cardiovascular malformations in the fetus with distended abdomen, multiple cardiovascular abnormalities in one fetus
At 100 mg/kg/day: internal hydrocephaly in one fetus, malpositioned testis and tetralogy of Fallot in one fetus, several cardiovascular defects in two fetuses
Controls: lung cyst in one fetus or persistent truncus arteriosus in the fetus with anencephaly
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Variations reported ((no dose response and/or within historical controls) included:
Visceral: Left carotid originating from branchiocephalic trunk, absent/small gall bladder, supernumerary artery from aortic arch, absent accessory lung lobe;
Skeletal: 13th full/rudimentary ribs, caudal shift of pelvic gridle, unossified sternabrae, malaligned sternabrae
Details on embryotoxic / teratogenic effects:
No substance-related changes were noted in sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations.
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:
Remarks on result:
other: no adverse effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no





























































































































































Dose (mg/kg bw)/


Endpoint



0



100



300



1000



Treatment related



Mortality



1/22*



1/22



1/22#



0/22



no



Clinical signs


- reduced faeces production



 


+



 


+



 


+



 


+



no



Body weight (gain)



NTRE



no



Food consumption day 0-29



NTRE



no



Uterus weight



NTRE



no



Macroscopy


- Oviducts, Cyst(s)


- Ectopic splenic tissue



 


6/22


5/22



 


4/22


8/22



 


6/22


4/22



 


4/22


4/22



no



Pregnancy rate



20/22



18/22



20/22



19/22



no



Corpora lutea (mean/dam)



11.7



11.3



11.3



11.7



no



Implantations (mean/dam)



10.4



9.6



9.8



10.0



no



Early resorptions (mean/dam)



0.6



0.4



0.3



0.4



no



Late resorptions (mean/dam)



0.5



0.1



0.1



0.1



no



Pre-implementation loss (%)



9.9



15.8



13.0



12.6



no



Post-implementation loss (%)



9.3



4.1



3.5



4.0



no



Live young/Litter



9.4



9.1



9.4



9.6



no



Sex ratio young (% males)



54.5



50.0



43.6



52.5



no



Fetal weight



NTRE



no



Fetal major abnormalities External (no. affected)**



1



1



1



3



no



Fetal malformations Visceral (no. affected)**



2



4



3



1



no



Fetal malformations Skeletal (no. affected)**



2



0



0



3



no



Fetal variations##



NTRE



no



NTRE= no treatment related effects


↑/↓= significantly increased/decreased


% compared to controls


*Sacrificed after early delivery on day 23


# Killed in extremis on day 28 (gavage error)


** At 1000 mg/kg bw: exencephaly, spina bifida and flexure of both tarsals in one fetus; short tail +mal-positioned and fused kidneys + vertebral centrum anomaly in one fetus;  mal-rotated hindlimbs in one fetus; fused skull bones in one fetus; vertebral anomaly in one fetus


      at 300 mg/kg bw: distended abdomen+ cardiovascular malformations in one fetus; multiple cardiovascular abnormalities in one fetus; mal-positioned kidneys  in one fetus


      at 100 mg/kg bw: meningocele, brachydactyly, malpositioned digits and carpal flexure + several cardiovascular defects in one fetus; internal hydrocephaly in one fetus; mal-positioned testis and tetralogy of Fallot  in one fetus; several cardiovascular defects in one fetus


     in controls: anencephaly and carpal flexures + persistent truncus arteriosus in one fetus; lung cyst in one fetus; vertebral anomaly in one fetus; costal cartilage anomaly in one fetus


##  Findings without dose response included: Visceral: Left carotid originating from branchiocephalic trunk, absent/small gall bladder, supernumerary artery from aortic arch, absent accessory lung lobe; Skeletal: 13th full/rudimentary ribs, caudal shift of pelvic gridle, unossified sternabrae, malaligned sternabrae

Conclusions:
No substance-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).
Executive summary:

In a study according to OECD 414 female (22 time-mated/group) rabbits were exposed to 0, 100, 300, 1000 mg/kg/day during day 7 to 28 post-coitum. Females were necropsied at day 29 post-coitum and the uterus content was examined.


No effects on maternal toxicity were seen on mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.


For the fetuses no effects were reported on the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations


Two females did not survive until scheduled necropsy (one in the control group, one at 300 mg/kg/day). As these premature deaths were considered to be incidental or have occurred as the result of the gavage procedure, they were unrelated to treatment with the test item.


In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for the substance of 1000 mg/kg/day was established.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 November 2019 to 19 December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
request by ECHA (decision CCH-D-2114440086-55-01/F )
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Strain: Wistar Han
- Age at study initiation: 10-14 weeks (females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating))
- Weight at study initiation: 192-258 g
- Fasting period before study: NA
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: Municipal tap water ad libitum
- Acclimation period: NA

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean 20.9-21.4°C
- Humidity (%): 52-54%
- Air changes (per hr): at least 10/hr
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% Aqueous carboxymethyl cellulose with 0.1% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: daily as a suspension
Substance dosing formulations (w/w) were homogenized using an ultra-turrax to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 24 hours after adding the vehicle to the test item.
Substance dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.

VEHICLE
- Justification for use and choice of vehicle:based on trial preparations
- Concentration in vehicle: 8, 40 and 200 mg/mL at 40, 200 and 1000 mg/kg bw
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
UPLC/UV

Instrument Acquity UPLC system (Waters, Milford, MA, USA)
Detector Acquity UPLC PDA detector (Waters)
Column Acquity UPLC BEH C18, 50 mm x 2.1 mm i.d., dp =1.7 µm (Waters)
Column temperature 40°C ± 1°C
Injection volume 1 and 2 µL
Mobile phase Methanol
Flow 0.6 mL/min
UV detection 220 nm

Method development
Calibration: at 80-600 mg/mL, r> 0.99 (see attached document)
QC samples: at 1 mg/ml 98-104% of nominal; at 200 mg/mL 99-102% of nominal
Accuracy: day of preparation: at 1 mg/mL 99% of nominal; at 200 mg/mL 93% of nominal
Homogeneity: confirmed CV at 1 mg/mL 5.4%; CV at 200 mg/mL 0.9 %
Stability in refrigerator: 3 days at 1 mg/mL 83% of nominal (CV 10%); 3 days at 200 mg/mL 97% of nominal (CV 0.94) --> not stable -->daily formulation

Sample analyses:
Calibration: at 80-600 mg/mL r> 0.99 (no details)
QC: at 8 mg/mL 104% of nominal; at 200 mg/mL 98% of nominal
Accuracy: at 40, 200 and 1000 mg/kg bw: 53, 96 and 98% of nominal, respectively
Homogeneity: at 40 and 1000 mg/kg bw: CV 3.8% and 0.66%, respectively

The accuracy at 40 mg/kg bw was below the 85% specification. Therefore the concentration tested at 40 mg/kg bw could have been lower than indicated. This does not affect the study outcome, as a NOAEL of 1000 mg/kg bw was derived. For this NOAEL all analytical specifications were met.
Details on mating procedure:
Purchased timed pregnant (arriving on Day 0 or Day 1 post-coitum)
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum, inclusive
Frequency of treatment:
daily
Duration of test:
21 days
Dose / conc.:
40 mg/kg bw/day
Remarks:
measured 53% of nominal
Dose / conc.:
200 mg/kg bw/day
Remarks:
measured 96% of nominal
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
measured 98% of nominal
No. of animals per sex per dose:
22 pregnant females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
In a OECD 422 study (28-day study combined with the reproduction and developmental screening test), Alpk:APfSD (Wister-derived) rats were dosed by gavage at 0 (corn oil only), 100, 350 or 1000 mg/kg/day. There were no mortalities or clinical symptoms, no adverse effects on body weight or food consumption and no adverse effects on reproductive or developmental parameters up to 1000 mg/kg/day. However, in both sexes at 1000 mg/kg/day, inflammatory changes were observed microscopically in a variety of tissues (overall incidence, severity and distribution was greater in females) in combination with an increase in total white blood cell counts (due to increases in large unstained cells, lymphocytes and monocytes), increased liver enzyme activity, increases in spleen, liver, kidney and ovary weight and splenic extramedullary haemopoiesis. In addition, the spleen and lymph nodes of females were enlarged at necropsy. The majority of these effects were seen at decreased severity and/or incidence in animals dosed at 350 or 100 mg/kg/day, but some of the microscopic changes were not observed in a dose-related relationship. Nevertheless, no NOAEL was established in this study.
Based on the results of the OECD 422 study, Alpk:APfSD (Wister-derived) rats received 0, 65, 650 or 6500 ppm by dietary administration, resulting in dose levels of 0, 6-7, 63-70 or 645-691 mg/kg/day, during an OECD 408 study (90-day study). There were no mortalities or clinical symptoms up to 6500 ppm. A decrease in body weight and food consumption was noted in animals at 6500 ppm. Similar effects were described at 6500 ppm as observed during the OECD 422 study, consisting of a decrease in red blood cell parameters, increased white blood cell counts, increased liver enzyme activity, increased spleen and liver weights and treatment-related inflammatory changes in a variety of tissues in both sexes. The inflammatory changes included foci of inflammation and histiocytosis. In addition, kidney weight was increased at this dose level in females only. At 650 ppm, there were similar findings but of lesser severity and/or incidence when compared with the 6500 ppm group and at 65 ppm there were no changes of biological or toxicological relevance when compared with controls.
In the current study, the duration of exposure was only 14 days (Day 6-20 post-coitum). Considering this information and the results of the previously performed toxicity studies, 40 mg/kg/day was selected as low dose as this dose level is anticipated to be a ‘No Adverse Effect Level’ (NOAEL). A dose level of 1000 mg/kg/day was selected as high dose, as this was considered likely to result in some toxicity similar to the effects observed in previous studies. A mid dose level of 200 mg/kg/day was selected as this was at an appropriate interval between the low and high dose.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily from day 2 to 20

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18 21 post-coitum

WATER CONSUMPTION: Yes, visual inspection
- Time schedule for examinations: on regular basis

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy: Day 21
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 10 females
- Parameters examined: White blood cells (WBC),Neutrophils (absolute),Lymphocytes (absolute),Monocytes (absolute),Eosinophils (absolute),Basophils (absolute),Large unstained cells (LUC), (absolute),Red blood cells, Reticulocyte (absolute),Red Blood Cell Distribution Width (RDW),Haemoglobin,Haematocrit,Mean corpuscular volume (MCV),Mean corpuscular haemoglobin (MCH),Mean corpuscular haemoglobin concentration (MCHC),Platelets

BIOCHEMISTRY : Yes
- Time schedule for collection of blood: prior to necropsy: Day 21
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: 10 females
- Parameters examined: Triiodothyronine (T3), Thyroxine (T4), Thyroid-Stimulating Hormone (TSH)

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: external, thoracic and abdominal examination

ORGAN WEIGHTS: Yes
- Organs examined: uterus, thyroid

HISTOPATHOLOGY: yes, thyroid
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not for female that delivered early)
- Number of corpora lutea: Yes
- Number of implantations: Yes (using Salewski staining)
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The sex of each fetus based on the anogenital distance.
Fetal examinations:
- Fetal body weight
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litte]

Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Dunnett test, Steel-test, Mann Whitney test, Kruskal-Wallis nonparametric ANOVA test
Indices:
Preimplantation loss (%):
Postimplantation loss (%):
Viable fetuses affected/litter (%):

Historical control data:
For fetal examinations included in the report
Thyroid hormones:
TSH (ulU/mL); mean (P5-P95): 0.359 (0.1280-0.7140)
Total T3 (ng/dL); T3 mean (P5-P95): 58.0 (40.50-81.90)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection at 200 mg/kg bw and 1000 mg/kg bw at the end of the treatment period. No dose response
Alopecia incidental at 40 mg/kg bw and 200 mg/kg bw (within range of background findings)
Mortality:
no mortality observed
Description (incidence):
One female at 40 mg/kg/day was sacrificed on the day of scheduled necropsy as this female delivered her offspring prior to necropsy.
Body weight and weight changes:
no effects observed
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
Description (incidence and severity):
no abnormalities reported
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significantly lower T3 levels in females at 40 and 200 mg/kg/day were observed, whereas T3 levels at 1000 mg/kg bw/day were unaffected. Total T3 concentrations of 16/22 at 40 mg/kg/day and 11/20 females at 200 mg/kg/day were below the lower limit of quantification (40 ng/dL), compared to 7/21 females in the control group and 3/22 females at 1000 mg/kg/day. Mean values were below available historical control range at both 40 and 200 mg/kg/day, .
Decreased serum levels of thyroid-stimulating hormone (TSH) were noted at 1000 mg/kg/day (0.65x of controls). This decrease was not statistically significant, however, based on the magnitude of change, a test item related effect could not be excluded. As the mean value remained within the available historical control data , the decrease was considered non adverse.
Serum levels of total T4 were considered to be unaffected by treatment up to 1000 mg/kg/day.
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
incidental: Hypertrophy follicular cells of thyroid gland
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
see attached tables

No maternal toxicity was observed up to 1000 mg/kg bw/day.
Decreased thyroid-stimulating hormone levels were noted at 1000 mg/kg bw/day. Based on the magnitude of change, although not statistically significant, a test-item related effect could not be excluded. However, as the mean value remained within the available historical control data, this was considered to be non-adverse. In the animals at 1000 mg/kg bw/day showing a TSH concentration below historical control ranges (7/22), no effects on thyroid weight and/or histopathology of the thyroid was reported with the exception of Female No. 71 that was observed with lower thyroid weights and Female No. 79 that showed minimal follicular cell hypertrophy of the thyroid.
Additionally, statistically significantly lower triiodothyronine (T3) levels were observed at 40 and 200 mg/kg bw/day, whereas. T3 levels at 1000 mg/kg bw/day were unaffected. T3 concentrations below the lower limit of quantification (LLOQ; 40 ng/dL) were reported as LLOQ/2 (20 ng/dL). It should be noted that the relevance of this finding remains unclear, in absence of a distinct dose relationship, unaffected T4 levels and the fact that the findings on thyroid parameters (including TSH, weight and histopathology) were scattered among animals, showing no consistency. Besides that, the downstream biological consequences of thyroid hormone changes are not assessed within this type of study and was therefore not taken into account when determining the maternal NOAEL.
No test item-related changes were noted in the remaining maternal parameters investigated in this study (i.e. mortality/moribundity, clinical appearance, body weight, food consumption, total thyroxine levels, haematology, organ weights, uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss).
Number of abortions:
no effects observed
Description (incidence and severity):
at 40 mg/kg bw: one early delivery on day of scheduled necropsy
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implementation loss (%) 3.7, 4.9, 3.9 and 5.3% at 0, 40, 200 and 1000 mg/kg bw
Post-implementation loss (%) 3.1, 5.1, 3.0 and 1.8% at 0, 40, 200 and 1000 mg/kg bw
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
Early resorptions: 0.4, 0, 0.6, 0.3 and 0.2 mean/dam at 0, 40, 200 and 1000 mg/kg bw
Late resorptions: 0.0, 0.0, 0.0 and 0.0 mean/dam at 0, 40, 200 and 1000 mg/kg bw
Dead fetuses:
no effects observed
Description (incidence and severity):
Live young/Litter 10.4, 10.9, 10.5 and 10.7 at 0, 40, 200 and 1000 mg/kg bw
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Details on maternal toxic effects:
see attached tables
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: absence of treatment related effects
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean fetal weight 5.3 g in all dose groups
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetuses in any of the dose groups
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio young (% males): 48.3, 51.3, 50.5 and 51.1% at 0, 40, 200 and 1000 mg/kg bw
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
exencephaly, lower jaw and cleft palate in two foetuses at 1000 mg/kg bw (incidental)
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Vertebral anomaly and/or polydactyly in two foetuses at 1000 mg/kg bw and bent limb bones in one foetus at 200 mg/kg bw (within historical controls)

Variations:
Malaligned sternebrae: 7/21 (8/108 fetuses (6.7% per litter)) in control, 5/21 (6/116 fetuses (6.0% per litter)) at 40 mg/kg bw; 6/22 (7/117 fetuses(5.7% per litter)) at 200 mg/kg bw and 12/22 (16/120 fetuses (12.6% per litter)
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
situs inversus in one foetus at 1000 mg/kg bw (spontaneous)
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No developmental toxicity was observed up to 1000 mg/kg bw/day.
At 1000 mg/kg bw/day, the incidence of the variation “malaligned sternebrae” was just outside the historical control data and therefore, a test item-related effect could not be excluded. However, as this increased incidence concerns a variation, this was considered to be non adverse.
No test item-related changes were noted in the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and/or developmental variations).
Details on embryotoxic / teratogenic effects:
see attached tables
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: absence of treatment related malformations an variations
Abnormalities:
effects observed, non-treatment-related
Description (incidence and severity):
malaligned sternabrae slightly above historical control values at 1000 mg/kg bw
Developmental effects observed:
no








































































































































































































Dose (mg/kg bw)/


Endpoint



0



40



200



1000



Treatment related



Mortality



0/22



1/22*



0/22



0/22



 



Clinical signs


-piloerection


-alopecia



 



 


 


+



 


 


+



 


+


 



no



Body weight



 



 



↓ day 2-15 <5%



 



no



Body weight gain



NTRE



 



Food consumption day 0-20



NTRE



 



Haematology



NTRE



 



Thyroid hormones


-TSH



 


 



 


 



 


 



 


↓ (35% ns)



Value within historical controls



-T3



 



↓ (39%)



↓ (31%)



 



No dose response



-T4



NTRE



 



Thyroid weight



NTRE



 



Uterus weight



NTRE



 



Macroscopy



NTRE



 



Histopathology thyroid


-Hypertrophy follicular cells



 


3/22



 


5/22



 


6/22



 


4/22



 



Pregnancy rate



21/22



22/22



22/22



22/22



 



Corpora lutea (mean/dam)



11.2



12.1



11.3



11.5



 



Implantations (mean/dam)



10.8



11.5



10.9



11.0



 



Early resorptions (mean/dam)



0.4



0.6



0.3



0.2



 



Late resorptions (mean/dam)



0.0



0.0



0.0



0.0



 



Pre-implementation loss (%)



3.7



4.9



3.9



5.3



 



Post-implementation loss (%)



3.1



5.1



3.0



1.8



 



Live young/Litter



10.4



10.9



10.5



10.7



 



Sex ratio young (% males)



48.3



51.3



50.5



51.1



 



Anogenital distance (M/F)



NTRE



 



Litter weight



NTRE



 



Fetal weight



NTRE



 



Fetal major abnormalities


External, visceral and skeletal#



 


NTRE



Values within historical controls



Fetal variations Visceral



NTRE



 



Fetal variations Skeletal


-Malaligned sternebrae **



 


7/21 (8/108 fetuses


(6.7% per litter))



 


5/21 (6/116 fetuses (6.0% per litter))



 


6/22 (7/117 fetuses


(5.7% per litter))



 


12/22 (16/120 fetuses (12.6% per litter)



 


 



NTRE= no treatment related effects


↑/↓= significantly increased/decreased


% compared to controls


*Killed in extremis on day of scheduled necropsy (early delivery)


# exencephaly, lower jaw and cleft palate in two foetuses at 1000 mg/kg bw (incidental); situs inversus in one foetus at 1000 mg/kg bw (spontaneous); vertebral anomaly and/or polydactyly in two foetuses at 1000 mg/kg bw and bent limb bones in one foetus at 200 mg/kg bw (within historical controls)


** historical controls: 3.0 - 11.7% per litter

Conclusions:
Based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for the substance of 1000 mg/kg bw/day was established.
Executive summary:

In a test according to OECD 414 female rats (time-mated) were exposed to the substance at 0, 40, 200 and 1000 mg/kgbw by gavage from day 6 to 20 post coitum. Test substance formulations prepared were considered homogeneous at the concentrations tested, but target concentration at 40 mg/kg bw was below specifications. This does not affect the study results, as the NOAEL is 1000 mg/kg bw.
No maternal and developmental adverse effects were observed up to 1000 mg/kg bw/day.


There were no effects on mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, thyroid weights, uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss for maternal animals.
Decreased thyroid-stimulating hormone levels were noted at 1000 mg/kg bw/day. Based on the magnitude of change, although not statistically significant, a test-item related effect could not be excluded. However, as the mean value remained within the available historical control data, this was considered to be non-adverse. Additionally, statistically significantly lower T3 levels were observed at 40 and 200 mg/kg bw/day, whereas T3 levels at 1000 mg/kg bw/day were unaffected. It should be noted that the relevance of this finding remains unclear, in absence of a distinct dose relationship, unaffected T4 levels and the fact that the findings on thyroid parameters (including TSH, weight and histopathology), if any, were scattered among animals, showing no consistency.


For  the F1-generation the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations were unaffected. At 1000 mg/kg bw/day, the incidence of the variation “malaligned sternebrae” was just outside the historical control data and therefore, a test item-related effect could not be excluded. However, as this increased incidence concerns a variation, this was considered to be non-adverse.
In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for the substance of 1000 mg/kg bw/day was established.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
24 July 2001 - 28 January 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to internationally accepted guidelines and under GLP. No CoA attached to study report. A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in the rat was performed. For a full summary of this study refer to section 7.8.1.
Remarks:
Doses / Concentrations:

Basis:

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Available studies


Effects on reproductive performance were evaluated in a combined repeated dose toxicity study with reproductive and developmental toxicity screening in Wistar-derived rats (10/sex/dose) according to OECD Test Guideline 422. The test was performed with Aquapel 364 at doses of 100, 350 or 1000 mg/kg bw/day, which was administered by oral gavage two weeks prior to mating until day four of lactation. There was no effect on male and female reproductive performance and no effect on male reproductive organs. Based on data from five females from each dose group, AKD caused inflammation of the ovaries, uterus and cervix, which was associated with a statistically significant, but not dose related, increase in pre-implantation losses (the ratio of corpora lutea to implantation sites was reduced in all treated groups compared to control group). Few animals, however, showed


The authors of the study conclude that a NOAEL for reproductive performance could not be achieved in this study due to pre-implantation losses at the lowest dose tested. However, the increased pre-implantation losses (the number of implantation sites compared to corpora lutea was reduced), that are observed at all doses, are, are most likely a secondary to the general organ inflammation and therefore not a specific reproductive effect. No direct effects on fertility were observed. No effects on male reproductive organs and pup development were observed.
In a test according to OECD 414 female Wistar rats (time-mated) were exposed to the substance at 0, 40, 200 and 1000 mg/kg bw by gavage in 1% CMC with 0.1% Tween 80 from day 6 to 20 post coitum. There were no effects on mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, thyroid weights, uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and post-implantation loss for maternal animals. Decreased thyroid-stimulating hormone levels were noted at 1000 mg/kg bw/day. Based on the magnitude of change, although not statistically significant, a test-item related effect could not be excluded. However, as the mean value remained within the available historical control data, this was considered to be non-adverse. Additionally, statistically significantly lower T3 levels were observed at 40 and 200 mg/kg bw/day, whereas T3 levels at 1000 mg/kg bw/day were unaffected. It should be noted that the relevance of this finding , in absence of a distinct dose relationship, unaffected T4 levels and the fact that the findings on thyroid parameters (including TSH, weight and histopathology), if any, were scattered among animals, showing no consistency. In addition, in absence of information on the downstream biological consequences of thyroid hormone changes within this type of study, the findings were not taken into account when determining the maternal NOAEL.
For  the fetuses the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations were unaffected. At 1000 mg/kg bw/day, the incidence of the variation “malaligned sternebrae” was just outside the historical control data and therefore, a test item-related effect could not be excluded. However, as this increased incidence concerns a variation, this was considered to be non-adverse.
In conclusion, based on the results in this prenatal developmental toxicity study a  developmental No Observed Adverse Effect Level (NOAEL) for the substance of 1000 mg/kg bw/day was established. The maternal NOAEL was set at 1000 mg/kg bw/day in absence of adverse effects at any dose levels.


In a study according to OECD 414 female (22 time-mated/group) rabbits were exposed to 0, 100, 300, 1000 mg/kg/day in 1% CMC with 0.1% Tween 80 during day 7 to 28 post-coitum. Females were necropsied at day 29 post-coitum and the uterus content was examined. No effects on maternal toxicity were seen on mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents.
For the fetuses no effects were reported on the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.
In conclusion, based on the results in this prenatal developmental toxicity study a maternal and developmental No Observed Adverse Effect Level (NOAEL) for the substance of 1000 mg/kg/day was established.


In the EOGRTS according to OECD 443 rats received 0, 10, 50 and 250 mg/kg bw in 1% CMC with 0.1% Tween 80 during 10 weeks pre-mating. Males were dosed during mating and females were dosed during mating, gestation and lactation. The F1 animals were dosed from weaning day 21 post-natal until day 89-97 (3 cohorts). 
No treatment related adverse effects were observed up to the highest dose level tested in the F0 generation (250 mg/kg/day). No test item-related changes were noted in any of the developmental parameters investigated up to the highest dose level tested (250 mg/kg/day) during the gestation and lactation phases. There were no treatment related effects on estrous cycle, sperm analysis, gestation index and duration, post-implantation survival index, parturition, live births and maternal care.
For pups there were no treatment related effects on pup mortality, viability and weaning indices, sex ratio, litter size, early postnatal pup development consisting of mortality, pup clinical signs, pup body weight, anogenital distance, areola/nipple retention, thyroid hormone levels of pups (T4 of PND 4 pups,  T4 and TSH of PND 22 pups), macroscopic examination and brain, thymus and spleen weight of pups sacrificed at the end of the lactation period.
For the F1 as dosed after weaning no developmental toxicity was observed up to the highest dose level tested (250 mg/kg/day). No treatment-related effects on F1-animals were recorded for mortality, clinical signs, body weight and food consumption, balanopreputial separation (prepuce opening), vaginal patency (vaginal opening), occurrence of first estrous, time between vaginal opening and first estrous, length and regularity of the estrous cycle, sperm motility, sperm concentration and morphology, haematology, coagulation parameters, clinical pathology, total T4 and TSH levels, urinalysis, splenic lymphocyte subpopulation data (other than NK-cell counts), follicular and corpora lutea count, macroscopy, organ weights and stage-dependent qualitative evaluation of spermatogenesis in the testis).
Based on the findings the NOAEL for reproduction toxicity and developmental toxicity is 250 mg/kg bw.


In conclusion, there is no indication of effects on development or teratogenicity in the available studies on both rats and rabbits. The NOAEL for developmental toxicity is therefore concluded to be 1000 mg/kg bw in absence of adverse effects in any of the studies at the highest dose tested.

Justification for classification or non-classification

The substance is not classified for reproductive and developmental toxicity  based on the available studies.
The substance is not classified for effects on fertility since existing studies give no indication of toxicity.

Additional information